SAMPLE PREPARATION FOR MASS SPECTROMETRY

Abstract

The present invention provides a method of preparing a sample for an analytic procedure, said sample comprising at least one protein, polypeptide or peptide molecule, and said method comprising fragmenting said molecule using at least one moving magnetic body.

Claims

1. A method of preparing a sample for an analytic procedure, said sample comprising at least one molecule selected from a protein, a polypeptide and a peptide molecule, and said method comprising: (a) fragmenting said molecule using at least one moving magnetic body to produce fragments of said molecule, wherein said at least one magnetic body performs a fluctuating or oscillating motion triggered by a fluctuating or oscillating magnetic field.

2. The method of claim 1, wherein said magnetic field is generated by an electric current and/or an electromagnet.

3. The method of claim 1, wherein: (i) said fragmenting is a non-enzymatic and non-chemical process; or (ii) said fragmenting is a chemical process comprising adding a chemical selected from CNBr, formic acid, hydroxylamine, 2-nitro-5-thiocyano benzoic acid and a protease to said sample.

4. The method of claim 1, wherein said magnetic body collides with said molecule; and/or at least one non-magnetic particle is present, wherein said motion of said magnetic body triggers collision of said at least one non-magnetic particle with said molecule.

5. The method of claim 1, wherein said sample is of biological origin and comprises: (i) a solution or suspension of said molecule; (ii) a cell selected from a prokaryotic and an eukaryotic cell; (iii) a suspension of viruses; and/or (iv) a tissue selected from muscle tissue and brain tissue.

6. The method of claim 1, further comprising: (b) exposing said sample to heat, denaturing said sample, adding detergent to said sample, and/or adding a chaotropic agent to said sample, wherein step (b) is performed prior to or concomitantly with step (a).

7. The method of claim 1, further comprising: (c) chemically modifying said molecule and/or the fragments of said molecule.

8. The method of claim 1, wherein said analytic procedure is mass spectrometry (MS).

9. The method of claim 7, wherein said chemically modifying said molecule and/or the fragments of said molecule is selected from: (ca) reducing a disulfide; (cb) alkylating a thiol group; (cc) cross-linking; or (cd) any combination of (ca), (cb) and (cc).

10. The method of claim 1, further comprising adding at least one of an inert viscous liquid; a polyacrylamide gel; agarose gel; an aerogel; and a zeolith to said sample.

11. The method of claim 1, further comprising: (d) cleaning and/or enriching the obtained fragments.

12. The method of claim 1, further comprising: (e) labeling said molecule and/or the fragments of said molecule.

13. The method of claim 12, wherein said labeling comprises reacting a functional group of said molecule with a reagent capable of forming a conjugate with said functional group, wherein said reagent capable of forming a conjugate is a tag which is detectable by mass spectrometry.

14. An analytic method comprising the method of claim 1, and a step of performing mass spectrometry of the fragments of said molecule.

15. A method of identifying a site on a first protein which is capable or suspected to be capable of binding to a second protein or a binding partner, said method comprising: fragmenting said first protein using at least one moving magnetic body, adding said second protein or said binding partner, separating fragments of said first protein which bind to said second protein or said binding partner from non-binding fragments, and identifying said fragments which bind said second protein or binding partner to identify said site, wherein said at least one magnetic body performs a fluctuating or oscillating motion triggered by a fluctuating or oscillating magnetic field, and wherein said first protein is an antigen and said second protein is an antibody.

16. (canceled)

17. A kit comprising: (i) at least one magnetic body; (ii) a vessel or array of vessels each configured to receive said magnetic body; and (iii) a sample comprising at least one molecule selected from a protein, a polypeptide and a peptide molecule.

18. The kit of claim 17, further comprising: (iv) (a reducing agent; and (v) an alkylating agent; and optionally: (vi) at least one of a surfactant, a chaotropic agent, a denaturing agent, and an organic solvent; (vii) at least one buffer; (viii) non-magnetic particles; and/or (ix) an instruction manual.

19. A device comprising: (i) a coil; and (ii) a vessel or an array of vessels; wherein the opening of said coil is configured to accommodate said vessel or said array of vessels; (iii) at least one magnetic body; and (iv) a control unit configured to cause said at least one magnetic body to perform a fluctuating or oscillating motion when in use.

20. A computer-implemented method of analyzing a mass spectrum obtained from a sample which has been prepared by the method of claim 1, said computer-implemented method comprising the step of assembling sequences of the fragments of said molecule to obtain a sequence of said molecule.

Description

[0202] The Figures show:

[0203] FIG. 1: Number of peptide or fragment identifications (ID). Yeast and Liver samples were either process by a regular sample preparation (Reg./Standard) or by magnetic fragmentation (Mag./Magnetic System). “Tryptic” peptides contain a K- or R-amino acid at their C-terminus while “Fragment” peptides can end with any other amino acid.

[0204] FIG. 2: Sequence coverage as observed with different implementation of the invention.

[0205] FIG. 3: Exemplary distribution of fragment sizes as obtained with the method described in Example 3.

[0206] The Examples illustrate the invention.

EXAMPLE 1

[0207] Improvement of Lysis and Digestion when Applying the Method of the Invention in Addition to Proteolytic Digestion

[0208] Materials

[0209] Fresh Saccharomyces cerevisiae and Mouse liver samples were used at a quantity corresponding to 100 μg protein content. Buffers and enzymes from the iST kit (P.O.00001, PreOmics GmbH) were used throughout this experiment.

[0210] Methods

[0211] Standard iST Sample Preparation:

[0212] Sample preparation was carried out according to the PreOmics standard protocol for yeast samples and according to the mammalian tissue protocol for liver samples. For cell lysis and protein denaturation, yeast pellets (approx. 100 μg protein content) were resuspended in 50 μl lyse buffer, boiled at 95° C. and 1000 rpm for 10 min and sheared in a Diagenode Bioruptor (10 cycles, 30 sec on, 30 sec off). Liver samples (1-2 mg wet weight with approx. 100 μg protein content) were resuspended in 100 μl lyse buffer, sheared in the Diagenode Bioruptor (10 cycles, 30 sec on, 30 sec off) with glass beads to facilitate tissue lysis and boiled at 95° C. and 1000 rpm for 10 min. All samples were further processed according to the manufacturer instructions. After elution, purified peptides were dried in a vacuum centrifuge and resuspended in LC-Load. Samples were analyzed on a ThermoFisher Scientific Easy n-LC 1000 system coupled with a Thermo LTQ Orbitrap XL. Peptides were separated on a home-made C18 column applying a 45 min gradient and tandem mass spectrometry was performed using a DDA Top 10 method. The MS/MS data was searched against a yeast database using the MaxQuant software with default settings, except that the unspecific digestion mode was selected.

[0213] iST sample preparation with cell lysis and digestion on novel Magnetic System:

[0214] Yeast samples containing 100 μg of protein were resuspended in 50 μl lyse buffer and either first boiled at 95° C. and 1000 rpm for 10 min or directly mixed with 50 μl trypsin/LysC solution. For cell lysis and protein digestion, samples were incubated on the magnetic system with a 3 mm round Neodymium magnet at a magnetic flux density of approx. 1 mT and 120 Hz for 60 min. Next, 100 μl stop buffer were added and peptides were purified and analyzed as described in Standard iST samples preparation.

[0215] Results

[0216] See FIG. 1.

[0217] Discussion

[0218] The magnetic system clearly improves the overall peptide identifications for yeast cells as well as tissue samples. When comparing the process in conjunction to additional boiling, the process appears to be free standing and additional boiling is not required to achieve best results. The system can directly be employed and can replace traditional lysis plus mixing during digestion. Best results were achieved when using the magnetic system for lysis and digestion in a directly combined manner.

EXAMPLE 2

[0219] Fragmentation of an individual protein by the method of the invention (in absence of any protease)

[0220] Materials

[0221] 50 μg Carbonic anhydrase (bovine erythrocytes; C7025-1VL) at 10 mg/ml in ddH2O was used for the following experiments. For protein extraction and fragmentation, a magnetic system prototype with a Helmholtz coil of 50 windings was developed and run with cylindric 3 mm×2 mm Samarium Cobalt magnets (MagnetExpert, F412SC-250). 1.2 mm steel beads or 1.4 mm ceramic beads were added where indicated. For peptide clean-up, the iST kit was used (PreOmics GmbH, P.O.00001).

[0222] Methods

[0223] Samples were incubated on the magnetic system prototype with a cylindric samarium cobalt magnet and steel beads or ceramic beads where indicated. The system was used at a magnetic flux density of approx. 1 mT and 120 Hz for 60 minutes. Fragment peptides were directly analyzed on a ThermoFisher Scientific Easy n-LC 1200 system coupled to a LTQ Orbitrap XL. Peptides were purified according to the iST manual and separated on a home-made C18 column applying a 45 min gradient and tandem mass spectrometry was performed using a DDA Top 10 method. The MS/MS data was searched against a carbonic anhydrase database using the MaxQuant software with default settings, except that the unspecific digestion mode was selected.

[0224] Results

[0225] Peptides of various lengths were generated. Fragments with differences of exactly one amino acid length are commonly observed. This provides a near perfect coverage of every possible peptide fragment option in the given analytical range. For example, fragments of sequence ANGERQSP, ANGERQSPV, ANGERQSPVD, ANGERQSPVDI, ANGERQSPVDID, ANGERQSPVDIDT, ANGERQSPVDIDTK, etc. were measured as individual peptides.

[0226] See FIG. 2.

[0227] Discussion

[0228] The magnetic system can be used to fragment a protein in nearly every peptide composition possible. With only 1.5 h sample preparation from start to measurement, the carbonic anhydrase could be fragmented and generated a peptide based protein sequence coverage of >95%. Four Experiments combined generated a complete sequence coverage. The peptide fragments obtained with the method of the invention can be used to sequence a protein, since fragments of every length combination are generated, differing only in the mass of a single amino acid.

[0229] By using these differences, the original sequence can furthermore be deduced.

EXAMPLE 3

[0230] Protein fragmentation on a proteome-wide scale

[0231] Materials

[0232] Fresh Saccharomyces cerevisiae cell pellets containing approximately 100 μg were used for the following experiments. For protein extraction and fragmentation, a magnetic system prototype with a coil of 250 windings and inner diameter of 12 mm was developed and run with 2 mm round Neodymium magnets. Peptide purification and preparation for LC-MS measurement was performed with iST cartridges and buffers from PreOmics GmbH (iST Kit, P.O.00001). Water was commercially purchased from Fisher Scientific (W6-212).

[0233] Methods

[0234] Yeast pellets containing 100 μg yeast proteins were resuspended in 100 μl ddH20 with pH of 1,4,7, or 10 in 90 μl ddH20 with pH of 1,4,7, or 10 and 10 μl acetonitrile. For protein extraction and fragmentation, samples were incubated on the magnetic system prototype with a 2 mm round Neodymium magnet applying a magnetic flux density of approx. 1.5 mT and 120 Hz for 60 minutes. Next, 100 μl iST stop buffer were added and peptides were purified according to the manufacturer protocol. Peptides were dried in the vacuum centrifuge and resuspended in LC-Load to a final concentration of 2.5 μg/μl. Samples were analyzed on a ThermoFisher Scientific Easy n-LC 1200 system coupled to a LTQ Orbitrap XL. Peptide loads of 5 μg were separated on a home-made C18 column applying a 45 min gradient and tandem mass spectrometry was performed using a DDA Top 10 method. The MS/ MS data was searched against a yeast database using the MaxQuant software with default settings, except that the unspecific digestion mode was selected.

[0235] Results

[0236] See FIG. 3.

[0237] Discussion

[0238] The magnetic system as used in this experiment generated peptides of a mean length of 13.2 amino acids which is longer then standard tryptic digestion (approx. 12 amino acid length). The system generates suitable peptide fragments at various pH ranges, also in the presence of organic solvent.