Method for characterizing at least one microorganism by means of mass spectrometry

Abstract

A method for characterizing at least one microorganism from a sample includes identifying the at least one microorganism and determining the properties of typing, potential resistance to at least one antimicrobial, and virulence factor. The properties of typing, resistance to at least one antimicrobial, and virulence factor for the at least one microorganism are determined by implementing mass spectrometry using proteins, peptides and/or metabolites as markers of the properties of typing, resistance to at least one antimicrobial, and virulence factor.

Claims

1. A method for characterizing a microorganism, comprising: identifying the microorganism; and subjecting the microorganism identified to mass spectrometry with a spectrometer in MS/MS mode using multiple reaction monitoring (MRM) to identify markers of properties of typing, potential resistance to at least one antimicrobial, and virulence factor, of the microorganism, wherein: the markers are selected from the group consisting of proteins, peptides, and metabolites, and the microorganism is a bacterium, a virus, a protozoan, or a yeast.

2. The method for characterizing a microorganism of claim 1, wherein the microorganism is Staphylococcus aureus.

3. The method for characterizing a microorganism of claim 2, wherein the markers identified comprise at least one peptide that has an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 13, 15, 21, and 24.

4. The method for characterizing a microorganism of claim 2, wherein the markers identified comprise at least one peptide that has an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 and 15.

5. The method for characterizing a microorganism of claim 2, wherein the markers identified comprise at least one peptide that has an amino acid sequence selected from the group consisting of SEQ ID NOs: 21 and 24.

6. The method for characterizing a microorganism of claim 1, wherein the microorganism is Escherichia coli.

7. The method for characterizing a microorganism of claim 6, wherein the markers identified comprise at least one peptide that has an amino acid sequence selected from the group consisting of SEQ ID NOs: 64, 66, and 85 to 87.

8. The method for characterizing a microorganism of claim 6, wherein the markers identified comprise at least one peptide that has an amino acid sequence selected from the group consisting of SEQ ID NOs: 64 and 66.

9. The method for characterizing a microorganism of claim 6, wherein the markers identified comprise at least one peptide that has an amino acid sequence selected from the group consisting of SEQ ID NOs: 85, 86, and 87.

10. The method for characterizing a microorganism of claim 1, wherein the microorganism is Candida albicans.

11. The method for characterizing a microorganism of claim 1, wherein the markers are identified simultaneously.

12. The method for characterizing a microorganism of claim 1, wherein the markers are identified in the same mass spectrometry apparatus.

13. The method for characterizing a microorganism of claim 1, wherein the microorganism is identified by mass spectrometry.

14. The method for characterizing a microorganism of claim 13, wherein the mass spectrometry for identifying the microorganism is selected from the group consisting of MS type, MS/MS type, and MS type followed by MS/MS type.

15. The method for characterizing a microorganism of claim 1, further comprising: confirming, by mass spectrometry, the identification of the microorganism.

16. The method for characterizing a microorganism of claim 15, wherein the mass spectrometry for confirming the identification of the microorganism is MS/MS type.

17. The method for characterizing a microorganism of claim 16, wherein the mass spectrometry for confirming the identification of the microorganism is MRM type.

18. The method for characterizing a microorganism of claim 1, wherein: the markers are peptides, and the microorganism is a bacterium or a yeast.

19. A method for characterizing a microorganism, comprising: identifying the microorganism; and subjecting the microorganism identified to mass spectrometry with a spectrometer in MS/MS mode using multiple reaction monitoring (MRM) to identify at least one marker of a property of typing, potential resistance to at least one antimicrobial, or virulence factor, of the microorganism, wherein: the marker is a protein or a peptide, and the microorganism is a bacterium, a virus, a protozoan, or a yeast.

20. A method for characterizing a microorganism, comprising: identifying the microorganism; and subjecting the microorganism identified to mass spectrometry with a spectrometer in MS/MS mode using multiple reaction monitoring (MRM) to identify at least one marker of a property of typing, potential resistance to at least one antimicrobial, or virulence factor, of the microorganism, wherein: the marker is selected from the group consisting of proteins, peptides, and metabolites, and the microorganism is a virus, a protozoan, or a yeast.

Description

EXAMPLE 1: IDENTIFICATION OF MICROORGANISMS FROM A SAMPLE BY MEANS OF BIOCHEMICAL PROFILE

(1) 1. Culturing the Sample on a Culture Medium

(2) The optimum culture media and the optimum culture conditions are different according to the species of microorganism. By default, the sample is inoculated onto various media: Columbia agar with sheep blood (bioMrieux ref 43041) for 18 to 24 h at 35 C., with or without anaerobic conditions; TSA agar (bioMrieux reference 43011) for 18 to 24 h at 37 C.

(3) 2. Identification of Microorganisms

(4) The identification is implemented as follows: 1. Selection of isolated colonies. 2. While observing aseptic conditions, transfer of 3.0 ml of aqueous sterile saline solution (containing 0.45-0.50% of NaCl, at pH 4.5 to 7.0) into a transparent plastic (polystyrene) test tube. 3. Using a sterile cotton bud or a sterile swab, transfer of a sufficient number of identical colonies into the tube of saline solution prepared in step 2 and adjustment of the bacterial suspension to between 0.50 and 0.63 McFarland with a calibrated VITEK 2 DENSICHEK. 4. Positioning of the tube of bacterial suspension and of a VITEK 2 identification card on a VITEK 2 cassette. 5. Loading of the cassette into the VITEK 2 instrument. 6. The filling, sealing, incubation and reading operations are automatic. 7. Acquisition of a biochemical profile. 8. Identification with the VITEK 2 system, carried out by comparison with biochemical profiles of known strains.

EXAMPLE 2: IDENTIFICATION OF MICROORGANISMS FROM A SAMPLE BY MEANS OF MALDI-TOF

(5) The identification is implemented as follows: 1. Transfer, using a 1 l loop, of a portion of microorganism colony obtained according to example 1, and uniform deposition on a plate for mass spectrometry by MALDI-TOF. 2. Covering of the deposit with 1 l of matrix. The matrix used is a saturated solution of HCCA (alpha-cyano-4-hydroxycinnamic acid) in an organic solvent (50% acetonitrile and 2.5% trifluoroacetic acid). 3. Drying at ambient temperature. 4. Introduction of the plate into the mass spectrometer. 5. Acquisition of a mass spectrum. 6. Comparison of the spectrum obtained with the spectra contained in a knowledge base. 7. Identification of the microorganism by comparison of the peaks obtained with those of the knowledge base.

EXAMPLE 3: IDENTIFICATION OF MICROORGANISMS FROM A SAMPLE BY MEANS OF ESI-MS

(6) The identification is implemented as follows: 1. Sampling of a microorganism colony, obtained according to example 1, and suspension in 100 l of demineralized water. 2. Centrifugation at 3000 g for 5 minutes. 3. Removal of the supernatant. 4. Resuspension in 100 l of demineralized water. 5. Centrifugation at 3000 g for 5 minutes. 6. Removal of the supernatant. 7. Resuspension in 100 l of a mixture of acetonitrile, demineralized water and formic acid (50/50/0.1%). 8. Filtration with a filter having a pore size of 0.45 m. 9. Injection into a mass spectrometer in single MS mode. 10. Acquisition of a mass spectrum. 11. Comparison of the spectrum obtained with the spectra contained in a knowledge base. 12. Identification of the microorganism by reference to reference spectra.

EXAMPLE 4: OBTAINING OF DIGESTED PROTEINS FROM MICROORGANISMS

(7) Conventionally, the following protocol is implemented in 11 steps: 1. Sampling of a microorganism colony, obtained according to example 1, and suspension in 10 to 100 l of a solution of 6M guanidine hydrochloride, 50 mM Tris-HCl, pH=8.0. 2. Addition of dithiothreitol (DTT) in order to obtain a final concentration of 5 mM. 3. Reduction for 20 minutes at 95 C. in a water bath. 4. Cooling of the tubes to ambient temperature. 5. Addition of iodoacetamide in order to obtain a final concentration of 12.5 mM. 6. Alkylation for 40 minutes at ambient temperature and in the dark. 7. Dilution by a factor of 6 with a 50 mM, NH.sub.4HCO.sub.3 solution, pH=8.0 in order to obtain a final guanidine hydrochloride concentration of 1M. 8. Addition of 1 g of trypsin. 9. Digestion at 37 C. for 6 hours overnight. 10. Addition of formic acid to a pH of less than 4 in order to stop the reaction. 11. Ultracentrifugation at 100 000 g for 30 minutes.

EXAMPLE 5: CHARACTERIZATION OF S. AUREUS SAMPLES

(8) After having established the one or more species of the samples according to any one of the methods described in examples 1 to 3, the species listed below are analyzed.

(9) Thirteen strains of S. aureus are analyzed in order to confirm their identification and to establish their characteristics:

(10) TABLE-US-00019 ID2 ID3 ID4 ID2a ID3a ID4a AST7 AST8 AST13 AST14 VIR5 VIR6 VIR7

(11) The same method of analysis is applied to species that do not belong to the S. aureus species, in order to serve as a negative control: E. coli

(12) TABLE-US-00020 ID7 AST2 S. pneumoniae

(13) VIR21 C. difficile

(14) VIR26

(15) Each sample is treated according to example 4, then a volume of 5 l of digested proteins is injected and analyzed according to the following conditions: Agilent 1100 series chromatographic system from the company Agilent Technologies (Massy, France). Waters Symmetry C18 column, 2.1 mm internal diameter, 100 mm long, particle size 3.5 m. Solvent A: H.sub.2O+0.1% formic acid. Solvent B: ACN+0.1% formic acid. HPLC gradient defined in TABLE 1 hereinafter:

(16) TABLE-US-00021 TABLE 1 Time Flow rate (l) Solvent A (%) Solvent B (%) 0 300 95 5 25 300 60 40 27 300 0 100 35 300 0 100 35.1 300 95 5 45 300 95 5 The eluate at the output of the chromatographic column is directly injected into the ionization source of the QTRAP 5500 mass spectrometer from the company Applied Biosystems (Foster City, United States of America). The peptides, resulting from the digestion of the proteins of the microorganism, are analyzed by mass spectrometry in MRM mode. Only the peptides indicated in TABLE 2 are detected. For this, the first-generation fragment(s) indicated in TABLE 2 is (are) detected. The application, to which each transition, i.e. each peptide associated with its first-generation fragment, makes it possible to respond, is specified in the clinical interest column of TABLE 2 with the letters I, T, R and V. I denotes the confirmation of the identification of the microorganism, T the typing, R the resistance to at least one antibiotic and V the detection of virulence factors.

(17) TABLE-US-00022 TABLE 2 Transition First-generation Clinical number Protein Peptide (SEQ ID NO.) fragment ion interest 1 protein A DDPSQSANVLGEAQK (2) y9 singly charged T 2 protein A DDPSQSANVLGEAQK (2) y13 singly charged T 3 protein A DQQSAFYEILNMPNLNEEQR (3) y8 singly charged T 4 protein A DQQSAFYEILNMPNLNEEQR (3) y8 doubly charged T 5 protein A DDPSQSTNVLGEAK (4) y9 singly charged T 6 protein A DDPSQSTNVLGEAK (4) y12 singly charged T 7 protein A EQQNAFYEILNMPNLNEEQR (5) y8 singly charged T 8 protein A EQQNAFYEILNMPNLNEEQR (5) y8 doubly charged T 9 protein A DDPSQSANLLAEAK (6) y9 singly charged T 10 protein A DDPSQSANLLAEAK (6) y12 singly charged T 11 protein A DDPSVSK (7) y5 singly charged T 12 protein A DDPSVSK (7) y4 singly charged T 13 protein A IAADNK (8) y4 singly charged T 14 protein A IAADNK (8) y5 singly charged T 15 PBP2a IYNSLGVK (10) y6 singly charged R + T 16 PBP2a IYNSLGVK (10) y7 singly charged R + T 17 PBP2a DINIQDR (11) y5 singly charged R + T 18 PBP2a DINIQDR (11) y4 singly charged R + T 19 PBP2a ELSISEDYIK (12) y6 singly charged R + T 20 PBP2a ELSISEDYIK (12) y8 singly charged R + T 21 PBP2a FQITTSPGSTQK (13) y9 singly charged R + T 22 PBP2a FQITTSPGSTQK (13) y6 singly charged R + T 23 PBP2a ILTAMIGLNNK (14) y8 singly charged R + T 24 PBP2a ILTAMIGLNNK (14) y9 singly charged R + T 25 PBP2a YEVVNGNIDLK (15) y8 singly charged R + T 26 PBP2a YEVVNGNIDLK (15) y9 singly charged R + T 27 PBP2a VALELGSK (16) y6 singly charged R + T 28 PBP2a VALELGSK (16) y7 singly charged R + T 29 PBP2a SYANLIGK (17) y6 singly charged R + T 30 LukS TNDPNVDLINYLPK (19) y8 singly charged V + T 31 LukS TNDPNVDLINYLPK (19) y11 singly charged V + T 32 LukS SVQWGIK (20) y5 singly charged V + T 33 LukS SVQWGIK (20) y4 singly charged V + T 34 LukS ANSFITSLGK (21) y8 singly charged V + T 35 LukS ANSFITSLGK (21) y6 singly charged V + T 36 LukF MPVLSR (23) y5 singly charged V + T 37 LukF MPVLSR (23) y3 singly charged V + T 38 LukF GNFNPEFIGVLSR (24) y8 singly charged V + T 39 LukF GNFNPEFIGVLSR (24) y9 singly charged V + T 40 RL30 LQITLTR (26) y5 singly charged I + T 41 RL30 LQITLTR (26) y6 singly charged I + T 42 RL30 TNSSVVVEDNPAIR (27) y8 singly charged I + T 43 RL30 TNSSVVVEDNPAIR (27) y8 singly charged I + T 44 RL331 VNVTLAC[CAM]TEC[CAM]GDR (29) y8 singly charged I + T 45 RL331 NYITTK (30) y5 singly charged I + T 46 RL331 NYITTK (30) y4 singly charged I + T 47 SSAA2 AGYTVNNTPK (32) y6 singly charged I + T 48 SSAA2 AGYTVNNTPK (32) y7 singly charged I + T 49 SSAA2 AGYTVNNTPK (32) y8 singly charged I + T 50 SSAA2 AGYTVNNTPK (32) y9 singly charged I + T 51 Y772 ATDFIDKVK (35) y6 singly charged I + T 52 Y772 ATDFIDKVK (35) y8 singly charged I + T 53 ATL AYLAVPAAPK (38) y7 singly charged I + T 54 ATL AYLAVPAAPK (38) y8 singly charged I + T 55 EFTU TVGSGVVTEIIK (40) y7 singly charged I + T 56 EFTU TVGSGVVTEIIK (40) y8 singly charged I + T 57 EFTU TVGSGVVTEIIK (40) y9 singly charged I + T 58 Y772 EFVENAKEK (34) y6 singly charged I + T 59 Y772 EFVENAKEK (34) y7 singly charged I + T 60 Y772 EFVENAKEK (34) y8 singly charged I + T 61 ISAA LSNGNTAGATGSSAAQIMAQR (42) y7 singly charged I + T 62 ISAA LSNGNTAGATGSSAAQIMAQR (42) y8 singly charged I + T 63 ATL LYSVPWGTYK (37) y6 singly charged I + T 64 ATL LYSVPWGTYK (37) y7 singly charged I + T 65 ATL LYSVPWGTYK (37) y8 singly charged I + T 66 Y197A NITQDQDIHAVPK (44) y8 singly charged I + T 67 Y197A NITQDQDIHAVPK (44) y7 singly charged I + T 68 Y197A NITQDQDIHAVPK (44) y6 singly charged I + T 69 Y197A LDSKDVSR (45) y6 singly charged I + T 70 Y197A LDSKDVSR (45) y7 singly charged I + T
The charge state of the precursor peptide, its retention time and the transitions, i.e. the ratios (m/z).sub.1 in Q1 and (m/z).sub.2 in Q3, are indicated in TABLE 3. The collision energy used to fragment the precursor ion is also indicated in TABLE 3.

(18) TABLE-US-00023 TABLE 3 Precursor (m/z) Transition charge Retention filtered (m/z) filtered Collision number state time in Q1 in Q3 energy 1 2 8.2 779.87 929.51 39 2 2 8.2 779.87 664.84 39 3 3 17.9 813.71 999.49 45 4 3 17.9 813.71 500.24 45 5 2 7.9 730.85 918.49 37 6 2 7.9 730.85 615.82 37 7 3 16.5 827.39 999.49 45 8 3 16.5 827.39 500.25 34 9 2 10 729.86 916.51 37 10 2 10 729.86 614.83 37 11 2 11 374.18 517.3 21 12 2 11 374.18 420.25 21 13 2 8.9 316.17 447.22 19 14 2 8.9 316.17 518.26 19 15 2 6.7 447.26 617.36 25 16 2 6.7 447.26 780.43 25 17 2 6.2 437.22 645.33 24 18 2 6.2 437.22 531.3 24 19 2 11.1 598.81 754.36 31 20 2 11.1 598.81 954.48 31 21 2 7.4 647.84 906.45 34 22 2 7.4 647.84 617.3 26 23 2 11.8 594.34 860.47 31 24 2 11.8 594.34 961.51 31 25 2 10.1 632.33 872.48 33 26 2 10.1 632.33 971.55 33 27 2 7.4 408.74 646.38 23 28 2 7.4 408.74 717.41 23 29 2 8.2 433.24 615.38 24 30 2 14.9 808.42 975.55 41 31 2 14.9 808.42 643.4 41 32 2 8.2 409.23 631.36 23 33 2 8.2 409.23 503.3 23 34 2 10.3 519.28 852.48 28 35 2 10.3 519.28 618.4 28 36 2 6.4 351.7 571.36 20 37 2 6.4 351.7 375.24 20 38 2 14.3 725.38 920.52 37 39 2 14.3 725.38 509.29 37 40 2 9.2 422.77 603.38 24 41 2 9.2 422.77 731.44 24 42 2 8 750.89 913.47 38 43 3 8 500.93 913.47 29 44 2 7.9 747.84 968.36 38 45 2 2.8 370.2 625.36 21 46 2 2.8 370.2 462.29 21 47 2 3.9 532.77 672.37 28 48 2 3.9 532.77 773.42 28.442 49 2 3.9 532.77 936.48 28.442 50 2 3.9 532.77 993.5 28.442 51 2 7.2 518.79 749.46 28 52 2 7.2 518.79 965.53 27.827 53 2 9 500.79 653.4 27 54 2 9 500.79 766.48 27.035 55 2 11.2 601.85 801.51 31 56 2 11.2 601.85 858.53 31.482 57 2 11.2 601.85 945.56 31.482 58 2 2.3 547.28 718.37 29 59 2 2.3 547.28 817.44 29.08 60 2 2.3 547.28 964.51 29.08 61 3 8.7 669.33 817.43 37 62 3 8.7 669.33 888.47 37.466 63 2 12.2 607.32 751.38 32 64 2 12.2 607.32 850.45 31.722 65 2 12.2 607.32 937.48 31.722 66 2 6.3 739.88 907.5 38 67 2 6.3 739.88 779.44 37.555 68 2 6.3 739.88 664.41 37.555 69 2 2 460.25 691.37 25.251 70 2 2 460.25 806.4 25.251 The other machine parameters used are the following:

(19) Scan type: MRM

(20) Polarity: Positive

(21) Ionization source: Turbo V (Applied BioSystems)

(22) Q1 setting: Filtering with unit resolution

(23) Q3 setting: Filtering with unit resolution

(24) Inter-scan pause: 5.00 msec

(25) Scan speed: 10 Da/s

(26) Curtain gas: 50.00 psi

(27) Cone voltage: 5500.00 V

(28) Source temperature: 500.00 C.

(29) Nebulizing gas: 50.00 psi

(30) Heating gas: 40.00 psi

(31) Dynamic filling: activated

(32) Declustering potential (DP): 100.00 V

(33) Entry potential before Q0 (EP): 6.00 V

(34) Collision cell exit potential (CXP): 11 V

(35) Total cycle time: 1.6 sec

(36) The areas obtained for each of the transitions and for each of the microorganisms studied were measured. All the transitions with an area greater than or equal to 1000 (arbitrary units) are considered to be positive and have been denoted 1 in tables 4A and 4B. All the transitions with an area less than 1000 are considered to be negative and have been denoted 0 in tables 4A and 4B. When no signal peak was observed, the transition was noted as negative.

(37) The positive-transition number is then summed for the applications I, R and V and reported in TABLE 5:

(38) TABLE-US-00024 TABLE 4A ID2 ID3 ID4 ID2a ID3a ID4a AST7 AST8 Transition S. aureus S. aureus S. aureus S. aureus S. aureus S. aureus S. aureus S. aureus 1 0 1 1 0 1 1 0 0 2 0 1 1 0 1 1 0 0 3 0 1 1 0 1 1 0 0 4 0 1 1 0 1 1 0 0 5 1 1 1 1 1 1 0 0 6 1 1 1 1 1 1 0 1 7 1 1 1 1 1 1 0 0 8 1 1 1 1 1 1 0 0 9 1 1 1 1 1 1 0 0 10 1 1 1 1 1 1 0 0 11 0 0 0 0 0 0 1 0 12 0 0 0 0 0 0 0 0 13 1 1 1 0 1 1 0 0 14 1 1 1 1 1 1 1 0 15 1 0 1 1 0 1 0 0 16 0 0 1 0 0 1 0 0 17 1 0 1 1 0 1 0 0 18 1 0 1 1 0 1 0 0 19 1 0 1 1 0 1 0 0 20 1 0 1 1 0 1 0 0 21 1 0 1 1 0 1 0 0 22 1 0 1 1 0 1 0 0 23 1 0 1 1 0 1 0 0 24 1 0 1 1 0 1 0 0 25 1 0 1 1 0 1 0 0 26 1 0 1 1 0 1 0 0 27 1 1 1 1 1 1 0 0 28 1 0 1 1 0 1 0 0 29 1 0 1 1 0 1 0 0 30 0 0 0 0 0 0 0 0 31 0 0 0 0 0 0 0 0 32 0 0 0 0 0 0 0 0 33 0 0 1 0 0 0 0 0 34 0 0 0 0 0 0 0 0 35 0 0 0 0 0 0 0 1 36 0 0 0 0 0 0 1 1 37 0 0 0 0 0 1 0 0 38 0 0 0 0 0 1 0 0 39 0 0 0 0 0 0 0 0 40 1 1 1 1 1 1 1 1 41 1 1 1 1 1 1 1 1 42 1 1 1 1 1 1 1 1 43 1 1 1 1 1 1 1 1 44 1 1 1 1 1 1 1 1 45 1 1 1 1 1 1 1 1 46 1 1 1 1 1 1 1 1 47 1 1 1 1 1 1 1 1 48 1 1 1 1 1 1 1 1 49 1 1 1 1 1 1 1 1 50 1 1 1 1 1 1 1 1 51 1 1 1 1 1 1 1 1 52 1 1 1 1 1 1 1 1 53 1 1 1 1 1 1 1 1 54 1 1 1 1 1 1 1 1 55 1 1 1 1 1 1 1 1 56 1 1 1 1 1 1 1 1 57 1 1 1 1 1 1 1 1 58 1 1 1 1 1 1 1 1 59 1 1 1 1 1 1 1 1 60 1 1 1 1 1 1 1 1 61 1 1 1 1 1 1 1 1 62 1 1 1 1 1 1 1 1 63 1 1 1 1 1 1 1 1 64 1 1 1 1 1 1 0 0 65 1 1 1 1 1 1 1 1 66 1 1 1 1 1 1 1 1 67 1 1 1 1 1 1 1 1 68 1 1 1 1 1 1 1 1 69 1 1 1 1 1 1 1 1 70 1 1 1 1 1 1 1 1

(39) TABLE-US-00025 TABLE 4B AST13 AST14 VIR5 VIR6 VIR7 ID7 AST2 VIR21 VIR26 Transition S. aureus S. aureus S. aureus S. aureus S. aureus E. coli E. coli S. pneumoniae C. difficile 1 0 0 0 0 0 0 0 0 0 2 0 0 0 1 0 0 0 0 0 3 0 0 0 0 0 0 0 0 0 4 0 0 0 0 0 0 0 0 0 5 0 0 0 1 0 0 0 0 0 6 1 0 0 1 0 0 0 0 0 7 0 0 0 1 0 0 0 0 0 8 0 0 0 1 0 0 0 0 0 9 0 0 0 1 0 0 0 0 0 10 0 0 0 1 0 0 0 0 0 11 0 1 1 1 0 0 0 0 0 12 0 1 1 0 0 0 0 0 0 13 0 0 0 0 0 0 0 0 0 14 1 0 0 1 0 0 0 0 0 15 1 0 0 0 0 0 0 0 0 16 1 0 0 0 0 0 0 0 0 17 1 1 0 0 0 0 0 0 0 18 1 0 0 0 0 0 1 0 0 19 1 1 0 0 0 0 0 0 0 20 1 1 0 0 0 0 0 0 0 21 1 1 0 0 0 0 0 0 0 22 1 0 0 0 0 0 0 0 0 23 1 0 0 0 0 0 1 0 0 24 1 1 0 1 0 0 0 0 0 25 1 1 0 0 0 0 0 0 0 26 1 0 0 0 0 0 0 0 0 27 1 1 0 0 0 0 0 0 1 28 1 1 0 0 0 0 0 0 0 29 1 1 0 0 0 0 0 0 0 30 0 0 1 0 1 0 0 0 0 31 0 0 1 0 1 0 0 0 0 32 0 0 1 0 1 0 0 0 0 33 0 0 1 0 1 0 0 0 0 34 0 0 1 0 1 0 0 0 0 35 0 0 1 0 1 0 0 0 0 36 1 1 1 1 1 0 0 0 0 37 0 0 1 0 1 0 0 0 0 38 0 0 1 0 1 0 0 0 0 39 0 0 1 0 1 0 0 0 0 40 1 1 1 1 1 0 0 0 0 41 1 1 1 1 1 0 0 1 0 42 1 1 1 1 1 0 0 0 0 43 0 1 0 1 0 0 0 0 0 44 1 1 1 1 1 0 0 0 0 45 1 1 0 1 1 0 0 0 0 46 1 1 1 1 1 0 0 0 0 47 1 1 1 1 1 0 0 0 0 48 1 1 0 1 1 0 0 0 0 49 1 1 0 1 0 0 0 0 0 50 1 0 0 1 0 0 0 0 0 51 1 1 0 1 1 0 0 0 0 52 1 1 0 1 1 1 0 0 0 53 1 1 1 1 1 0 0 0 0 54 1 1 1 1 1 0 0 0 0 55 1 1 1 1 1 0 0 0 0 56 1 1 1 1 1 0 0 0 0 57 1 1 1 1 1 0 0 0 0 58 1 1 0 1 1 0 0 0 0 59 1 1 0 1 1 0 0 0 0 60 1 1 0 1 1 0 0 0 0 61 1 1 0 1 1 0 0 0 0 62 1 1 0 1 1 0 0 0 0 63 1 1 1 1 1 0 0 0 0 64 1 0 0 0 0 0 0 0 0 65 1 1 1 1 1 0 0 0 0 66 1 1 0 1 1 0 0 0 0 67 0 1 0 1 0 0 0 0 0 68 1 1 0 1 0 0 0 0 0 69 1 1 1 1 1 0 0 0 0 70 1 1 0 1 1 0 0 0 0

(40) TABLE-US-00026 TABLE 5 I R V ID2 S. aureus 30 14 0 ID3 S. aureus 30 1 0 ID4 S. aureus 30 15 1 ID2a S. aureus 30 14 0 ID3a S. aureus 30 1 0 ID4a S. aureus 30 15 2 AST7 S. aureus 29 0 1 AST8 S. aureus 29 0 2 AST13 S. aureus 28 15 1 AST14 S. aureus 28 9 1 VIR5 S. aureus 13 0 10 VIR6 S. aureus 29 1 1 VIR7 S. aureus 24 0 10 ID7 E. coli 1 0 0 AST2 E. coli 0 2 0 VIR21 S. pneumoniae 1 0 0 VIR26 C. difficile 0 1 0

(41) All the S. aureus samples exhibit more than 12 positive transitions in the I category. All these samples are therefore confirmed as indeed belonging to the S. aureus species.

(42) On the other hand, the samples ID7, AST2, VIR21 and VIR26 exhibit less than two positive transitions in the I category, these samples are therefore confirmed as not belonging to the S. aureus species.

(43) The ID2, ID4, ID2a, ID4a, AST13 and AST14 strains of S. aureus exhibit more than eight positive transitions for the R category, they therefore express the penicillin binding protein (PBP2a), this being synonymous with a mechanism of resistance to group M penicillins (e.g. methicillin).

(44) On the other hand, the ID3, ID3a, AST7, AST8, VIR5, VIR6 and VIR7 strains of S. aureus exhibit less than three positive transitions for the V category, they therefore do not express PBP2a. These strains are therefore sensitive to an antibiotic such as a group M penicillin

(45) The ID7, AST2, VIR21 and VIR26 strains which do not belong to the S. aureus species also exhibit less than three transitions for the R category, they do not therefore express PBP2a, thereby confirming the specificity of the method.

(46) The VIR5 and VIR7 samples of S. aureus exhibit more than nine positive transitions in the V category, they therefore express the Panton-Valentine-Leukocidin (PVL) protein.

(47) On the other hand, the ID2, ID3, ID4, ID2a, ID3a, ID4a, AST7, AST8, AST13, AST14 and VIR6 strains of S. aureus exhibit less than three positive transitions, they do not therefore express PVL. These strains do not therefore have the virulence properties related to PVL.

(48) The ID7, AST2, VIR21 and VIR26 strains which do not belong to the S. aureus species also exhibit less than three transitions for the V category, they do not therefore express PVL, thereby confirming the specificity of the method.

(49) For the typing, the T-category transitions of each strain are compared with the transitions of the other strains considered as reference strains. In practice, a value 0 is assigned when the transitions between the two strains are classified in the same category (positive or negative) and a value 1 is assigned when the transitions between the two strains are classified in different categories (a positive transition and a negative transition). The values are summed for all the T-category transitions of each strain pair in order to establish a score. The scores are shown in TABLE 6:

(50) TABLE-US-00027 TABLE 6 ID2 ID3 ID4 ID2a ID3a ID4a AST7 AST8 AST13 AST14 VIR5 VIR6 VIR7 ID7 AST2 VIR21 VIR26 ID2 0 17 6 1 17 7 24 24 10 18 51 18 38 52 51 52 52 ID3 17 0 15 18 0 16 15 15 27 25 42 9 29 43 46 43 43 ID4 6 15 0 7 15 3 30 30 14 24 55 22 42 58 57 58 58 ID2a 1 18 7 0 18 8 23 23 9 17 50 17 37 51 50 51 51 ID3a 17 0 15 18 0 16 15 15 27 25 42 9 29 43 46 43 43 ID4a 7 16 3 8 16 0 31 31 15 25 54 23 41 59 58 59 59 AST7 24 15 30 23 15 31 0 4 20 12 27 8 16 32 35 32 34 AST8 24 15 30 23 15 31 4 0 20 14 27 10 14 32 35 32 34 AST13 10 27 14 9 27 15 20 20 0 14 43 24 30 46 45 46 46 AST14 18 25 24 17 25 25 12 14 14 0 33 18 24 40 43 40 40 VIR5 51 42 55 50 42 54 27 27 43 33 0 35 13 27 28 25 27 VIR6 18 9 22 17 9 23 8 10 24 18 35 0 24 40 43 40 42 VIR7 38 29 42 37 29 41 16 14 30 24 13 24 0 34 37 34 36 ID7 52 43 58 51 43 59 32 32 46 40 27 40 34 AST2 51 46 57 50 46 58 35 35 45 43 28 43 37 VIR21 52 43 58 51 43 59 32 32 46 40 25 40 34 VIR26 52 43 58 51 43 59 34 34 46 40 27 42 36

(51) The strains which have a score less than or equal to 4 are of the same type, the strains which have a score strictly above 4 are of different type.

(52) Thus, the ID2 and ID2a, ID3 and ID3a, ID4 and ID4a and AST7 and AST8 strains are of the same type. All the other strains taken in pairs are of different type. The high sums obtained between the ID7, AST2, VIR21 and VIR26 strains, which are not S. aureus, and all the other strains which are S. aureus, should be noted. These results confirm the specificity of the method.

(53) The ID7, AST2, VIR21 and VIR26 strains are not of course of the same type; this would be absurd, they are of different species. These strains cannot therefore be compared with one another and no value is reported in TABLE 6.

(54) Extremely advantageously, scores above 25, for instance between ID2 and VR7, reflect a great divergence between strains. Scores between 15 and 25, as between ID2 and AST14, reflect a moderate divergence and scores between 5 and 15, as between ID2 and ID4a, a weak divergence.

(55) The method thus implemented therefore makes it possible not only to establish whether two strains are of the same type, which is important for identifying a common seat of infection, but also to estimate the proximity of two strains, which is extremely important for epidemiological studies.

(56) This example shows that, very advantageously, the present invention makes it possible, in a time of less than one hour, which is very short, to confirm the identity of a species such as S. aureus and to determine, simultaneously within the same analysis, the properties of typing and potential resistance to at least one antibiotic and to establish the existence of a virulence factor. These properties were established with the same instrument, which greatly facilitates the analysis and the reporting of the results. Finally, the characteristics of the bacteria are established using bacterial proteins, which reflects the existence of live and viable microorganisms, unlike characterizations using bacterial DNA which can be distorted by the existence of dead bacteria.

EXAMPLE 6: PROTOCOL FOR DIGESTION OF MICROORGANISMS WITH A DESALIFYING STEP

(57) Conventionally, the following protocol is implemented in 17 steps:

(58) Steps 1 to 10: idem example 4. 11. The sample volume is made up to 1 ml with water/0.1% (v/v) formic acid. 12. Equilibration of Waters Oasis HLB columns with 1 ml of methanol then 1 ml of H.sub.2O/0.1% (v/v) formic acid. 13. Loading of the sample which flows by gravity. 14. Washing with 1 ml of H.sub.2O/0.1% (v/v) formic acid. 15. Elution with 1 ml of a mixture of 80% methanol and 20% water/0.1% (v/v) formic acid. 16. The eluate is evaporated with a SpeedVac SPD2010 evaporator (Thermo Electron Corporation, Waltham, Mass., United States of America), for two hours, in order to obtain a volume of approximately 100 l. 17. The eluate is then taken up in a water/0.5% (v/v) formic acid solution, quantity sufficient for (QS) 250 l.

EXAMPLE 7: CHARACTERIZATION OF E COLI SAMPLES

(59) After having established the one or more species of the samples according to any one of the methods described in examples 1 to 3, the species listed below are analyzed.

(60) Fifteen E. coli strains are analyzed in order to confirm their identification and to establish their characteristics:

(61) TABLE-US-00028 AST1 AST2 AST3 AST4 AST5 VIR41 VIR42 VIR43 VIR44 VIR45 ID6 ID7 ID8 ID9 ID10

(62) The same method of analysis is applied to species not belonging to the E. coli species in order to serve as a negative control: S. aureus

(63) VIR10 S. pneumoniae

(64) VIR19 C. difficile

(65) VIR28

(66) Each sample is treated according to example 6, then a volume of 5 l of digested proteins is injected and analyzed according to the following conditions: Agilent 1100 series chromatographic system from the company Agilent Technologies (Massy, France). Waters Symmetry C18 column, 2.1 mm internal diameter, 100 mm long, particle size 3.5 m. Solvent A: H.sub.2O+0.1% formic acid. Solvent B: ACN+0.1% formic acid. HPLC gradient defined in TABLE 7 hereinafter:

(67) TABLE-US-00029 TABLE 7 Flow rate Time (min) (l) Solvent A (%) Solvent B (%) 0 300 95 5 3 300 95 5 28 300 60 40 30 300 0 100 38 300 0 100 38.1 300 95 5 45 300 95 5 The eluate at the output of the chromatographic column is directly injected into the ionization source of the QTRAP 5500 mass spectrometer from the company Applied Biosystems (Foster City, United States of America). The peptides, resulting from the digestion of the proteins of the microorganism, are analyzed using the mass spectrometer in MRM mode. Only the peptides indicated in TABLE 8 are detected. For this, the first-generation fragment(s) indicated in TABLE 8 is (are) detected. The application, to which each transition, i.e. each peptide associated with its first-generation fragment, makes it possible to respond, is specified in the clinical interest column of TABLE 8 with the letters I, T, R and V. I denotes the confirmation of the identification of the microorganism, T the typing, R the resistance to at least one antibiotic and V the detection of virulence factors.

(68) TABLE-US-00030 TABLE 8 Transition first-generation fragment Clinical number Protein Peptide (SEQ. ID NO.) ion interest 1 ACON2 ILEIEGLPDLK (46) y7 singly charged I + T 2 ACON2 ILEIEGLPDLK (46) y8 singly charged I + T 3 ACON2 ILEIEGLPDLK (46) y9 singly charged I + T 4 ACON2 VADGATVVSTSTR (47) y7 singly charged I + T 5 ACON2 VADGATVVSTSTR (47) y8 singly charged I + T 6 ACON2 VADGATVVSTSTR (47) y9 singly charged I + T 7 ASPG2 TNTTDVATFK (48) y6 singly charged I + T 8 ASPG2 TNTTDVATFK (48) y7 singly charged I + T 9 ASPG2 TNTTDVATFK (48) y8 singly charged I + T 10 FABB LDTTGLIDR (49) y6 singly charged I + T 11 FABB LDTTGLIDR (46) y7 singly charged I + T 12 FABB LDTTGLIDR (46) y8 singly charged I + T 13 FABB VGLIAGSGGGSPR (50) y8 singly charged I + T 14 FABB VGLIAGSGGGSPR (50) y9 singly charged I + T 15 FABB VGLIAGSGGGSPR (50) y10 singly charged I + T 16 GLNH AIDFSDGYYK (51) y6 singly charged I + T 17 GLNH AIDFSDGYYK (51) y7 singly charged I + T 18 GLNH AIDFSDGYYK (51) y8 singly charged I + T 19 MODA LGAWDTLSPK (52) y7 singly charged I + T 20 MODA LGAWDTLSPK (52) y8 singly charged I + T 21 MODA LGAWDTLSPK (52) y9 singly charged I + T 22 ODP2 FGEIEEVELGR (53) y7 singly charged I + T 23 ODP2 FGEIEEVELGR (53) y8 singly charged I + T 24 ODP2 FGEIEEVELGR (53) y9 singly charged I + T 25 OMPC INLLDDNQFTR (54) y7 singly charged I + T 26 OMPC INLLDDNQFTR (54) y8 singly charged I + T 27 OMPC INLLDDNQFTR (54) y9 singly charged I + T 28 PFLB LATAWEGFTK (55) y6 singly charged I + T 29 PFLB LATAWEGFTK (55) y7 singly charged I + T 30 PFLB LATAWEGFTK (55) y8 singly charged I + T 31 SUCD DSILEAIDAGIK (56) y8 singly charged I + T 32 SUCD DSILEAIDAGIK (56) y9 singly charged I + T 33 SUCD DSILEAIDAGIK (56) y10 singly charged I + T 34 SUCD FAALEAAGVK (57) y7 singly charged I + T 35 SUCD FAALEAAGVK (57) y8 singly charged I + T 36 SUCD FAALEAAGVK (57) y9 singly charged I + T 37 SUCD SLADIGEALK (58) y6 singly charged I + T 38 SUCD SLADIGEALK (58) y7 singly charged I + T 39 SUCD SLADIGEALK (58) y8 singly charged I + T 40 TKT1 TEEQLANIAR (59) y7 singly charged I + T 41 TKT1 TEEQLANIAR (59) y8 singly charged I + T 42 TKT1 TEEQLANIAR (59) y9 singly charged I + T 43 YFCZ AEAEQTLAALTEK (60) y8 singly charged I + T 44 YFCZ AEAEQTLAALTEK (60) y9 singly charged I + T 45 YFCZ AEAEQTLAALTEK (60) y10 singly charged I + T 46 YGAU SGDTLSAISK (61) y6 singly charged I + T 47 YGAU SGDTLSAISK (61) y7 singly charged I + T 48 YGAU SGDTLSAISK (61) y8 singly charged I + T 49 TEM-2 LLTGELLTLASR (62) y7 singly charged R + T 50 TEM-2 LLTGELLTLASR (62) y8 singly charged R + T 51 TEM-2 LLTGELLTLASR (62) y9 singly charged R + T 52 TEM-2 SALPAGWFIADK (63) y7 singly charged R + T 53 TEM-2 SALPAGWFIADK (63) y8 singly charged R + T 54 TEM-2 SALPAGWFIADK (63) y9 singly charged R + T 55 TEM-2 VAGPLLR (64) y4 singly charged R + T 56 TEM-2 VAGPLLR (64) y5 singly charged R + T 57 TEM-2 VAGPLLR (64) y6 singly charged R + T 58 TEM-2 VGYIELDLNSGK (65) y8 singly charged R + T 59 TEM-2 VGYIELDLNSGK (65) y9 singly charged R + T 60 TEM-2 VGYIELDLNSGK (65) y10 singly charged R + T 61 TEM-2 VLLCGAVLSR (66) y7 singly charged R + T 62 TEM-2 VLLCGAVLSR (66) y8 singly charged R + T 63 TEM-2 VLLCGAVLSR (66) y9 singly charged R + T 64 ASPA ISDIPEFVR (67) y5 singly charged T 65 ASPA ISDIPEFVR (67) y6 singly charged T 66 ASPA ISDIPEFVR (67) y7 singly charged T 67 ASPA IEEDLLGTR (68) y6 singly charged T 68 ASPA IEEDLLGTR (68) y7 singly charged T 69 ASPA IEEDLLGTR (68) y8 singly charged T 70 ASPA LVDAINQLR (69) y6 singly charged T 71 ASPA LVDAINQLR (69) y7 singly charged T 72 ASPA LVDAINQLR (69) y8 singly charged T 73 ATPA TALAIDAIINQR (70) y7 singly charged T 74 ATPA TALAIDAIINQR (70) y8 singly charged T 75 ATPA TALAIDAIINQR (70) y9 singly charged T 76 ATPA VVNTLGAPIDGK (71) y7 singly charged T 77 ATPA VVNTLGAPIDGK (71) y8 singly charged T 78 ATPA VVNTLGAPIDGK (71) y9 singly charged T 79 CH10 SAGGIVLTGSAAAK (72) y9 singly charged T 80 CH10 SAGGIVLTGSAAAK (72) y10 singly charged T 81 CH10 SAGGIVLTGSAAAK (72) y11 singly charged T 82 CH60 AVTAAVEELK (73) y6 singly charged T 83 CH60 AVTAAVEELK (73) y7 singly charged T 84 CH60 AVTAAVEELK (73) y8 singly charged T 85 DBHB ALDAIIASVTESLK (74) y8 singly charged T 86 DBHB ALDAIIASVTESLK (74) y9 singly charged T 87 DBHB ALDAIIASVTESLK (74) y10 singly charged T 88 DCEB YWDVELR (75) y5 singly charged T 89 DCEB YWDVELR (75) y6 singly charged T 90 DCEB YWDVELR (75) y4 singly charged T 91 DHSA LPGILELSR (76) y6 singly charged T 92 DHSA LPGILELSR (76) y7 singly charged T 93 DHSA LPGILELSR (76) y8 singly charged T 94 DPS SKATNLLYTR (77) y6 singly charged T 95 DPS SKATNLLYTR (77) y7 singly charged T 96 DPS SKATNLLYTR (77) y8 singly charged T 97 HNS SEALKILNNIR (78) y7 singly charged T 98 HNS SEALKILNNIR (78) y8 singly charged T 99 HNS SEALKILNNIR (78) y9 singly charged T 100 MDH LFGVTTLDIIR (79) y6 singly charged T 101 MDH LFGVTTLDIIR (79) y7 singly charged T 102 MDH LFGVTTLDIIR (79) y8 singly charged T 103 PGK ASLPTIELALK (80) y7 singly charged T 104 PGK ASLPTIELALK (80) y8 singly charged T 105 PGK ASLPTIELALK (80) y9 singly charged T 106 PUR7 LLSDTECLVK (81) y6 singly charged T 107 PUR7 LLSDTECLVK (81) y7 singly charged T 108 PUR7 LLSDTECLVK (81) y8 singly charged T 109 RL4 SILSELVR (82) y5 singly charged T 110 RL4 SILSELVR (82) y6 singly charged T 111 RL4 SILSELVR (82) y7 singly charged T 112 RS1 GGFTVELNGIR (83) y7 singly charged T 113 RS1 GGFTVELNGIR (83) y8 singly charged T 114 RS1 GGFTVELNGIR (83) y9 singly charged T 115 YJGF TGEVPADVAAQAR (84) y8 singly charged T 116 YJGF TGEVPADVAAQAR (84) y9 singly charged T 117 YJGF TGEVPADVAAQAR (84) y10 singly charged T 118 stx1A TYVDSLNVIR (85) y6 singly charged V + T 119 stx1A TYVDSLNVIR (85) y7 singly charged V + T 120 stx1A TYVDSLNVIR (85) y8 singly charged V + T 121 stx1A-2A FVTVTAEALR (86) y7 singly charged V + T 122 stx1A-2A FVTVTAEALR (86) y8 singly charged V + T 123 stx2A ISNVLPEYR (87) y6 singly charged V + T 124 stx2A ISNVLPEYR (87) y7 singly charged V + T 125 stx2A ISNVLPEYR (87) y8 singly charged V + T
The charge state of the precursor peptide, its retention time and the transitions, i.e. the ratios (m/z).sub.1 in Q1 and (m/z).sub.2 in Q3, are indicated in TABLE 9. The collision energy used to fragment the precursor ion is also indicated in TABLE 9.

(69) TABLE-US-00031 TABLE 9 (m/z) (m/z) Transition Precursor Retention filtered in filtered in Collision number charge state time Q1 Q3 energy 1 2 19.04 620.36 771.42 36 2 2 19.04 620.36 884.51 36 3 2 19.04 620.36 1013.55 36 4 2 8.93 632.33 749.42 37 5 2 8.93 632.33 850.46 37 6 2 8.93 632.33 921.5 37 7 2 9.99 549.28 680.36 32 8 2 9.99 549.28 781.41 32 9 2 9.99 549.28 882.46 32 10 2 12.89 502.27 674.38 30 11 2 12.89 502.27 775.43 30 12 2 12.89 502.27 890.46 30 13 2 10.63 564.31 674.32 33 14 2 10.63 564.31 745.36 33 15 2 10.63 564.31 858.44 33 16 2 14.12 589.77 732.32 34 17 2 14.12 589.77 879.39 34 18 2 14.12 589.77 994.42 34 19 2 15.09 544.29 846.44 32 20 2 15.09 544.29 917.47 32 21 2 15.09 544.29 974.49 32 22 2 15.61 639.32 831.42 37 23 2 15.61 639.32 944.5 37 24 2 15.61 639.32 1073.55 37 25 2 16.42 674.85 895.39 39 26 2 16.42 674.85 1008.47 39 27 2 16.42 674.85 1121.56 39 28 2 15.49 562.29 767.37 33 29 2 15.49 562.29 838.41 33 30 2 15.49 562.29 939.46 33 31 2 19.84 622.84 816.45 36 32 2 19.84 622.84 929.53 36 33 2 19.84 622.84 1042.61 36 34 2 13.35 488.78 687.4 29 35 2 13.35 488.78 758.44 29 36 2 13.35 488.78 829.48 29 37 2 15.56 508.78 630.38 30 38 2 15.56 508.78 745.41 30 39 2 15.56 508.78 816.45 30 40 2 11.4 572.8 785.46 34 41 2 11.4 572.8 914.51 34 42 2 11.4 572.8 1043.55 34 43 2 14.85 687.86 846.49 39 44 2 14.85 687.86 974.55 39 45 2 14.85 687.86 1103.59 39 46 2 9.8 489.76 618.38 29 47 2 9.8 489.76 719.43 29 48 2 9.8 489.76 834.46 29 49 2 19.38 643.89 773.49 33 50 2 19.38 643.89 902.53 33 51 2 19.38 643.89 959.55 33 52 2 18.43 638.34 836.43 33 53 2 18.43 638.34 907.47 33 54 2 18.43 638.34 1004.52 33 55 2 10.79 363.24 498.34 21 56 2 10.79 363.24 555.36 21 57 2 10.79 363.24 626.4 21 58 2 15.94 654.35 875.45 34 59 2 15.94 654.35 988.53 34 60 2 15.94 654.35 1151.59 34 61 2 14.8 544.32 762.39 29 62 2 14.8 544.32 875.48 29 63 2 14.8 544.32 988.56 29 64 2 12.8 538.29 647.35 29 65 2 12.8 538.29 760.44 29 66 2 12.8 538.29 875.46 29 67 2 12.9 523.28 674.38 28 68 2 12.9 523.28 803.43 28 69 2 12.9 523.28 932.47 28 70 2 15.31 521.31 714.43 28 71 2 15.31 521.31 829.45 28 72 2 15.31 521.31 928.52 28 73 2 17.37 649.87 829.45 34 74 2 17.37 649.87 942.54 34 75 2 17.37 649.87 1013.57 34 76 2 11.94 592.34 657.36 31 77 2 11.94 592.34 770.44 31 78 2 11.94 592.34 871.49 31 79 2 11.08 601.84 817.48 31 80 2 11.08 601.84 930.56 31 81 2 11.08 601.84 987.58 31 82 2 11.86 515.79 688.39 28 83 2 11.86 515.79 759.42 28 84 2 11.86 515.79 860.47 28 85 2 22.24 715.91 834.46 36 86 2 22.24 715.91 947.54 36 87 2 22.24 715.91 1060.62 36 88 2 15.35 490.75 631.34 27 89 2 15.35 490.75 817.42 27 90 2 15.35 490.75 516.31 27 91 2 17.1 499.31 730.45 27 92 2 17.1 499.31 787.47 27 93 2 17.1 499.31 884.52 27 94 2 10.02 583.83 779.44 31 95 2 10.02 583.83 880.49 31 96 2 10.02 583.83 951.53 31 97 2 14.94 635.88 870.55 33 98 2 14.94 635.88 983.64 33 99 2 14.94 635.88 1054.67 33 100 2 20.34 624.37 730.45 32 101 2 20.34 624.37 831.49 32 102 2 20.34 624.37 930.56 32 103 2 18.16 578.35 787.49 30 104 2 18.16 578.35 884.55 30 105 2 18.16 578.35 997.63 30 106 2 13.11 589.31 749.39 31 107 2 13.11 589.31 864.41 31 108 2 13.11 589.31 951.45 31 109 2 17.68 458.78 603.35 25 110 2 17.68 458.78 716.43 25 111 2 17.68 458.78 829.51 25 112 2 15.99 581.81 800.46 31 113 2 15.99 581.81 901.51 31 114 2 15.99 581.81 1048.58 31 115 2 10.34 642.83 801.42 33 116 2 10.34 642.83 898.47 33 117 2 10.34 642.83 997.54 33 118 2 14.85 590.32 701.43 31 119 2 14.85 590.32 816.46 31 120 2 14.85 590.32 915.53 31 121 2 14.15 553.81 759.44 29 122 2 14.15 553.81 860.48 29 123 2 12.9 545.8 776.43 29 124 2 12.9 545.8 890.47 29 125 2 12.9 545.8 977.51 29 The other machine parameters used are the following:

(70) Scan type: MRM

(71) Scheduled MRM: yes

(72) Polarity: Positive

(73) Ionization source: Turbo V (Applied BioSystems)

(74) Q1 setting: Filtering with unit resolution

(75) Q3 setting: Filtering with unit resolution

(76) Inter-scan pause: 5.00 msec

(77) Scan speed: 10 Da/s

(78) Curtain gas: 50.00 psi

(79) Cone voltage: 5500.00 V

(80) Source temperature: 550.00 C.

(81) Nebulizing gas: 50.00 psi

(82) Heating gas: 40.00 psi

(83) Dynamic filling: activated

(84) Declustering potential (DP): 100.00 V

(85) Entry potential before Q0 (EP): 9.00 V

(86) Collision cell exit potential (CXP): 35 V

(87) Total cycle time: 1.2 sec

(88) Detection window 80 sec

(89) The areas obtained for each of the transitions and for each of the microorganisms studied were measured. All the transitions with an area greater than or equal to 2500 (arbitrary units) are considered to be positive and have been denoted 1 in tables 10A and 10B. All the transitions with an area less than 2500 are considered to be negative and have been denoted 0 in tables 10A and 10B. When no signal peak was observed, the transition was noted as negative.

(90) The positive-transition number is then summed for the I, R and V applications and reported in TABLE 11:

(91) TABLE-US-00032 TABLE 10A Transition number AST1 AST2 AST3 AST4 AST5 VIR41 VIR42 VIR43 VIR44 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 3 1 1 1 1 1 1 1 1 1 4 1 1 1 1 1 1 1 1 1 5 1 1 1 1 1 1 1 1 1 6 1 1 1 1 1 1 1 1 1 7 1 1 1 1 1 1 1 1 1 8 1 1 1 1 1 1 1 1 1 9 1 1 1 1 1 1 1 1 1 10 1 1 1 1 1 1 1 1 1 11 1 1 1 1 1 1 1 1 1 12 1 1 1 1 1 1 1 1 1 13 1 1 1 1 1 1 1 1 1 14 1 1 1 1 1 1 1 1 1 15 1 1 1 1 1 1 1 1 1 16 1 1 1 1 1 1 1 1 1 17 1 1 1 1 1 1 1 1 1 18 1 1 1 1 1 1 1 1 1 19 1 1 1 1 1 1 1 1 1 20 1 1 1 1 1 1 1 1 1 21 1 1 1 1 1 1 1 1 1 22 1 1 1 1 1 1 1 1 1 23 1 1 1 1 1 1 1 1 1 24 1 1 1 1 1 1 1 1 1 25 1 1 1 1 1 1 1 1 1 26 1 1 1 1 1 1 1 1 1 27 1 1 1 1 1 0 0 0 0 28 1 1 1 1 1 1 1 1 1 29 1 1 1 1 1 1 1 1 1 30 1 1 1 1 1 1 1 1 1 31 1 1 1 1 1 1 1 1 1 32 1 1 1 1 1 1 1 1 1 33 1 1 1 1 1 1 1 1 1 34 1 1 1 1 1 1 1 1 1 35 1 1 1 1 1 1 1 1 1 36 1 1 1 1 1 1 1 1 1 37 1 1 1 1 1 1 1 1 1 38 1 1 1 1 1 1 1 1 1 39 1 1 1 1 1 1 1 1 1 40 1 1 1 1 1 1 1 1 1 41 1 1 1 1 1 1 1 1 1 42 1 1 0 0 0 1 1 1 1 43 1 1 1 1 1 1 1 1 1 44 1 1 1 1 1 1 1 1 1 45 1 1 1 1 1 1 1 1 1 46 1 1 1 1 1 1 1 1 1 47 1 1 1 1 1 1 1 1 1 48 1 1 1 1 1 1 1 1 1 49 0 1 1 1 1 1 1 1 1 50 0 1 1 1 1 0 0 0 0 51 0 1 1 1 1 1 1 1 1 52 0 1 1 1 1 0 0 1 1 53 0 1 1 1 1 0 0 0 0 54 0 1 1 1 1 0 0 0 0 55 0 1 1 1 1 1 1 1 1 56 1 1 1 1 1 0 0 0 1 57 0 1 1 1 1 0 0 0 0 58 0 1 1 1 1 0 0 0 0 59 0 1 1 1 1 0 0 0 0 60 0 1 1 1 1 0 0 0 0 61 0 1 1 1 1 0 0 0 0 62 0 1 1 1 1 0 1 1 1 63 0 1 1 1 1 0 0 0 0 64 0 0 0 0 0 0 0 0 1 65 0 0 0 0 0 0 0 0 1 66 0 0 0 0 0 1 1 1 1 67 1 1 1 1 1 1 1 1 1 68 1 1 1 1 1 1 1 1 1 69 1 1 1 1 1 1 1 1 1 70 1 1 1 1 1 1 1 1 1 71 1 1 1 1 1 1 1 1 1 72 1 1 1 1 1 1 1 1 1 73 1 1 1 1 1 1 1 1 1 74 1 1 1 1 1 1 1 1 1 75 1 1 1 1 1 1 1 1 1 76 1 1 1 1 1 1 1 1 1 77 1 1 1 1 1 1 1 1 1 78 1 1 1 1 1 1 1 1 1 79 1 1 1 1 1 1 1 1 1 80 1 1 1 1 1 1 1 1 1 81 1 1 1 1 1 1 1 1 1 82 1 1 1 1 1 1 1 1 1 83 1 1 1 1 1 1 1 1 1 84 1 1 1 1 1 1 1 1 1 85 1 1 1 1 1 1 1 1 1 86 1 1 1 1 1 1 1 1 1 87 1 1 1 1 1 1 1 1 1 88 1 1 1 1 1 1 1 1 1 89 0 1 1 1 1 1 1 1 1 90 1 1 1 1 1 1 1 1 1 91 1 1 1 1 1 1 1 1 1 92 1 1 1 1 1 1 1 1 1 93 1 1 1 1 1 1 1 1 1 94 1 1 1 1 1 1 1 1 0 95 1 1 1 1 1 1 1 1 0 96 1 1 1 1 1 1 1 1 1 97 0 0 0 0 0 1 1 1 1 98 0 0 0 0 0 1 1 1 0 99 0 0 0 0 0 1 0 0 0 100 1 1 1 1 1 1 1 1 1 101 1 1 1 1 1 1 1 1 1 102 1 1 1 1 1 1 1 1 1 103 1 1 1 1 1 1 1 1 1 104 1 1 1 1 1 1 1 1 1 105 1 1 1 1 1 1 1 1 1 106 1 1 1 1 1 1 1 1 1 107 1 1 1 1 1 1 1 1 1 108 1 1 1 1 1 1 1 1 1 109 1 1 1 1 1 1 1 1 1 110 1 1 1 1 1 1 1 1 1 111 1 1 1 1 1 1 1 1 1 112 1 1 1 1 1 1 1 1 1 113 1 1 1 1 1 1 1 1 1 114 0 0 0 0 0 0 0 0 1 115 1 1 1 1 1 1 1 1 1 116 1 1 1 1 1 1 1 1 1 117 1 1 1 1 1 1 1 1 1 118 0 0 0 0 0 1 0 1 0 119 1 0 0 0 0 1 0 1 0 120 0 1 1 0 1 1 0 1 0 121 0 0 0 0 0 1 1 1 1 122 1 0 0 0 0 1 1 1 1 123 0 0 0 0 0 1 1 0 1 124 0 1 0 0 1 1 1 1 1 125 0 0 0 0 0 1 1 0 1

(92) TABLE-US-00033 TABLE 10B Transition number VIR45 ID6 ID7 ID8 ID9 ID10 VIR10 VIR19 VIR28 1 1 1 1 1 1 1 0 0 0 2 1 1 1 1 1 1 0 0 0 3 1 1 1 1 1 1 0 0 0 4 1 1 1 1 1 1 0 0 0 5 1 1 1 1 1 1 0 0 0 6 1 1 1 1 1 1 0 0 0 7 1 1 1 1 1 1 0 0 0 8 1 1 1 1 1 1 0 0 0 9 1 1 1 1 1 1 0 0 0 10 1 1 1 1 1 1 0 0 0 11 1 1 1 1 1 1 0 0 1 12 1 1 1 1 1 1 0 0 0 13 1 1 1 1 1 1 0 0 0 14 1 1 1 1 1 1 0 0 1 15 1 1 1 1 1 1 0 0 0 16 1 1 1 1 1 1 0 0 0 17 1 1 1 1 1 1 0 0 0 18 1 1 1 1 1 1 0 0 0 19 1 1 1 1 1 1 0 0 0 20 1 1 1 1 1 1 0 0 0 21 1 1 1 1 1 1 0 0 0 22 1 1 1 1 1 1 0 0 0 23 1 1 1 1 1 1 0 0 0 24 1 1 1 1 1 1 0 0 0 25 1 1 1 1 1 1 0 0 0 26 1 1 1 1 1 1 0 0 0 27 1 1 1 1 1 1 0 0 0 28 1 1 1 1 1 1 0 0 0 29 1 1 1 1 1 1 0 0 0 30 1 1 1 1 1 1 0 0 0 31 1 1 1 1 1 1 0 0 0 32 1 1 1 1 1 1 0 0 0 33 1 1 0 0 0 0 0 0 0 34 1 1 1 1 1 1 1 0 0 35 1 1 1 1 1 1 0 1 0 36 1 1 1 1 1 1 0 0 0 37 1 1 1 1 1 1 0 0 0 38 1 1 1 1 1 1 0 0 0 39 1 1 1 1 1 1 0 0 0 40 1 1 1 1 1 1 0 0 0 41 1 1 1 1 1 1 0 0 0 42 1 1 0 0 0 0 0 0 0 43 1 1 1 1 1 1 0 0 0 44 1 1 1 1 1 1 0 0 0 45 1 1 0 0 0 0 0 0 0 46 1 1 1 1 1 1 0 0 0 47 1 1 1 1 1 1 0 0 0 48 1 1 1 1 1 1 0 0 0 49 1 0 0 0 0 0 0 0 0 50 0 0 0 0 0 0 0 0 0 51 1 0 0 0 0 0 0 0 0 52 1 0 0 0 0 0 0 0 0 53 0 0 0 0 0 0 0 1 0 54 0 0 0 0 0 0 0 0 0 55 1 0 0 0 0 0 0 0 0 56 0 0 0 0 0 0 0 0 0 57 0 0 0 0 0 0 0 0 0 58 0 0 0 0 1 1 1 0 0 59 0 0 0 0 0 0 0 0 0 60 0 0 0 0 0 0 0 0 0 61 0 0 1 1 1 0 0 0 0 62 1 0 1 1 1 0 0 0 0 63 0 0 0 0 0 0 0 0 0 64 1 0 0 0 0 0 0 0 0 65 1 0 0 0 0 0 0 0 0 66 1 0 0 0 0 0 0 0 0 67 1 1 1 1 1 1 0 0 0 68 1 1 1 1 1 1 1 0 0 69 1 1 1 1 1 1 0 1 0 70 1 1 1 1 1 1 0 0 0 71 1 1 1 1 1 1 0 0 0 72 1 1 0 0 0 0 0 0 0 73 1 1 1 1 1 1 0 0 0 74 1 1 1 1 1 1 0 0 0 75 1 1 1 1 1 1 0 0 0 76 1 1 1 1 1 1 0 0 0 77 1 1 1 1 1 1 0 0 0 78 1 1 1 1 1 1 0 0 0 79 1 1 1 1 1 1 1 1 0 80 1 1 1 1 1 1 0 0 0 81 1 1 1 1 1 1 0 0 0 82 1 1 1 1 1 1 0 0 0 83 1 1 1 1 1 1 0 0 0 84 1 1 1 1 1 1 0 0 0 85 1 1 1 1 1 1 0 0 0 86 1 1 1 1 1 1 0 0 0 87 1 1 0 0 0 0 0 0 0 88 1 0 1 1 1 1 0 0 0 89 1 0 1 1 1 1 0 0 0 90 1 1 1 1 1 1 0 0 0 91 1 1 1 1 1 1 1 0 0 92 1 1 1 1 1 1 0 0 0 93 1 1 1 1 1 1 0 0 0 94 0 1 1 1 1 1 0 0 0 95 1 1 1 1 1 1 0 0 0 96 1 1 1 1 1 1 0 0 0 97 1 1 1 1 1 1 0 0 0 98 1 0 1 0 1 0 0 0 0 99 0 0 0 0 0 0 0 0 0 100 1 1 1 1 1 1 0 0 0 101 1 1 1 1 1 1 1 0 0 102 1 1 1 1 1 1 0 0 0 103 1 1 1 1 1 1 0 0 0 104 1 1 1 1 1 1 0 0 0 105 1 1 1 1 1 1 0 0 0 106 1 1 1 1 1 1 0 0 0 107 1 1 1 1 1 1 0 0 0 108 1 1 1 1 1 1 0 0 0 109 1 1 1 1 1 1 0 0 0 110 1 1 1 1 1 1 0 0 0 111 1 1 1 1 1 1 0 0 0 112 1 1 1 1 1 1 0 0 0 113 1 1 1 1 1 1 0 0 0 114 1 0 0 0 0 0 0 0 0 115 1 1 1 1 1 1 0 0 0 116 1 1 1 1 1 1 0 0 0 117 1 1 1 1 1 1 0 0 0 118 0 0 0 0 0 0 0 0 0 119 0 0 0 0 0 0 0 0 0 120 0 0 0 0 0 0 0 0 0 121 0 0 0 0 0 0 0 0 0 122 0 1 0 1 1 1 0 0 0 123 1 0 0 0 0 0 0 0 0 124 1 0 0 0 0 0 0 0 0 125 0 0 0 0 0 0 0 0 0

(93) TABLE-US-00034 TABLE 11 Strains Species Transitions I Transitions R Transitions V AST1 E. coli 48 1 2 AST2 E. coli 48 15 2 AST3 E. coli 47 15 1 AST4 E. coli 47 15 0 AST5 E. coli 47 15 2 VIR41 E. coli 47 3 8 VIR42 E. coli 47 4 5 VIR43 E. coli 47 5 6 VIR44 E. coli 47 6 5 VIR45 E. coli 48 5 2 ID6 E. coli 48 0 1 ID7 E. coli 45 2 0 ID8 E. coli 45 2 1 ID9 E. coli 45 3 1 ID10 E. coli 45 1 1 VIR10 S. aureus 1 1 0 VIR19 S. pneumoniae 1 1 0 VIR28 C. difficile 2 0 0

(94) All the E. coli samples exhibit more than 44 positive transitions in the I category. All these samples are therefore confirmed as indeed belonging to the E. coli species.

(95) On the other hand, the VIR10, VIR19 and VIR28 samples exhibit less than 3 positive transitions in the I category, these samples are therefore confirmed as not belonging to the E. coli species.

(96) The AST2, AST3, AST4 and AST5 strains of E. coli exhibit more than 14 positive transitions for the R category, they therefore express the plasmid-mediated penicillinase TEM-2, this being synonymous with a mechanism of resistance to penicillins.

(97) On the other hand, the AST1, VIR41, VIR42, VIR43, VIR44, VIR45, ID6, ID7, ID8, ID9 and ID10 strains of E. coli exhibit less than 7 positive transitions for the R category, they do not therefore express the plasmid-mediated penicillinase TEM-2. These strains are therefore sensitive to penicillins, in particular aminopenicillins or A penicillins (ampicillin), carboxypenicillins or C penicillins (ticarcillin) and ureidopenicillin or penicillin U (piperacillin).

(98) The VIR10, VIR19 and VIR28 strains which do not belong to the E. coli species exhibit less than two transitions for the R category, they do not therefore express TEM-2, thereby confirming the specificity of the method.

(99) The VIR41, VIR42, VIR43 and VIR44 samples of E. coli exhibit more than four positive transitions in the V category, they therefore express the shigatoxin 1 or 2 (STX1 or STX2) toxins. More specifically, for VIR41, transitions 118 to 125 are positive, VIR41 therefore simultaneously expresses shigatoxin 1 and shigatoxin 2. VIR42 and VIR44 are positive for transitions 121 to 125, they therefore express shigatoxin 2. The same is true for VIR45, which exhibits transitions 123 and 124. VIR43, which exhibits transitions 118 to 122, expresses shigatoxin 2. On the other hand, the AST1, AST2, AST3, AST4, AST5, VIR45, ID6, ID7, ID8, ID9 and ID10 strains of E. coli exhibit less than three positive transitions, they do not therefore express shigatoxin. These strains do not therefore have the properties of virulence related to shigatoxins.

(100) The VIR10, VIR19 and VIR28 strains which do not belong to the E. coli species also exhibit less than three transitions for the V category, they do not therefore express stx1 or stx2, thereby confirming the specificity of the method.

(101) For the typing, the T-category transitions of each strain are compared with the transitions of the other strains considered as reference strains. In practice, a value 0 is assigned when the transitions between the two strains are classified in the same category (positive or negative) and a value of 1 is assigned when the transitions between the two strains are classified in different categories (a positive transition and a negative transition). The values are summed for all the T-category transitions of each strain pair in order to establish a score. The scores are given in TABLE 12:

(102) TABLE-US-00035 TABLE 12 AST1 AST2 AST3 AST4 AST5 VIR41 VIR42 VIR43 VIR44 VIR45 AST1 0 19 18 18 19 17 12 15 17 16 AST2 19 0 3 3 2 24 21 18 22 19 AST3 18 3 0 0 1 25 22 21 23 20 AST4 18 3 0 0 1 25 22 21 23 20 AST5 19 2 1 1 0 24 23 20 24 21 VIR41 17 24 25 25 24 0 5 6 12 13 VIR42 12 21 22 22 23 5 0 7 7 8 VIR43 15 18 21 21 20 6 7 0 10 9 VIR44 17 22 23 23 24 12 7 10 0 5 VIR45 16 19 20 20 21 13 8 9 5 0 ID6 3 22 19 19 20 16 13 16 18 17 ID7 11 22 19 19 20 22 19 20 22 19 ID8 12 23 20 20 21 23 20 21 23 20 ID9 11 22 19 19 20 20 17 18 22 19 ID10 9 22 19 19 20 20 17 20 22 19 VIR10 92 109 106 106 107 105 102 105 103 102 VIR19 92 109 106 106 107 105 102 105 103 102 VIR28 95 112 109 109 110 108 105 108 106 105 ID6 ID7 ID8 ID9 ID10 VIR10 VIR19 VIR28 AST1 3 11 12 11 9 92 92 95 AST2 22 22 23 22 22 109 109 112 AST3 19 19 20 19 19 106 106 109 AST4 19 19 20 19 19 106 106 109 AST5 20 20 21 20 20 107 107 110 VIR41 16 22 23 20 20 105 105 108 VIR42 13 19 20 17 17 102 102 105 VIR43 16 20 21 18 20 105 105 108 VIR44 18 22 23 22 22 103 103 106 VIR45 17 19 20 19 19 102 102 105 ID6 0 10 11 10 8 91 91 94 ID7 10 0 1 4 4 89 89 92 ID8 11 1 0 2 3 90 92 92 ID9 10 4 2 0 3 90 94 94 ID10 8 4 3 3 0 87 91 91 VIR10 91 89 90 90 87 ~ ~ ~ VIR19 91 89 92 94 91 ~ ~ ~ VIR28 94 92 92 94 91 ~ ~ ~

(103) The strains which have a score less than or equal to 4 are of the same type, the strains which have a score strictly greater than 4 are of different type.

(104) Thus, the AST2, AST3, AST4 and AST5 strains are of the same type. The same is true, respectively, for the AST1 and ID6 strains and for the ID7, ID8, ID9 and ID10 strains. All the other strains taken in pairs are of different types. The high sums obtained between the VIR10, VIR19 and VIR28 strains, which are not S. aureus, and all the other strains which are S. aureus, should be noted. These results confirm the specificity of the method.

(105) The VIR10, VIR19 and VIR28 strains are of different species. These strains cannot therefore be compared with one another and no value is reported in TABLE 12. Extremely advantageously, scores greater than 20, for instance between AST4 and VIR41, reflect a great divergence between strains. Scores between 12 and 20, as between ID7 and VIR42, reflect a moderate divergence and scores between 4 and 12, as between ID10 and AST1, a weak divergence.

(106) The method thus implemented therefore makes it possible not only to establish whether two strains are of the same type, which is important for identifying a common seat of infection, but also to estimate the proximity of two strains, which is extremely important for epidemiological studies.

(107) This example shows that, very advantageously, the present invention makes it possible, in a time of less than one hour, which is very short, to confirm the identity of a species such as E. coli and to determine, simultaneously within the same analysis, the properties of typing and potential resistance to at least one antibiotic and to establish the existence of a virulence factor. These properties were established with the same instrument, which greatly facilitates the analysis and the reporting of the results. Finally, the characteristics of the bacteria are established using bacterial proteins, which reflects the existence of live and viable microorganisms, unlike characterizations using bacterial DNA which can be distorted by the existence of dead bacteria.

EXAMPLE 8: PROTOCOL FOR DIGESTION OF MICROORGANISMS IN THE PRESENCE OF METHANOL

(108) Conventionally, the following protocol is implemented in 17 steps: 1. Sampling of a microorganism colony, obtained according to example 1, and suspension in 400 l of a 50 mM ammonium bicarbonate solution, pH=8.0. 2. Addition of 600 l of methanol. Steps 3 to 7: idem steps 2 to 6 of example 4. Steps 8 to 17: idem steps 8 to 17 of example 6.

EXAMPLE 9: CHARACTERIZATION OF C. ALBICANS SAMPLES

(109) After having established the one or more species of the samples according to any one of the methods described in examples 1 to 3, the species listed below are analyzed.

(110) Seventeen C. albicans strains are analyzed in order to confirm their identification and to establish their characteristics:

(111) TABLE-US-00036 ATF1 ATF2 ATF3 ATF4 ATF5 ATF6 ATF7 VIR31 VIR32 VIR33 VIR34 VIR35 VIR36 VIR37 VIR38 VIR39 CA16

(112) The same method of analysis is applied to species not belonging to the C. albicans species in order to serve as a negative control: E. coli

(113) VIR43 S. aureus

(114) VIR5 E. faecium

(115) AST-VAN8

(116) Each sample is treated according to example 8, then a volume of 5 l of digested proteins is injected and analyzed according to the same conditions as in example 7.

(117) The peptides, resulting from the digestion of the proteins of the microorganism, are analyzed using the mass spectrometer in MRM mode. Only the peptides indicated in TABLE 13 are detected. For this, the first-generation fragment(s) indicated in TABLE 13 is (are) detected. The application, to which each transition, i.e. each peptide associated with its first-generation fragment, makes it possible to respond, is specified in the clinical interest column of TABLE 14 with the letters I, T, R and V. I denotes the confirmation of the identification of the microorganism, T the typing, R the resistance to at least one antibiotic and V the detection of virulence factors.

(118) TABLE-US-00037 TABLE 13 Transition First-generation Clinical number Protein Peptide (SEQ ID NO.) fragment ion interest 1 ALF ADEEFFAK (88) y5 singly charged I + T 2 ALF ADEEFFAK (88) y6 singly charged I + T 3 ALF ADEEFFAK (88) y7 singly charged I + T 4 HSP75 AFDDESVQK (89) y6 singly charged I + T 5 HSP75 AFDDESVQK (89) y7 singly charged I + T 6 HSP75 AFDDESVQK (89) y8 singly charged I + T 7 ENO1 AKIDVVDQAK (90) y6 singly charged I + T 8 ENO1 AKIDVVDQAK (90) y7 singly charged I + T 9 ENO1 AKIDVVDQAK (90) y8 singly charged I + T 10 RL13 AVEVPEQTAYR (91) y6 singly charged I + T 11 RL13 AVEVPEQTAYR (91) y7 singly charged I + T 12 RL13 AVEVPEQTAYR (91) y8 singly charged I + T 13 RS22 CGVIQPR (92) y5 singly charged I + T 14 RS22 CGVIQPR (92) y6 singly charged I + T 15 RS22 CGVIQPR (92) y4 singly charged I + T 16 RLA4 DLQELIAEGNTK (93) y8 singly charged I + T 17 RLA4 DLQELIAEGNTK (93) y9 singly charged I + T 18 RLA4 DLQELIAEGNTK (93) y7 singly charged I + T 19 PPIA FADENFVKR (94) y6 singly charged I + T 20 PPIA FADENFVKR (94) y7 singly charged I + T 21 PPIA FADENFVKR (94) y8 singly charged I + T 22 TPIS FALDTGVK (95) y6 singly charged I + T 23 TPIS FALDTGVK (95) y7 singly charged I + T 24 TPIS FALDTGVK (95) y5 singly charged I + T 25 RL28 GRLPEVPVIVK (96) y8 singly charged I + T 26 RL28 GRLPEVPVIVK (96) y5 singly charged I + T 27 ALF IAEALDIFHTK (97) y6 singly charged I + T 28 ALF IAEALDIFHTK (97) y7 singly charged I + T 29 ALF IAEALDIFHTK (97) y8 singly charged I + T 30 ENO1 IDVVDQAK (98) y5 singly charged I + T 31 ENO1 IDVVDQAK (98) y6 singly charged I + T 32 ENO1 IDVVDQAK (98) y7 singly charged I + T 33 ENO1 IEEELGSEAIYAGKDFQK (99) y8 singly charged I + T 34 ENO1 IEEELGSEAIYAGKDFQK (99) y9 singly charged I + T 35 PPIA IESFGSGSGATSK (100) y9 singly charged I + T 36 PPIA IESFGSGSGATSK (100) y10 singly charged I + T 37 PPIA IESFGSGSGATSK (100) y11 singly charged I + T 38 NTF2 LASLPFQK (101) y5 singly charged I + T 39 NTF2 LASLPFQK (101) y6 singly charged I + T 40 NTF2 LASLPFQK (101) y7 singly charged I + T 41 MBF1 LDATDDVVAVK (102) y7 singly charged I + T 42 MBF1 LDATDDVVAVK (102) y8 singly charged I + T 43 MBF1 LDATDDVVAVK (102) y9 singly charged I + T 44 PGK NVEHLVEK (103) y6 singly charged I + T 45 PGK NVEHLVEK (103) y7 singly charged I + T 46 PGK NVEHLVEK (103) y5 singly charged I + T 47 RL36 SGIAAGVNK (104) y5 singly charged I + T 48 RL36 SGIAAGVNK (104) y6 singly charged I + T 49 RL36 SGIAAGVNK (104) y7 singly charged I + T 50 G3P SGVDYVIESTGVFTK (105) y9 singly charged I + T 51 G3P SGVDYVIESTGVFTK (105) y10 singly charged I + T 52 G3P SGVDYVIESTGVFTK (105) y8 singly charged I + T 53 EF1B SLNEFLADK (106) y6 singly charged I + T 54 EF1B SLNEFLADK (106) y7 singly charged I + T 55 RL13 SQETFDANVAR (107) y7 singly charged I + T 56 RL13 SQETFDANVAR (107) b8 singly charged I + T 57 RL13 SQETFDANVAR (107) y9 singly charged I + T 58 CS111 SSSSTTKK (108) y6 singly charged I + T 59 CS111 SSSSTTKK (108) y7 singly charged I + T 60 HSP71 STLDPVGK (109) y6 singly charged I + T 61 HSP71 STLDPVGK (109) y7 singly charged I + T 62 HSP71 STLDPVGK (109) y5 singly charged I + T 63 PGK SVELFQQAVAK (110) y7 singly charged I + T 64 PGK SVELFQQAVAK (110) y8 singly charged I + T 65 PGK SVELFQQAVAK (110) y6 singly charged I + T 66 KPYK TANDVLELR (111) y5 singly charged I + T 67 KPYK TANDVLELR (111) y6 singly charged I + T 68 KPYK TANDVLELR (111) y7 singly charged I + T 69 ENO1 VGDKIQIVGDDLTVTNPTR (112) y7 singly charged I + T 70 ENO1 VGDKIQIVGDDLTVTNPTR (112) y8 singly charged I + T 71 ENO1 VGDKIQIVGDDLTVTNPTR (112) y9 singly charged I + T 72 ADH1 VVAIDGGDEK (113) y7 singly charged I + T 73 ADH1 VVAIDGGDEK (113) y8 singly charged I + T 74 ADH1 VVAIDGGDEK (113) y9 singly charged I + T 75 RS22 WTDNLLPAR (114) y6 singly charged I + T 76 RS22 WTDNLLPAR (114) y7 singly charged I + T 77 RS22 WTDNLLPAR (114) y8 singly charged I + T 78 G3P YKGEVTASGDDLVIDGHK (115) y10 singly charged I + T 79 G3P YKGEVTASGDDLVIDGHK (115) y11 singly charged I + T 80 G3P YKGEVTASGDDLVIDGHK (115) y9 singly charged I + T 81 ADH1 YVLDTSK (116) y5 singly charged I + T 82 ADH1 YVLDTSK (116) y6 singly charged I + T 83 ADH1 YVLDTSK (116) y4 singly charged I + T 84 CP51 AVIYDCPNSR (117) y6 singly charged R + T 85 CP51 AVIYDCPNSR (117) y7 singly charged R + T 86 CP51 GHYVLVFPGYAHTSER (177) 10 singly charged R + T 87 CP51 GHYVLVFPGYAHTSER (177) y9 singly charged R + T 88 CP51 GHYVLVSPGYAHTSER (118) y9 singly charged R + T 89 CP51 GHYVLVSPGYAHTSER (118) 10 singly charged R + T 90 CP51 GVIYDCPNSR (119) y6 singly charged R + T 91 CP51 GVIYDCPNSR (119) y7 singly charged R + T 92 CP51 GVIYDCPNSR (119) y8 singly charged R + T 93 CP51 GVSSPYLPFGGGR (120) y9 singly charged R + T 94 CP51 GVSSPYLPFGGGR (120) y6 singly charged R + T 95 CP51 GVSSPYLPFSGGK (121) y9 doubly charged R + T 96 CP51 GVSSPYLPFSGGK (121) y9 singly charged R + T 97 CP51 GVSSPYLPFSGGK (121) y6 singly charged R + T 98 CP51 GVSSPYLPFSGGR (122) y9 doubly charged R + T 99 CP51 GVSSPYLPFSGGR (122) y9 singly charged R + T 100 CP51 GVSSPYLPFSGGR (122) y6 singly charged R + T 101 LIPASE8 AAVGDILQSR (123) y7 singly charged V + T 102 LIPASE8 AAVGDILQSR (123) y8 singly charged V + T 103 LIPASE8 ITPDDLR (124) y5 singly charged V + T 104 LIPASE8 ITPDDLR (124) y6 singly charged V + T 105 LIPASE8 ITPDDLR (124) y4 singly charged V + T 106 LIPASE8 TGWDILK (125) y5 singly charged V + T 107 LIPASE8 TGWDILK (125) y6 singly charged V + T 108 LIPASE8 TGWDILK (125) y4 singly charged V + T
The charge state of the precursor peptide, its retention time and the transitions, i.e. the ratios (m/z).sub.1 in Q1 and (m/z).sub.2 in Q3, are indicated in TABLE 14. The collision energy used to fragment the precursor ion is also indicated in TABLE 14.

(119) TABLE-US-00038 TABLE 14 Precursor (m/z) (m/z) Transition charge Retention filtered filtered Collision number state time in Q1 in Q3 energy 1 2 11.9 478.72 641.33 26 2 2 11.9 478.72 770.37 26 3 2 11.9 478.72 885.4 26 4 2 7.8 519.74 705.34 28 5 2 7.8 519.74 820.37 28 6 2 7.8 519.74 967.44 28 7 2 8.5 543.81 659.37 29 8 2 8.5 543.81 774.4 29 9 2 8.5 543.81 887.48 29 10 2 11 631.82 767.37 33 11 2 11 631.82 864.42 33 12 2 11 631.82 963.49 33 13 2 6.4 415.22 612.38 23 14 2 6.4 415.22 669.4 23 15 2 6.4 415.22 513.31 23.3 16 2 15.5 665.85 845.47 34 17 2 15.5 665.85 974.52 34 18 2 15.5 665.85 732.39 34.3 19 2 10.5 563.29 792.44 30 20 2 10.5 563.29 907.46 30 21 2 10.5 563.29 978.5 30 22 2 12.5 425.74 632.36 24 23 2 12.5 425.74 703.4 24 24 2 12.5 425.74 519.28 23.7 25 2 14.8 603.88 880.55 32 26 2 14.8 603.88 555.39 31.6 27 2 17.8 629.35 760.4 33 28 2 17.8 629.35 873.48 33 29 2 17.8 629.35 944.52 33 30 2 9 444.25 560.3 25 31 2 9 444.25 659.37 25 32 2 9 444.25 774.4 25 33 3 18.6 676.34 956.48 38 34 3 18.6 676.34 1069.57 38 35 2 8.4 614.3 751.36 32 36 2 8.4 614.3 898.43 32 37 2 8.4 614.3 985.46 32 38 2 14.1 452.27 632.38 25 39 2 14.1 452.27 719.41 25 40 2 14.1 452.27 790.45 25 41 2 12.1 573.31 745.41 30 42 2 12.1 573.31 846.46 30 43 2 12.1 573.31 917.49 30 44 2 7 484.26 754.41 26 45 2 7 484.26 853.48 26 46 2 7 484.26 625.37 26.3 47 2 4.5 408.73 488.28 23 48 2 4.5 408.73 559.32 23 49 2 4.5 408.73 672.4 23 50 2 18 801.41 981.53 40 51 2 18 801.41 1080.59 40 52 2 18 801.41 868.44 40.3 53 2 14.3 518.77 722.37 28 54 2 14.3 518.77 836.41 28 55 2 10.1 619.29 792.4 32 56 2 10.1 619.29 893.36 32 57 2 10.1 619.29 1022.49 32 58 2 10.6 413.22 651.37 23 59 2 10.6 413.22 738.4 23 60 2 8.7 408.73 628.37 23 61 2 8.7 408.73 729.41 23 62 2 8.7 408.73 515.28 23 63 2 14.6 610.34 791.44 32 64 2 14.6 610.34 904.53 32 65 2 14.6 610.34 644.37 31.9 66 2 12.9 515.78 629.4 28 67 2 12.9 515.78 744.43 28 68 2 12.9 515.78 858.47 28 69 3 15.3 681.04 788.43 38 70 3 15.3 681.04 901.51 38 71 3 15.3 681.04 1016.54 38 72 2 7.7 501.76 733.34 27 73 2 7.7 501.76 804.37 27 74 2 7.7 501.76 903.44 27 75 2 14.9 543.29 683.42 29 76 2 14.9 543.29 798.45 29 77 2 14.9 543.29 899.49 29 78 2 11.4 952.47 1068.53 47 79 2 11.4 952.47 1155.56 47 80 2 11.4 952.47 1011.51 46.9 81 2 8.5 413.22 563.3 23 82 2 8.5 413.22 662.37 23 83 2 8.5 413.22 450.22 23.2 84 2 9.3 597.78 748.3 31 85 2 9.3 597.78 911.37 31 86 2 14.3 916.96 1164.54 45 87 2 14.3 916.96 1017.47 45.3 88 2 11 886.94 1017.47 44 89 2 11 886.94 1104.51 44 90 2 9.4 590.77 748.3 31 91 2 9.4 590.77 911.37 31 92 2 9.4 590.77 1024.45 31 93 2 16.1 647.33 963.5 33 94 2 16.1 647.33 590.3 33.5 95 2 15.8 648.34 483.26 34 96 2 15.8 648.34 965.51 34 97 2 15.8 648.34 592.31 33.5 98 2 16 662.34 497.26 34 99 2 16 662.34 993.52 34 100 2 16 662.34 620.32 34.1 101 2 12.75 515.29 788.43 28 102 2 12.75 515.29 887.49 28 103 2 9.83 415.22 615.31 23 104 2 9.83 415.22 716.36 23 105 2 9.83 415.22 518.26 23.3 106 2 15.81 416.73 674.39 23 107 2 15.81 416.73 731.41 23 108 2 15.81 416.73 488.31 23.3

(120) The other machine parameters used are the same as in example 7. The areas obtained for each of the transitions and for each of the microorganisms studied were measured. All the transitions with an area greater than or equal to 2000 (arbitrary units) are considered to be positive and have been denoted 1 in tables 15A and 15B. All the transitions with an area less than 2000 are considered to be negative and have been denoted 0 in tables 15A and 15B. When no signal peak was observed, the transition was noted as negative.

(121) TABLE-US-00039 TABLE 15A Transition number ATF1 ATF2 ATF3 ATF4 VIR31 ATF5 ATF6 ATF7 VIR32 VIR33 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 3 1 1 1 1 1 1 1 1 1 1 4 1 1 1 1 1 1 1 1 1 1 5 1 1 1 1 1 1 1 1 1 1 6 1 1 1 1 1 1 1 1 1 1 7 1 1 1 1 1 1 1 1 1 1 8 1 1 1 1 1 1 1 1 1 1 9 1 1 1 1 1 1 1 1 1 1 10 1 1 1 1 0 1 1 1 0 0 11 1 1 1 1 0 1 1 1 0 1 12 1 1 1 1 0 1 1 1 0 0 13 1 1 1 1 0 1 1 1 1 1 14 1 1 1 1 0 1 1 1 1 1 15 1 1 1 1 1 1 1 1 1 1 16 1 1 1 1 1 1 1 1 1 1 17 1 1 1 1 1 1 1 1 1 1 18 1 1 1 1 1 1 1 1 1 1 19 1 1 1 1 0 1 1 1 0 1 20 1 1 1 1 0 1 1 1 1 1 21 1 1 1 1 0 1 1 1 0 1 22 1 1 1 1 1 1 1 1 1 1 23 1 1 1 1 1 1 1 1 1 1 24 1 1 1 1 1 1 1 1 1 1 25 1 1 1 1 1 1 1 1 1 1 26 1 1 1 1 1 1 1 1 1 1 27 0 0 0 0 1 0 0 0 1 1 28 0 0 0 0 1 0 0 0 1 1 29 0 0 0 0 0 0 0 0 0 1 30 1 1 1 1 1 1 1 1 1 1 31 1 1 1 1 1 1 1 1 1 1 32 1 1 1 1 1 1 1 1 1 1 33 0 0 0 0 1 0 0 0 1 1 34 0 0 0 0 0 0 0 0 0 1 35 1 1 1 1 1 1 1 1 1 1 36 1 1 1 1 1 1 1 1 1 1 37 1 1 1 1 1 1 1 1 1 1 38 1 1 1 1 0 1 1 1 0 0 39 1 1 1 1 0 1 1 1 0 1 40 1 1 1 1 0 1 1 1 0 0 41 1 1 1 1 1 1 1 1 1 1 42 1 1 1 1 1 1 1 1 1 1 43 1 1 1 1 1 1 1 1 1 1 44 1 1 1 1 1 1 1 1 1 1 45 1 1 1 1 1 1 1 1 1 1 46 1 1 1 1 1 1 1 1 1 1 47 1 1 1 1 1 1 1 1 1 1 48 1 1 1 1 1 1 1 1 1 1 49 0 0 0 0 1 0 0 0 1 1 50 1 1 1 1 1 1 1 1 1 1 51 1 1 1 1 1 1 1 1 1 1 52 1 1 1 1 1 1 1 1 1 1 53 1 1 1 1 0 1 1 1 1 1 54 1 1 1 1 1 1 1 1 1 1 55 1 1 1 1 0 1 1 1 0 0 56 1 1 1 1 0 1 1 1 0 0 57 1 1 1 1 0 1 1 1 0 0 58 0 0 0 0 1 0 0 0 1 1 59 0 0 0 0 1 0 0 0 1 1 60 1 1 1 1 1 1 1 1 1 1 61 1 1 1 1 0 1 1 1 0 1 62 1 1 1 1 1 1 1 1 1 1 63 1 1 1 1 1 1 1 1 1 1 64 1 1 1 1 0 1 1 1 0 0 65 1 1 1 1 0 1 1 1 1 1 66 1 1 1 1 1 1 1 1 1 1 67 1 1 1 1 1 1 1 1 1 1 68 1 1 1 1 1 1 1 1 1 1 69 1 1 1 1 1 1 1 1 1 1 70 1 1 1 1 1 1 1 1 1 1 71 1 1 1 1 0 1 1 1 1 1 72 1 1 1 1 1 1 1 1 1 1 73 1 1 1 1 1 1 1 1 1 1 74 1 1 1 1 1 1 1 1 1 1 75 1 1 1 1 1 1 1 1 1 1 76 1 1 1 1 1 1 1 1 1 1 77 1 1 1 1 1 1 1 1 1 1 78 1 1 1 1 1 1 1 1 1 1 79 1 1 1 1 1 1 1 1 1 1 80 1 1 1 1 0 1 1 1 1 1 81 1 1 1 1 1 1 1 1 1 1 82 1 1 1 1 1 1 1 1 1 1 83 1 1 1 1 1 1 1 1 1 1 84 0 0 0 0 0 0 1 1 0 0 85 0 0 0 0 0 0 1 1 0 0 86 0 0 1 0 0 0 0 0 0 0 87 0 1 1 0 0 0 0 0 0 0 88 0 0 0 1 0 0 0 0 0 0 89 0 0 0 1 0 0 0 0 0 0 90 1 1 0 1 0 1 1 0 0 1 91 1 1 0 1 0 1 1 0 0 0 92 0 0 0 1 0 0 0 0 0 0 93 1 1 1 1 0 0 1 0 0 0 94 1 1 1 1 0 0 1 1 0 0 95 0 0 0 0 1 1 0 0 1 1 96 0 0 0 0 0 1 0 0 0 0 97 1 0 0 1 1 1 0 1 1 1 98 0 0 0 0 0 0 0 1 0 1 99 0 0 0 0 0 0 0 1 0 0 100 0 0 0 0 0 0 0 1 0 0 101 0 0 0 0 1 0 0 0 0 0 102 0 0 0 0 1 0 0 0 0 0 103 0 0 0 0 1 0 0 0 1 1 104 0 0 0 1 1 0 0 0 1 1 105 0 0 0 0 1 0 0 0 1 0 106 0 0 0 0 1 0 0 0 1 0 107 0 0 0 0 1 0 0 0 1 0 108 0 0 0 0 1 0 0 0 1 0

(122) TABLE-US-00040 TABLE 15B Transition AST- number VIR34 VIR35 VIR36 VIR37 VIR38 VIR39 CA16 VIR43 VIR5 VAN8 1 1 1 1 1 1 1 1 0 0 0 2 1 1 1 1 1 1 1 0 0 0 3 1 1 1 1 1 1 1 0 0 0 4 1 1 1 1 1 1 1 0 0 0 5 1 1 1 1 1 1 1 0 0 0 6 1 1 1 1 1 1 1 0 0 0 7 1 1 1 1 1 1 1 1 0 0 8 1 1 1 1 1 1 1 0 0 0 9 1 1 1 1 1 1 1 0 0 0 10 0 0 0 0 0 0 1 0 0 0 11 1 1 1 1 1 1 1 0 0 0 12 0 0 0 0 0 0 1 1 0 0 13 1 1 1 1 1 0 1 0 0 0 14 1 1 0 1 1 1 1 0 0 0 15 1 1 1 1 1 1 1 0 0 0 16 1 1 1 1 1 1 1 0 0 0 17 1 1 1 1 1 1 1 0 0 0 18 1 1 1 1 1 1 1 0 0 0 19 1 1 1 1 1 1 1 0 0 0 20 1 1 1 1 1 1 1 1 0 0 21 1 1 1 1 1 1 1 0 0 0 22 1 1 1 1 1 1 1 0 0 0 23 1 1 1 1 1 1 1 1 0 0 24 1 1 1 1 1 1 1 1 0 0 25 1 1 1 1 1 1 1 0 0 0 26 1 1 1 1 1 1 1 0 0 0 27 1 1 1 1 1 1 1 0 0 0 28 1 1 1 1 1 1 1 0 0 0 29 1 1 1 1 1 1 1 0 0 0 30 1 1 1 1 1 1 1 0 0 0 31 1 1 1 1 1 1 1 0 0 0 32 1 1 1 1 1 1 1 1 1 1 33 1 1 1 1 1 1 1 0 0 0 34 1 1 0 1 1 1 1 0 0 0 35 1 1 1 1 1 1 1 0 0 0 36 1 1 1 1 1 1 1 0 0 0 37 1 1 1 1 1 1 1 0 0 0 38 0 0 0 0 0 0 1 0 0 0 39 1 1 1 0 0 1 1 0 0 0 40 0 0 0 0 0 0 1 0 0 0 41 1 1 1 1 1 1 1 0 0 0 42 1 1 1 1 1 1 1 0 0 0 43 1 1 1 1 1 1 1 0 0 0 44 1 1 1 1 1 1 1 0 0 0 45 1 1 1 1 1 1 1 0 0 0 46 1 1 1 1 1 1 1 0 0 0 47 1 1 1 1 1 1 1 0 0 0 48 1 1 1 1 1 1 1 0 0 0 49 1 1 1 1 1 1 0 0 0 0 50 1 1 1 1 1 1 1 0 0 0 51 0 1 1 1 1 1 1 0 0 0 52 1 1 1 1 1 1 1 0 0 0 53 1 1 1 1 1 1 1 0 0 0 54 1 1 1 1 1 1 1 0 0 0 55 0 0 0 0 0 0 1 0 0 0 56 0 0 0 0 0 0 1 0 0 0 57 0 0 0 0 0 0 1 0 0 0 58 1 1 1 1 1 1 1 0 0 0 59 1 1 1 1 1 1 1 0 0 0 60 1 1 1 1 1 1 1 0 0 0 61 1 0 0 0 1 1 0 0 0 0 62 1 1 1 1 1 1 1 1 0 0 63 0 1 1 1 1 1 1 1 0 0 64 0 1 1 1 1 1 1 0 0 0 65 0 1 1 1 1 1 1 1 0 0 66 1 1 1 1 1 1 1 0 0 0 67 1 1 1 1 1 1 1 0 0 0 68 1 1 1 1 1 1 1 0 0 0 69 1 1 1 1 1 1 1 0 0 0 70 1 1 1 1 1 1 1 1 0 0 71 1 1 1 1 1 1 1 0 0 0 72 1 1 1 1 1 1 1 0 0 0 73 1 1 1 1 1 1 1 0 0 0 74 1 1 1 1 1 1 1 0 0 0 75 1 1 1 1 1 1 1 0 0 0 76 1 1 1 1 1 1 1 1 0 0 77 1 1 1 1 1 1 1 0 0 0 78 1 1 1 1 1 1 1 0 0 0 79 1 1 1 1 1 1 1 0 0 0 80 1 1 1 1 1 1 0 0 0 0 81 1 1 1 1 1 1 1 0 1 0 82 1 1 1 1 1 1 1 1 0 0 83 1 1 1 1 1 1 1 0 0 0 84 0 0 0 0 0 0 0 0 0 0 85 0 0 0 0 0 0 0 0 0 0 86 0 0 0 0 0 0 0 0 0 0 87 0 0 0 0 0 0 0 0 0 0 88 0 0 0 0 0 0 0 0 0 0 89 0 0 0 0 0 0 0 0 0 0 90 0 0 0 0 0 0 0 0 0 0 91 0 0 0 0 0 0 0 0 0 0 92 0 0 0 0 0 0 0 1 0 0 93 0 0 0 0 0 0 0 0 0 0 94 1 0 0 0 0 0 0 0 0 0 95 0 1 1 1 1 1 1 0 0 0 96 0 0 0 0 0 0 0 0 0 0 97 1 0 0 1 1 1 0 0 0 0 98 1 1 1 1 1 0 0 0 0 0 99 0 0 0 0 0 0 0 0 0 0 100 0 0 0 0 0 0 0 0 0 0 101 0 0 0 0 0 0 0 0 0 0 102 0 0 0 0 0 0 0 0 0 0 103 0 1 1 0 0 1 0 1 0 0 104 0 1 1 1 1 0 1 0 0 0 105 0 0 0 0 0 0 0 0 0 0 106 0 0 0 0 0 0 0 0 0 0 107 0 0 0 0 0 0 0 0 0 0 108 0 0 0 0 0 0 0 0 0 0

(123) The positive-transition number is then summed for the I, R and V applications and reported in TABLE 16:

(124) TABLE-US-00041 TABLE 16 Strains Species Transitions I ATF1 C. albicans 75 ATF2 C. albicans 75 ATF3 C. albicans 75 ATF4 C. albicans 75 VIR31 C. albicans 61 ATF5 C. albicans 75 ATF6 C. albicans 75 ATF7 C. albicans 75 VIR32 C. albicans 68 VIR33 C. albicans 75 VIR34 C. albicans 72 VIR35 C. albicans 75 VIR36 C. albicans 73 VIR37 C. albicans 74 VIR38 C. albicans 75 VIR39 C. albicans 75 CA16 C. albicans 80 VIR43 E coli 12 VIR5 S. aureus 2 AST- E. faecium 1 VAN8

(125) All the C. albicans samples exhibit more than 60 positive transitions in the I category. All these samples are therefore confirmed as indeed belonging to the C. albicans species.

(126) On the other hand, the VIR43, VIR5 and AST-VANS samples exhibit less than 13 positive transitions in the I category, these samples are therefore confirmed as not belonging to the C. albicans species.

(127) The observation of transitions 84 and 85 indicates the presence of the mutated peptide and reflects the resistance of the ATF6 and ATF7 strains.

(128) The observation of transitions 86 and 87 indicates the presence of the mutated peptide and reflects the resistance of the ATF2 and ATF3 strains.

(129) The observation of transition 96 indicates the presence of the mutated peptide and reflects the resistance of the ATF5 strain.

(130) The observation of transitions 99 and 100 indicates the presence of the mutated peptide and reflects the resistance of the ATF7 strain.

(131) The observation of transitions 88 and 89 indicates the presence of the native peptide and reflects the sensitivity of the ATF4 strain.

(132) The observation of transitions 93 and 94 indicates the presence of the native peptide and reflects the sensitivity of the ATF1 and ATF4 strains.

(133) All the areas of the I transitions are summed to give the sum SI. All the areas of the V transitions are summed to give the sum SV. The SV/SI ratio is then calculated and multiplied by a multiplication factor MF. The results obtained are given in TABLE 17. TABLE 17:

(134) TABLE-US-00042 TABLE 17 Ratio of the Sum (SI) of Sum (SV) of sums SV/SI the areas the areas of multipled by of the I the V the multiplication Multiplication Strains transitions transitions factor (MF) factor (MF) ATF1 1.8E+07 1.6E+03 1.9E+03 2.0E+07 ATF2 1.3E+07 1.6E+03 2.4E+03 ATF3 2.0E+07 2.3E+03 2.3E+03 ATF4 2.9E+07 3.3E+03 2.3E+03 VIR31 1.6E+07 1.4E+06 1.8E+06 ATF5 1.7E+07 2.3E+03 2.6E+03 ATF6 2.4E+07 4.8E+03 4.0E+03 ATF7 2.9E+07 2.4E+03 1.7E+03 VIR32 2.9E+07 5.3E+05 3.6E+05 VIR33 1.9E+07 4.2E+04 4.4E+04 VIR34 4.2E+07 1.8E+03 8.9E+02 VIR35 1.1E+07 8.8E+03 1.5E+04 VIR36 8.3E+06 3.5E+04 8.5E+04 VIR37 2.3E+07 3.9E+03 3.4E+03 VIR38 2.4E+07 3.4E+03 2.8E+03 VIR39 1.6E+07 9.2E+03 1.1E+04 CA16 1.2E+07 5.4E+03 9.1E+03

(135) The VIR31 and VIR32 strains, which have a (SV/SI) ratioMF greater than 910.sup.4, overexpress lipase 8, these strains are therefore virulent. All the other strains have a ratio less than 910.sup.4, they do not overexpress lipase 8 and are not therefore virulent. Interestingly, since the (SV/SI) ratioMF is higher for the VIR31 strain than for the VIR32 strain, the VIR31 strain is characterized as more virulent than the VIR32 strain.

(136) For the typing, the T-category transitions of each strain are compared with the transitions of the other strains considered as reference strains. In practice, a value 0 is assigned when the transitions between the two strains are classified in the same category (positive or negative) and a value of 1 is assigned when the transitions between the two strains are classified in different categories (a positive transition and a negative transition). The values are summed for all the T-category transitions of each strain pair in order to establish a score. The scores are given in TABLE 18:

(137) TABLE-US-00043 TABLE 18 Strain ATF1 ATF2 ATF3 ATF4 ATF5 ATF6 ATF7 VIR31 VIR32 VIR33 VIR34 ATF1 0 2 5 4 4 3 8 39 30 23 23 ATF2 2 0 3 6 6 3 10 41 32 25 25 ATF3 5 3 0 9 9 6 9 40 31 26 24 ATF4 4 6 9 0 8 7 12 41 32 25 27 ATF5 4 6 9 8 0 7 10 37 28 21 25 ATF6 3 3 6 7 7 0 7 42 33 26 26 ATF7 8 10 9 12 10 7 0 41 32 25 23 VIR31 39 41 40 41 37 42 41 0 9 22 26 VIR32 30 32 31 32 28 33 32 9 0 13 19 VIR33 23 25 26 25 21 26 25 22 13 0 8 VIR34 23 25 24 27 25 26 23 26 19 8 0 VIR35 25 25 24 27 23 26 25 22 13 4 10 VIR36 25 25 24 27 23 26 25 20 13 6 12 VIR37 24 26 25 26 22 27 24 21 12 5 9 VIR38 23 25 24 25 21 26 23 22 13 4 8 VIR39 22 24 23 26 20 25 24 21 14 5 9 CA16 16 16 15 18 14 17 18 29 22 15 19 VIR43 70 70 69 72 70 71 72 65 66 71 71 VIR5 78 78 77 82 78 79 80 69 74 79 73 AST- 79 79 78 83 79 80 81 70 75 80 74 VAN8 Strain VIR35 VIR36 VIR37 VIR38 VIR39 CA16 VIR43 VIR5 AST-VAN8 ATF1 25 25 24 23 22 16 70 78 79 ATF2 25 25 26 25 24 16 70 78 79 ATF3 24 24 25 24 23 15 69 77 78 ATF4 27 27 26 25 26 18 72 82 83 ATF5 23 23 22 21 20 14 70 78 79 ATF6 26 26 27 26 25 17 71 79 80 ATF7 25 25 24 23 24 18 72 80 81 VIR31 22 20 21 22 21 29 65 69 70 VIR32 13 13 12 13 14 22 66 74 75 VIR33 4 6 5 4 5 15 71 79 80 VIR34 10 12 9 8 9 19 71 73 74 VIR35 0 2 3 4 5 11 69 77 78 VIR36 2 0 5 6 7 13 67 75 76 VIR37 3 5 0 1 6 12 70 76 77 VIR38 4 6 1 0 5 13 71 77 78 VIR39 5 7 6 5 0 14 68 76 77 CA16 11 13 12 13 14 0 72 80 81 VIR43 69 67 70 71 68 72 ~ ~ ~ VIR5 77 75 76 77 76 80 ~ ~ ~ AST- 78 76 77 78 77 81 ~ ~ ~ VAN8

(138) The strains which have a score less than or equal to 2 are of the same type, the strains which have a score strictly greater than 2 are of different type. Thus, the ATF1 and ATF2, VIR35 and VIR36, and VIR37 and VIR38 strains are respectively of the same type. All the other strains taken in pairs are of different types.

(139) The high sums obtained between the VIR43, VIR5 and AST-VANS strains, which are not S. aureus, and all the other strains, which are S. aureus, should be noted. These results confirm the specificity of the method.

(140) The VIR43, VIR5 and AST-VANS strains are of different species. These strains cannot therefore be compared with one another and no value is reported in TABLE 18. Extremely advantageously, scores greater than 20, for instance between ATF1 and VIR39, reflect a great divergence between strains. Scores between 14 and 20, as between CA16 and ATF1, reflect a moderate divergence and scores between 4 and 14, as between ATF1 and ATF7, a weak divergence.

(141) The method thus implemented therefore makes it possible not only to establish whether two strains are of the same type, which is important for identifying a common seat of infection, but also to estimate the proximity of two strains, which is extremely important for epidemiological studies.

(142) This example shows that, very advantageously, the present invention makes it possible, in a time of less than one hour, which is very short, to confirm the identity of a species such as C. albicans and to determine, simultaneously within the same analysis, the properties of typing and potential resistance to at least one antibiotic and to establish the existence of a virulence factor. The present invention also allows quantitative assaying, which is particularly advantageous when the properties of resistance to at least one antibiotic, or virulence, are linked to the level of expression of a protein or of a metabolite. This is what we illustrate here with the quantitative assaying of lipase 8. These properties were established with the same instrument, which greatly facilitates the analysis and the reporting of the results. Finally, the characteristics of yeasts are established using fungal proteins, which reflects the existence of live and viable microorganisms, unlike characterizations using fungal DNA which can be distorted by the existence of dead yeasts.

EXAMPLE 10: PROTOCOL FOR DIGESTION OF MICROORGANISMS IN THE PRESENCE OF METHANOL, SUITABLE FOR ASSAYING AT LEAST ONE METABOLITE

(143) Conventionally, the following protocol is implemented in 19 steps: 1. Sampling of five microorganism colonies, obtained according to example 1, and suspension in 100 l of a 6M guanidine hydrochloride, 50 mM Tris-HCl solution, pH=8.0. 2. Centrifugation at 15000 g for 5 minutes. 3. Pellet taken up in 400 l of a 50 mM ammonium bicarbonate solution, pH=8.0. 4. Addition of 600 l of methanol. Steps 5 to 9: idem steps 2 to 6 of example 4. Steps 10 to 19: idem steps 8 to 17 of example 6.

EXAMPLE 11: CHARACTERIZATION OF C. ALBICANS SAMPLES BY SIMULTANEOUS ANALYSIS OF PROTEINS AND OF METABOLITES

(144) After having established the one or more species of the samples according to any one of the methods described in examples 1 to 3, the species listed below are analyzed.

(145) Five strains of C. albicans are analyzed in order to confirm their identification and to establish their characteristics:

(146) TABLE-US-00044 ATF1 ATF2 ATF3 ATF4 ATF6
Each sample is treated according to example 10, then a volume of 20 l of digested proteins is injected and analyzed according to the same conditions as in example 7. Tables 13 and 14 are identical, with one exception. The transition number 109 is added to the method. It corresponds to the molecule, ergosterol, with clinical interest I and R. The charge state of the precursor is 1, the retention time is 36.3 minutes, the m/z filtered in Q1 is 379.4, the m/z filtered in Q3 is 69.2, and the collision energy is 47. The mass parameters are identical to those of example 7, with the exception of those below: Curtain gas: 25.00 psi Source temperature: 450.00 C. Heating gas: 50.00 psi Entry potential before Q0 (EP): 4.00 V For transition 109, parameters differ: Declustering potential (DP): 200.00 V Entry potential before Q0 (EP): 10.00 V Collision cell exit potential (CXP): 8 V
All the transitions 1 to 108 are analyzed in a manner identical to example 9. All the areas of the I transitions are summed to give the sum SI. The area of transition 109 is called A109. The A109/SI ratio is then calculated and multiplied by a multiplication factor MF. The results obtained are given in table 19.

(147) TABLE-US-00045 TABLE 19 Area of Sum (SI) A109/SI ratio transition of the multiplied by the 109 areas of the I multiplication Multiplification Strains (A109) transitions factor (MF) factor (MF) ATF1 1.7E+03 9.3E+07 1.6E+03 9.0E+07 ATF2 7.9E+03 1.1E+08 6.4E+03 ATF3 3.9E+03 5.7E+07 6.3E+03 ATF4 5.7E+03 2.9E+08 1.8E+03 ATF6 6.4E+03 6.8E+07 8.5E+03
The ATF2, ATF3 and ATF6 strains, which have a (A109/SI) ratioMF greater than 210.sup.3, overexpress ergosterol; these strains are therefore resistant. All the other strains have a ratio less than 210.sup.3, they do not express ergosterol and are therefore not resistant.

(148) This example shows that, very advantageously, the present invention makes it possible to simultaneously assay, within the same analysis, in a time of less than one hour, which is very short, and quantitatively, various compounds such as proteins, peptides or metabolites. Even more advantageously than the assaying of a protein, the quantitative assaying of a metabolite resulting from the action of this protein characterizes the presence of a functional protein and more broadly of a functional synthesis pathway. Thus, the quantification of ergosterol makes it possible in this case to establish the existence of a mechanism of resistance to fluconazole linked to the existence of functional and strongly active lanosterol demethylase.

EXAMPLE 12: CHARACTERIZATION OF MICROORGANISMS PRESENT IN URINE SAMPLES

(149) After having established the one or more species of the samples according to any one of the methods described in examples 1 to 3, five urine samples contaminated with E. coli (Urine 1 to 5) are analyzed. A noncontaminated sixth urine (Urine 6) is also analyzed in order to serve as a negative control.

(150) The following protocol is implemented in 29 steps (steps 5 to 12 are optional and could be omitted without significantly altering the results): 1. Centrifugation of 5 ml of contaminated urine at 2000 g for 30 seconds. 2. Recovery of the supernatant. 3. Centrifugation at 15000 g for 5 minutes. 4. Removal of the supernatant. 5. Washing of the pellet with 3 ml of distilled water by resuspension. 6. Centrifugation at 15000 g for 5 minutes. 7. Removal of the supernatant. 8. Bringing the pellet into contact with solvent in a dilution to 1/10. 9. Leaving for one hour at 20 C. 10. Centrifugation at 15000 g for 5 minutes. 11. Removal of the supernatant. 12. Bringing the pellet into contact with solvent in a dilution to 1/10. 13. Leaving for one hour at 20 C. 14. Centrifugation at 15000 g for 5 minutes. 15. Removal of the supernatant. 12. Suspension of the pellet in 10 to 100 l of a 6M guanidine hydrochloride, 50 mM Tris-HCl solution, pH=8.0. 13. Steps 14 to 18: idem steps 2 to 6 of example 4. 19. Steps 19 to 28: idem steps 8 to 17 of example 6. 29. Injection of 100 l of acidified eluate onto the chromatographic system and mass spectrometer, according to the protocol described in example 7.

(151) The areas obtained for each of the transitions and for each of the microorganisms studied were measured. Since the urine samples have a bacterial load that is very different from one sample to another, the threshold used in example 7 for declaring a positive transition cannot be used here. It must be adjusted to the amount of bacteria present. In the previous example, the sampling of a colony resulted in a comparable amount of microorganisms. This is no longer the case in this example. Thus, the amount of bacteria is estimated by summing the area of all the I transitions. The sum of the areas of the I transitions is reported in TABLE 20.

(152) TABLE-US-00046 TABLE 20 Urine 1 Urine 2 Urine 3 Urine 4 Urine 5 Urine 6 1 509 251.3 828 079.5 5 349 271.0 11 918 946.9 1 054 480.6 0.0
Thus, sample 4 comprises a bacterial load that is higher than sample 3, which itself has a higher load than examples 1 and 5, which themselves have higher loads than sample 2. Samples 1 and 5 have a comparable bacterial load. Sample 6 does not comprise an E. coli and exhibits a zero load, thereby demonstrating the specificity of the technique.
The area of each transition is then standardized by dividing it by the area of all the I transitions. All the transitions of which the ratio is greater than or equal to 0.00015 (non-dimensional ratio) are considered to be positive and have been denoted 1 in TABLE 21. All the transitions of which the ratio is less than 0.00015 are considered to be negative and have been denoted 0 in TABLE 21. When no signal peak was observed, the transition was noted as negative.

(153) TABLE-US-00047 TABLE 21 Transition number Urine 1 Urine 2 Urine 3 Urine 4 Urine 5 Urine 6 1 1 1 1 1 1 0 2 1 1 1 1 1 0 3 1 1 1 1 1 0 4 0 0 1 1 1 0 5 0 0 1 1 1 0 6 0 0 0 1 1 0 7 0 0 0 0 0 0 8 0 0 0 0 0 0 9 0 0 0 0 0 0 10 1 1 1 1 1 0 11 1 1 1 1 1 0 12 1 1 1 1 1 0 13 1 1 1 1 1 0 14 1 1 1 1 1 0 15 1 1 1 1 1 0 16 1 1 1 1 1 0 17 1 1 1 1 1 0 18 1 1 1 1 0 0 19 1 1 1 1 0 0 20 1 0 1 0 0 0 21 1 1 1 1 1 0 22 1 1 1 1 1 0 23 1 1 1 1 1 0 24 1 1 1 1 1 0 25 1 1 0 1 1 0 26 1 1 0 1 1 0 27 1 1 0 1 1 0 28 1 1 1 1 1 0 29 1 1 1 1 1 0 30 1 1 1 1 1 0 31 0 1 1 1 1 0 32 0 0 1 1 1 0 33 0 0 0 1 0 0 34 1 1 1 1 1 0 35 1 1 1 1 1 0 36 1 1 1 1 1 0 37 1 1 1 1 1 0 38 1 1 1 1 1 0 39 1 1 1 1 1 0 40 1 1 1 1 1 0 41 1 1 1 1 1 0 42 1 1 1 1 1 0 43 1 1 1 1 1 0 44 1 1 1 1 1 0 45 1 1 1 1 1 0 46 0 0 1 1 1 0 47 0 0 1 1 1 0 48 0 0 1 1 1 0 49 0 0 0 0 0 0 50 0 0 0 0 0 0 51 0 0 0 0 0 0 52 0 0 0 0 0 0 53 0 0 0 0 0 0 54 0 0 0 0 0 0 55 1 1 1 0 1 0 56 1 1 1 0 1 0 57 1 1 1 0 1 0 58 1 1 1 0 1 0 59 1 1 1 0 1 0 60 1 1 1 0 0 0 61 0 0 0 0 0 0 62 0 0 0 0 0 0 63 0 0 0 0 0 0 64 0 0 0 0 0 0 65 0 0 0 0 0 0 66 1 0 0 0 0 0 67 1 1 1 1 1 0 68 1 1 1 1 1 0 69 1 1 1 1 1 0 70 1 0 0 1 1 0 71 1 0 0 1 1 0 72 0 0 0 0 0 0 73 1 0 1 1 1 0 74 1 0 1 1 1 0 75 1 0 1 1 1 0 76 1 1 1 1 1 0 77 1 1 1 1 1 0 78 1 1 1 1 1 0 79 1 1 1 1 1 0 80 1 1 1 1 1 0 81 1 1 1 1 1 0 82 1 1 1 1 1 0 83 1 1 1 1 1 0 84 1 1 1 1 1 0 85 1 1 1 1 1 0 86 1 1 1 1 1 0 87 1 1 1 1 1 0 88 1 1 1 1 1 0 89 1 1 1 1 1 0 90 1 1 1 1 1 0 91 1 0 1 1 1 0 92 1 1 1 1 1 0 93 1 0 1 1 1 0 94 0 0 0 0 0 0 95 0 0 0 0 0 0 96 0 0 0 0 0 0 97 0 0 0 0 0 0 98 0 0 0 0 0 0 99 0 0 0 0 0 0 100 0 0 0 0 0 0 101 0 0 0 0 0 0 102 0 1 0 0 0 0 103 1 1 1 1 1 0 104 1 1 1 1 1 0 105 1 1 1 1 1 0 106 1 1 1 1 1 0 107 1 1 1 1 1 0 108 1 1 1 1 1 0 109 1 1 1 1 1 0 110 1 1 1 1 1 0 111 1 1 1 1 1 0 112 1 1 1 1 1 0 113 1 1 1 1 1 0 114 0 0 0 1 1 0 115 1 1 1 1 1 0 116 1 1 1 1 1 0 117 1 1 1 1 1 0 118 0 0 0 0 0 0 119 0 0 0 0 0 0 120 0 0 0 0 0 0 121 1 1 1 0 0 0 122 0 0 0 0 0 0 123 0 0 0 0 0 0 124 0 0 0 0 0 0 125 0 0 0 0 0 0

(154) The positive-transition number is then summed for the I, R and V applications and reported in TABLE 22:

(155) TABLE-US-00048 TABLE 22 Urine 1 Urine 2 Urine 3 Urine 4 Urine 5 Urine 6 I 36 36 40 44 41 0 R 6 6 6 0 5 0 V 1 1 1 0 0 0

(156) In the same way as the transition positivity threshold had to be modified in order to take into account the concentration of the samples, the number of peptides necessary for characterizing a strain must be adjusted to the total concentration of bacteria. Certain weakly detected peptides in example 7 may be under the limit of detection if the amount of bacteria is less than one colony.

(157) Urines 1 to 5 exhibit more than 30 positive transitions in the I category. All these samples are therefore confirmed as contaminated with the E. coli species.

(158) On the other hand, urine 6 exhibits no positive transition in the I category. It is therefore confirmed as not being contaminated with the E. coli species.

(159) Urines 1, 2, 3 and 5 exhibit at least five positive transitions for the R category, they therefore express the plasmid-mediated penicillinase TEM-2, which is synonymous with a mechanism of resistance to penicillins, in particular aminopenicillins or A penicillins (ampicillin), carboxypenicillins or C penicillins (ticarcillin) and ureidopnicillin or penicillin U (piperacillin).

(160) On the other hand, urines 4 and 6 do not exhibit positive transitions for the R category, they do not therefore express the plasmid-mediated penicillinase TEM-2. These strains are therefore sensitive to penicillins

(161) These results of resistance to ampicillin, ticarcillin and piperacillin were confirmed with the VITEK2 automated device sold by the applicant and the AST-EXN and AST-N103 cards. The confirmation took a time of 6 to 8 hours on the VITEK2 automated device, which is slower than for the present invention.

(162) Urines 1 to 6 do not exhibit more than one positive transition for the V category. These urines are not therefore contaminated with toxins of shigatoxin type.

(163) For the typing, the T-category transitions of each strain are compared with the transitions of the other strains considered as reference strains. In practice, a value 0 is assigned when the transitions between the two strains are classified in the same category (positive or negative) and a value of 1 is assigned when the transitions between the two strains are classified in different categories (a positive transition and a negative transition). The values are summed for all the T-category transitions of each strain pair in order to establish a score. The scores are given in TABLE 23:

(164) TABLE-US-00049 TABLE 23 Urine 1 Urine 2 Urine 3 Urine 4 Urine 5 Urine 1 0 11 13 19 15 Urine 2 11 0 16 24 20 Urine 3 13 16 0 16 12 Urine 4 19 24 16 0 8 Urine 5 15 20 12 8 0

(165) No urine has a score less than or equal to 4. The strains infecting urines 1 to 5 are therefore of different types. These urine samples were collected in a local medical test laboratory where infections often occur in different environments. Under these conditions, it is uncommon to observe urine infections linked to the same strain.

(166) This example shows that, very advantageously, the present invention makes it possible, directly from the primary sample, which is very advantageous, to confirm the identity of a species such as E. coli and to determine, simultaneously within the same analysis, the properties of typing and potential resistance to at least one antibiotic.

LITERATURE REFERENCES

(167) [1] J. Anhalt & C. Fenselau, 1975, Anal. Chem., 47(2):219-225. [2] A. Fox et al., ed., 1990, Analytical microbiology methods: chromatography and mass spectrometry, Plenum Press, New York, N.Y. [3] M. Claydon et al., 1996, Nature Biotech. 14:1584-1586. [4] T. Krishnamurthy & P. Ross, 1996, Rapid Com. Mass Spec., 10:1992-1996. [5] P. Seng et al. 2009, Clin. Infect. Dis., 49:543-551. [6] C. Fenselau et al., 2008, Appl. Environ. Microbiol., 904-906. [7] D. Ding et al. 2009, J. Pharm. Biomed. Anal. 50:79-85. [8] R. Everley et al., 2009, J. Microbiol. Methods, 77:152-158. [9] S. Hofstadler et al., 2005, Int. J. Mass Spectrom., 242:23-41. [10] D. Ecker, 2008, Nat. Rev. Microbiol., 6(7):553-558. [11] W.-J. Chen et al., 2008, Anal. Chem., 80: 9612-9621. [12] D. Lopez-Ferrer et al., 2008, Anal. Chem., 80:8930-8936. [13] D. Lopez-Ferrer et al., 2005, J. Proteome res., 4(5): 1569-1574. [14] T. Fortin et al., 2009, Mol. Cell Proteomics, 8(5): 1006-1015. [15] H. Keshishian et al., 2007, Mol. Cell Proteomics, 2212-2229. [16] J. Stal-Zeng et al., 2007, Mol. Cell Proteomics, 1809-1817. [17] Gaskell, Electrospray: principles and practise, 1997, J. Mass Spectrom., 32, 677-688). [18] V. Fusaro et al., 2009, Nature Biotech. 27, 190-198. [19] J. Mead et al., 15 Nov. 2008, Mol. Cell Proteomics, E-pub. [20] F. Desiere et al., 2006, Nucleic Acids Res., 34 (database issue): D655-8. [21] L. Anderson & C. Hunter, 2006, Mol. Cell Proteomics, 573-588. [22] B. Han & R. Higgs, 2008, Brief Funct Genomic Proteomic., 7(5):340-54. [23] K.-Y. Wang et al., 2008, Anal Chem, 80(16) 6159-6167. [24] J. Bundy & C. Fenselau, 1999, Anal. Chem. 71: 1460-1463. [25] K-C Ho et al., 2004, Anal. Chem. 76: 7162-7268. [26] Y. S. Lin et al., 2005, Anal. Chem., 77: 1753-1760. [27] S. Vaidyanathan et al., 2001, Anal. Chem., 73:4134-4144. [28] P. Seng et al., 2009, Clin. Infect. Dis., 49:543-551. [29] Manes N. et al., 2007, Mol. & Cell. Proteomics, 6(4): 717-727. [30] R. Nandakumar et al., 2009, Oral Microbiology Immunology, 24:347-352. [31] L. Hernychova et al., 2008, Anal. Chem., 80:7097-7104. [32] J.-M. Pratt et al., 2006, Nat. Protoc., 1:1029-1043. [33] V. Brun et al., 2007, Mol. Cell Proteomics, 2139-2149.