PROTEOMIC ANALYSIS OF SUBCELLULAR COMPARTMENTS

Abstract

Some embodiments are directed to a method for the subcellular proteomic analysis of a test biological sample, including metabolic isotopic labelling of proteins of a test biological sample, fixing of the sample, labelling of the test subcellular compartment, laser microdissection of said subcellular compartment, extracting the proteins of said subcellular compartment, reversion of the fixing and proteolysis, analyzing the peptides obtained by mass spectrometry, and identifying the analyzed peptides.

Claims

1. A process for the subcellular proteomic analysis of a test biological sample, comprising: a) metabolic isotopic labeling of the proteins from a test biological sample; b) fixation of the sample; c) labeling of the test subcellular compartment; d) laser microdissection of the subcellular compartment; e) extracting the proteins from the subcellular compartment, reversion of the fixation, and proteolysis; f) analyzing the peptides resulting from step e) by mass spectrometry; and g) identifying the peptides analyzed.

2. The process as claimed in claim 1, wherein the fixation is performed with a crosslinking agent.

3. The process as claimed in claim 2, wherein the crosslinking agent is paraformaldehyde.

4. The process as claimed in claim 1, wherein the labeling of the subcellular compartment is performed with a fluorescent marker.

5. The process as claimed in claim 1, wherein the subcellular compartment is chosen from a group consisting of the nucleus, cytoplasmic membrane, nuclear membrane, vesicles, mitochondria, lysosome, centriole, proteasome, focal adhesions, lamellipodium, filopodia, invadosome rosette, endoplasmic reticulum, Golgi apparatus, and cell-cell junctions.

6. A process for the in vitro identification of a protein from a biological sample from a subject, comprising: a) metabolic isotopic labeling of the proteins from a biological sample from a subject; b) fixation of the sample; c) labeling of the test subcellular compartment; d) laser microdissection of the subcellular compartment; e) extracting the proteins from the subcellular compartment, reversion of the fixation, and proteolysis; f) analyzing the peptides resulting from step e) by mass spectrometry; g) identifying of the peptides analyzed; h) qualitative and/or quantitative comparison of the peptides resulting from step g) relative to the peptides present in a reference sample or to a reference value.

7. The process as claimed in claim 6, wherein the subcellular compartment is the invadosome rosette.

8. The process as claimed in claim 1, wherein the isotope labeling is performed by the SILAC method.

9. The process as claimed in claim 2, wherein the subcellular compartment is chosen from a group consisting of the nucleus, cytoplasmic membrane, nuclear membrane, vesicles, mitochondria, lysosome, centriole, proteasome, focal adhesions, lamellipodium, filopodia, invadosome rosette, endoplasmic reticulum, Golgi apparatus, and cell-cell junctions.

10. The process as claimed in claim 3, wherein the subcellular compartment is chosen from a group consisting of the nucleus, cytoplasmic membrane, nuclear membrane, vesicles, mitochondria, lysosome, centriole, proteasome, focal adhesions, lamellipodium, filopodia, invadosome rosette, endoplasmic reticulum, Golgi apparatus, and cell-cell junctions.

11. The process as claimed in claim 4, wherein the subcellular compartment is chosen from a group consisting of the nucleus, cytoplasmic membrane, nuclear membrane, vesicles, mitochondria, lysosome, centriole, proteasome, focal adhesions, lamellipodium, filopodia, invadosome rosette, endoplasmic reticulum, Golgi apparatus, and cell-cell junctions.

12. The process as claimed in claim 2, wherein the isotope labeling is performed by the SILAC method.

13. The process as claimed in claim 3, wherein the isotope labeling is performed by the SILAC method.

14. The process as claimed in claim 4, wherein the isotope labeling is performed by the SILAC method.

15. The process as claimed in claim 5, wherein the isotope labeling is performed by the SILAC method.

16. The process as claimed in claim 6, wherein the isotope labeling is performed by the SILAC method.

17. The process as claimed in claim 7, wherein the isotope labeling is performed by the SILAC method.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0084] FIG. 1 represents the main steps of an embodiment of the process of the invention.

[0085] FIG. 2 represents (A-C) a fluorescence micrograph of NIH-3T3-Src cells labeled with DAPI and phalloidin in order to reveal, respectively, nuclear DNA and filamentous actin (the invadosome rosettes are indicated by arrows). (D-E). These micrographs show the same cell type stably expressing the peptide Lifeact-RFP; the microdissected elements are outlined by dotted lines. (F) This diagram shows a rosette outlined manually by the experimenter and cut out by a first laser. (G-H) This diagram represents the process of recovering the microdissected elements by a second laser which catapults the cut-out rosette to the collector cap. (I) This micrograph shows the fluorescence of the microdissected elements in the collector cap; the associated diagram represents this same collector cap with the microdissected and recovered elements.

[0086] FIG. 3 represents (A) the procedure for automation of the step of laser microdissection, (B) a representative fluorescence micrograph showing the rosettes which were selected by the automation process and which were cut out by laser microdissection.

[0087] FIG. 4 represents (A) an analysis by mass spectrometry carried out on a range of amounts of proteins extracted from total NIH-3T3-Src cells labeled according to the SILAC method with a heavy isotope (.sup.C13R and .sup.C13K). For each amount of proteins injected there is a corresponding sum of the intensities of all the labeled peptides detected by mass spectrometry. The sum of the intensities of all the labeled peptides resulting from 40 000 rosettes cut out by laser microdissection was compared to this range, to deduce therefrom an amount of 72 ng of proteins extracted (table, cf. line indicated with an arrow, and corresponding graphical representation below indicated with an arrow). (B) The comparison of the relative intensities of the peptides identified in the sample of rosettes and the total lyzate sample (100 ng) after standardization over the total sum of intensities detected by MS made it possible to confirm enrichment of the identified proteins in the rosettes (Rosettes/Total ratio of 2, grayed-out area indicated with an arrow).

[0088] FIG. 5 represents a confocal micrograph of NIH-3T3-src cells constitutively expressing the Lifeact peptide and making it possible to visualize the filamentous actin (A); these cells were transfected with an expression plasmid encoding the fusion protein HA-eEF1A1, the use of an anti-HA antibody making it possible to visualize the localization of this protein (B). The fusion of the labeling of (A) and (B) is represented in (C). The elements within boxes are represented in close-up under each image, respectively.

[0089] FIG. 6 represents a confocal micrograph of NIH-3T3-src cells constitutively expressing the Lifeact peptide and making it possible to visualize the filamentous actin (A); these cells were transfected with an expression plasmid encoding the fusion protein HA-Eif2A, the use of an anti-HA antibody making it possible to visualize the localization of this protein (B). The fusion of the labeling of (A) and (B) is represented in (C). The elements within boxes are represented in close-up under each image, respectively.

EXAMPLES OR EMBODIMENTS

Example 1: Proteomic Analysis of Invadosome Rosettes

[0090] While the following protocol can be adapted to other subcellular compartments, the subcellular compartment associated with the capacity of cells to degrade elements of the extracellular matrix was chosen as the model for the present study, namely the invadosome rosette. This type of structure is formed in cells constitutively expressing the active form of the oncogene c-Src, and contributes to the capacity for cellular invasion. It is crucial to know the exact and detailed protein composition of these structures, in order to demonstrate therapeutic targets to attempt to inhibit the invasion of tumor cells. Filamentous actin is the structural and predominant component of these rosettes, and consequently the element which was chosen to pinpoint these rosettes at the time of the laser microdissection.

[0091] The experiments were carried out on a cell line generated from NIH-3T3 fibroblast cells which constitutively express the oncogene c-Src (NIH3T3-src) and which form invadosome rosettes [7]. Moreover, these cells constitutively express the yeast peptide Lifeact [8] coupled to a fluorophore, namely mCherry (FIG. 2D). This peptide has the capacity to bind specifically to actin but only in the filamentous form thereof.

[0092] Analysis by mass spectrometry of a very small amount of material pushes spectrometers to the limits of their sensitivity. In this context, a majority of the proteins identified actually result from contaminations originating from handling and ambient air. In order to discriminate between the proteins of the microdissected rosettes and the external contaminating elements (ambient air, solutions, etc.), metabolic isotopic labeling of the proteins of interest (C.sup.13Arginine (Arg.sup.6) and C.sup.13Lysine (Lys.sup.6)) was first performed according to the SILAC method (Stable Isotopic Labeling by Amino acids in Cell culture). This method consists in using a culture medium devoid of arginine and lysine, in which arginine and lysine labeled with carbon C.sup.13, and unlabeled proline to prevent metabolization of the labeled arginine to labeled proline, are added, and in incorporating this labeling for at least 6 cycles of cell doubling.

[0093] The cells expressing the peptide Lifeact coupled to the fluorophore mCherry (FIG. 2D) and labeled with the lysine and arginine isotopes were cultured on a silicone membrane ring (which is covered with gelatin in order to promote adhesion of the cells) placed in a lumox dish 50 (Zeiss).

[0094] After adhesion and rinsing with PBS, the cells were fixed with a crosslinking agent, paraformaldehyde (PFA) at 4% in a solution of PBS for 20 minutes. After two rinsing operations, the cells were kept in a solution of PBS at 4 C.

[0095] These cells were then labeled with DAPI which is a fluorescent DNA intercalator which makes it possible to visualize the nuclei.

[0096] The cells were then placed in a laser microdissector fitted with a dry 63 objective (Zeiss), and the cells are kept in a thin film of PBS.

[0097] The rosettes to be cut out were outlined by ways of a stylus on a graphics tablet (dotted circles) before microdissection (FIG. 2E). For the laser dissection, a Zeiss microscope (PALM MicroBeam) was used.

[0098] The rosettes were then collected in the cap of a support (collector tube) made of silicone (FIG. 2H).

[0099] The support was then washed with a 50 mM Tris-HCl solution, pH 6.8, 7.5% SDS, 20% glycerol, 5% beta-mercaptoethanol, 0.1% bromophenol blue for 2 hours at 95 C., which made it possible to extract the proteins and reverse the fixation thereof.

[0100] This extract was then loaded into a well of 10% SDS-PAGE gel and placed under a voltage of 100 volts until the bromophenol blue migrated to the limit between the stacking gel and the separating gel, using a molecular weight marker as a visual control in another well.

[0101] A square of gel was then cut out between the upper limit of the well and the migration front was treated for reduction/alkylation of the proteins then proteolysis by trypsin and extraction of the peptides resulting from this digestion.

[0102] The peptides were then analyzed by LC-MS/MS with a C18 chromatography gradient for 2 hours and analysis on a mass spectrometer of Q-Exactive (Thermo) type.

[0103] The databases were consulted with two different algorithms (Mascot and Sequest) using the Proteome Discoverer software, including the C.sup.13Arg and C.sup.13Lys labeling as variable modifications. Only the peptides with high scores, labeled and/or identified with one and/or the other of the two algorithms, were retained.

[0104] Table 1 represents the summary of the number of proteins identified during the different experiments. Increasing the number of pieces microdissected firstly made it possible to increase the number of proteins identified, to reach 101 proteins identified after cutting out 10 000 rosettes.

TABLE-US-00001 TABLE 1 Percentage of proteins Number of Number of strictly identical pieces proteins relative to the Experiment microdissected identified preceding experiment 1 350 9 2 3000 55 44% 3 10 000 101 60% + 20% (proteins very close) = 80%

[0105] It is also observed that, by increasing the amount of material, more peptides are identified corresponding to the proteins identified during the preceding experiment performed with less material. Thus, between experiment 2 and experiment 3, the identification of 60% of the proteins is confirmed and a further 20% of very similar proteins are identified (isoforms, different subunits of the same protein), listed in the following table 2. These elements demonstrate the robustness and reproducibility of this technique.

TABLE-US-00002 TABLE 2 Proteins from the 1st experiment found again in experiments 2 and 3 Vimentin OS = Mus musculus GN = Vim Actin, cytoplasmic 1 (Fragment) OS = Mus musculus GN = Actb Histone H4 OS = Mus musculus GN = Hist1h4a Tubulin alpha-1C chain OS = Mus musculus GN = Tuba1c Common proteins between experiments 2 and 3 Peroxiredoxin-1 (Fragment) OS = Mus musculus GN = Prdx1 40S ribosomal protein S3 OS = Mus musculus GN = Rps3 ATP synthase subunit alpha OS = Mus musculus GN = Atp5a1 Histone H3 (Fragment) OS = Mus musculus GN = H3f3a Protein Ahnak OS = Mus musculus GN = Ahnak Histone H2A OS = Mus musculus GN = Hist1h2al Heterogeneous nuclear ribonucleoprotein H OS = Mus musculus GN = Hnrnph1 Fructose-bisphosphate aldolase A OS = Mus musculus GN = Aldoa Tubulin alpha-1B chain OS = Mus musculus GN = Tuba1b Elongation factor 1-alpha 1 OS = Mus musculus GN = Eef1a1 Histone H2B type 1-F/J/L OS = Mus musculus GN = Hist1h2bf Heat shock protein HSP 90-beta OS = Mus musculus GN = Hsp90ab1 40S ribosomal protein SA OS = Mus musculus GN = Rpsa Glyceraldehyde-3-phosphate dehydrogenase OS = Mus musculus GN = Gapdh Vimentin OS = Mus musculus GN = Vim Stress-70 protein, mitochondrial OS = Mus musculus GN = Hspa9 Isoform C of Prelamin-A/C OS = Mus musculus GN = Lmna Heterogeneous nuclear ribonucleoprotein A1 OS = Mus musculus GN = Hnrnpa1 Pyruvate kinase PKM OS = Mus musculus GN = Pkm ATP synthase subunit beta, mitochondrial OS = Mus musculus GN = Atp5b Elongation factor 2 OS = Mus musculus GN = Eef2 Poly(rC)-binding protein 1 OS = Mus musculus GN = Pcbp1 Actin, cytoplasmic 1 OS = Mus musculus GN = Actb Histone H4 OS = Mus musculus GN = Hist1h4a Isoform 2 of 60 kDa heat shock protein, mitochondrial OS = Mus musculus GN = Hspd1 Tubulin alpha-1A chain OS = Mus musculus GN = Tuba1a Heat shock cognate 71 kDa protein OS = Mus musculus GN = Hspa8 Polyubiquitin-B (Fragment) OS = Mus musculus GN = Ubb Histone H2B type 2-E OS = Mus musculus GN = Hist2h2be Cytoskeleton-associated protein 4 OS = Mus musculus GN = Ckap4 Myosin-9 OS = Mus musculus GN = Myh9 Nucleophosmin OS = Mus musculus GN = Npm1 Phosphoglycerate kinase OS = Mus musculus GN = Pgk1 Proteins from experiment 2 which strongly resemble the proteins detected during experiment 3 60S ribosomal protein L31 OS = Mus musculus GN = Rpl31 Elongation factor 1-delta (Fragment) OS = Mus musculus GN = Eef1d T-complex protein 1 subunit gamma OS = Mus musculus GN = Cct3 Heterogeneous nuclear ribonucleoprotein U, isoform CRA_b OS = Mus musculus GN = Gm28062 Annexin A2 OS = Mus musculus GN = Anxa2 60S ribosomal protein L13 OS = Mus musculus GN = Rpl13 Alpha-actinin-4 OS = Mus musculus GN = Actn4 40S ribosomal protein S8 OS = Mus musculus GN = Rps8 Probable ATP-dependent RNA helicase DDX5 OS = Mus musculus GN = Ddx5 Elongation factor 1-gamma OS = Mus musculus GN = Eef1g 60S acidic ribosomal protein P0 (Fragment)OS = Mus musculus GN = Rplp0

Example 2: Validation of the Enrichment

[0106] The experiment aimed to demonstrate that the process of the invention (isotopic labeling+targeted laser microdissection+mass spectrometry) makes it possible to enrich the sample with the proteins specifically expressed by the subcellular compartment of interest (i.e. the rosettes).

[0107] A range of amounts of proteins from a total cell lyzate was used as a point of comparison, to reach the same amount as that collected with the 40 000 rosettes, and be able to serve as reference (72 ng).

[0108] The results are presented in FIG. 4.

Example 3: Validation by Labeling of the Presence of the Proteins Identified (e.g. Elongation Factor 1-1)

[0109] The NIH3T3-src cells were seeded onto a glass slide then transfected with the HA-eEF1A1 plasmid (provided by Dr. IRWIN MS, University of Toronto, Ontario, Canada) using a transfection agent, lipofectamine 2000 (thermo-Fisher), according to the protocol described by the manufacturer. The cells were then rinsed after 6 hours of incubation, and left to rest for 48 h. The cells were then fixed by the addition of a 4% paraformaldehyde-PBS solution for 10 minutes.

[0110] The immunofluorescence protocol was then carried out; the cells were permeabilized by a Triton solution. Finally, the cells were incubated with the primary antibody anti-HA (3F10, Roche) in a primary antibody-PBS-BSA solution, then after washing operations with a solution containing a secondary antibody coupled to a fluorophore before observation with a confocal microscope (Leica SP5).

[0111] The results are presented in FIG. 5.

Example 4: Validation by Labeling of the Presence of the Proteins Identified (e.g. HA-Eif2A)

[0112] The NIH3T3-src cells were seeded onto a glass slide then transfected with the Flag-EEF2 plasmid (provided by Dr. Huang Y S, Academia Sinica, Taipei, Taiwan) using a transfection agent, lipofectamine 2000 (thermo-Fisher), according to the protocol described by the manufacturer. The cells were then rinsed after 6 hours of incubation, and left to rest for 48 h. The cells were then fixed by the addition of a 4% paraformaldehyde-PBS solution for 10 minutes.

[0113] The immunofluorescence protocol was then carried out; the cells were permeabilized by a Triton solution. Finally, the cells were labeled with an anti-flag antibody (Sigma F1804) in an anti-flag antibody-PBS-BSA solution, then after washing operations with a solution containing a secondary antibody coupled to a fluorophore before observation with a confocal microscope (Leica SP5).

[0114] The results are presented in FIG. 6.

[0115] These experiments, described in examples 3 and 4, made it possible to demonstrate the presence of these proteins, eEF1A1 and HA-Eif2A, in the rosettes, and thereby to validate the procedure for identifying new markers in the rosettes.

[0116] Similarly, experiments were carried out which validate the presence of the proteins EEF2, EIF3H, eif4E and Reptin in the rosettes.

REFERENCE LIST

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