Quantitative Proteomic Analysis of Protein Complexes
2007; Elsevier BV; Volume: 7; Issue: 2 Linguagem: Inglês
10.1074/mcp.m700282-mcp200
ISSN1535-9484
AutoresDelphine Pflieger, Martin A. Jünger, Markus Müller, Oliver Rinner, Hookeun Lee, Peter Gehrig, Matthias Gstaiger, Ruedi Aebersold,
Tópico(s)Microbial Natural Products and Biosynthesis
ResumoProtein complexes have largely been studied by immunoaffinity purification and (mass spectrometric) analysis. Although this approach has been widely and successfully used it is limited because it has difficulties reliably discriminating true from false protein complex components, identifying post-translational modifications, and detecting quantitative changes in complex composition or state of modification of complex components. We have developed a protocol that enables us to determine, in a single LC-MALDI-TOF/TOF analysis, the true protein constituents of a complex, to detect changes in the complex composition, and to localize phosphorylation sites and estimate their respective stoichiometry. The method is based on the combination of fourplex iTRAQ (isobaric tags for relative and absolute quantification) isobaric labeling and protein phosphatase treatment of substrates. It was evaluated on model peptides and proteins and on the complex Ccl1-Kin28-Tfb3 isolated by tandem affinity purification from yeast cells. The two known phosphosites in Kin28 and Tfb3 could be reproducibly shown to be fully modified. The protocol was then applied to the analysis of samples immunopurified from Drosophila melanogaster cells expressing an epitope-tagged form of the insulin receptor substrate homologue Chico. These experiments allowed us to identify 14-3-3ε, 14-3-3ζ, and the insulin receptor as specific Chico interactors. In a further experiment, we compared the immunopurified materials obtained from tagged Chico-expressing cells that were either treated with insulin or left unstimulated. This analysis showed that hormone stimulation increases the association of 14-3-3 proteins with Chico and modulates several phosphorylation sites of the bait, some of which are located within predicted recognition motives of 14-3-3 proteins. Protein complexes have largely been studied by immunoaffinity purification and (mass spectrometric) analysis. Although this approach has been widely and successfully used it is limited because it has difficulties reliably discriminating true from false protein complex components, identifying post-translational modifications, and detecting quantitative changes in complex composition or state of modification of complex components. We have developed a protocol that enables us to determine, in a single LC-MALDI-TOF/TOF analysis, the true protein constituents of a complex, to detect changes in the complex composition, and to localize phosphorylation sites and estimate their respective stoichiometry. The method is based on the combination of fourplex iTRAQ (isobaric tags for relative and absolute quantification) isobaric labeling and protein phosphatase treatment of substrates. It was evaluated on model peptides and proteins and on the complex Ccl1-Kin28-Tfb3 isolated by tandem affinity purification from yeast cells. The two known phosphosites in Kin28 and Tfb3 could be reproducibly shown to be fully modified. The protocol was then applied to the analysis of samples immunopurified from Drosophila melanogaster cells expressing an epitope-tagged form of the insulin receptor substrate homologue Chico. These experiments allowed us to identify 14-3-3ε, 14-3-3ζ, and the insulin receptor as specific Chico interactors. In a further experiment, we compared the immunopurified materials obtained from tagged Chico-expressing cells that were either treated with insulin or left unstimulated. This analysis showed that hormone stimulation increases the association of 14-3-3 proteins with Chico and modulates several phosphorylation sites of the bait, some of which are located within predicted recognition motives of 14-3-3 proteins. The past few years have seen a growing interest in the characterization of protein complexes, the functional modules that catalyze many cellular processes. The formation of protein complexes and/or the modulation of their function(s) often appear to be correlated with the addition or removal of covalent modifications, among which phosphorylation is particularly important. This chemical group can indeed modulate the catalytic activity, subcellular localization, and interactions of proteins and protein complexes and is thus a key molecular mechanism controlling signaling pathways and other cellular functions. Methods allowing the detailed study of protein complexes by concurrently identifying genuine protein partners, detecting the variable composition of complexes upon cell stimulation, localizing phosphorylation sites, and determining their stoichiometry would therefore likely contribute to understanding the molecular basis of numerous cellular processes. Typically protein complexes are analyzed by mass spectrometry after affinity purification (1Gingras A.C. Aebersold R. Raught B. Advances in protein complex analysis using mass spectrometry.J. Physiol. 2005; 563: 11-21Crossref PubMed Scopus (152) Google Scholar). Although this approach has been successfully and widely used, it suffers from a number of limitations, some of which are addressed by specific methods. To distinguish true complex components from co-purified, nonspecific interactors the composition of an immunopurified sample of interest has been compared with a mock-purified sample either using differential isotope labeling followed by quantitative MS (2Ranish J.A. Yi E.C. Leslie D.M. Purvine S.O. Goodlett D.R. Eng J. Aebersold R. The study of macromolecular complexes by quantitative proteomics.Nat. Genet. 2003; 33: 349-355Crossref PubMed Scopus (311) Google Scholar, 3Wang T. Gu S. Ronni T. Du Y.C. Chen X. 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Gerrits B. Panse C. Schlapbach R. Mansuy I.M. Qualitative and quantitative analyses of protein phosphorylation in naive and stimulated mouse synaptosomal preparations.Mol. Cell. Proteomics. 2007; 6: 283-293Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). However, in this targeted approach only anticipated phosphorylation sites are quantified, and the stoichiometry can only be determined if the absolute protein abundance is also obtained in the same experiment. To estimate the level of phosphorylation on a more global scale, several studies compared the MS signals of phosphopeptides and their non-modified counterparts with (18Steen H. Jebanathirajah J.A. Springer M. Kirschner M.W. Stable isotope-free relative and absolute quantitation of protein phosphorylation stoichiometry by MS.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 3948-3953Crossref PubMed Scopus (182) Google Scholar) or without (19Carr S.A. Huddleston M.J. Annan R.S. 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Proteomics. 2006; 5: 1610-1627Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) compensation for differences in ionization and detection efficiencies. These methods, although individually quite successful, have failed so far to achieve the comprehensive analysis of protein complexes in a single analysis. Here we present a method to characterize protein complexes that combines the capabilities of some of the above methods in a single analysis. It consists of the following steps. First an affinity-purified sample of interest and a mock-purified control sample are isolated and digested, and the two samples are split into two halves and subjected to four-channel iTRAQ 1The abbreviations used are: iTRAQ, isobaric tags for relative and absolute quantification; A(114), area of the iTRAQ reporter group 114; A, angiotensin; pA, phosphorylated angiotensin; CIP, calf intestinal phosphatase; IP, immunoprecipitation; IR, insulin receptor; IRS (Chico), insulin receptor substrate; SAP, shrimp alkaline phosphatase; TAP, tandem affinity purification; DSP, dithiobis(succinimidyl propionate); TEV, tobacco etch virus; HA, hemagglutinin; S/N, signal to noise ratio; ACTH, adrenocorticotropic hormone; INS, insulin. isotope labeling. Second, two of the four iTRAQ-labeled samples are subjected to dephosphorylation via phosphatase treatment. Third, the samples are recombined and analyzed by liquid chromatography-tandem mass spectrometry using a MALDI-TOF/TOF instrument; and fourth, the data are analyzed to distinguish true complex components from nonspecific interactors and to determine the site(s) and stoichiometry of phosphorylation. Therefore, the method provides the potential to subject immunopurified protein complexes to comprehensive analysis in a single experiment. The method was tested on model samples, angiotensin II and its phosphorylated counterpart, as well as caseins. It was also applied to the study of a complex purified from yeast cells by tandem affinity purification (TAP) (23Puig O. Caspary F. Rigaut G. Rutz B. Bouveret E. Bragado-Nilsson E. Wilm M. Seraphin B. The tandem affinity purification (TAP) method: a general procedure of protein complex purification.Methods. 2001; 24: 218-229Crossref PubMed Scopus (1428) Google Scholar) and to single step-purified complexes built around the insulin receptor substrate, Chico, in Drosophila melanogaster cells (24Bohni R. Riesgo-Escovar J. Oldham S. Brogiolo W. Stocker H. Andruss B.F. Beckingham K. Hafen E. Autonomous control of cell and organ size by CHICO, a Drosophila homolog of vertebrate IRS1–4.Cell. 1999; 97: 865-875Abstract Full Text Full Text PDF PubMed Scopus (688) Google Scholar). The procedure proved efficient in identifying bona fide interactors of Chico and variable phosphorylation sites regulated by insulin. The iTRAQ reagent multiplex kit (reference 4352135, Applied Biosystems) contained the necessary reagents for resuspension (Dissolution Buffer: 500 mm triethylammonium bicarbonate, pH 8.5), reduction (Reducing Reagent: 50 mm tris-(2-carboxyethyl)phosphine), alkylation (Cysteine-Blocking Reagent: 200 mm methyl methanethiosulfonate), and differential labeling of protein samples. For all described samples, the protocol from Applied Biosystems was followed except for adjustments of the added volumes of reducing and alkylating reagents that were applied depending on the initial sample volume. Modified porcine trypsin was purchased from Promega (reference V5113), Rapigest detergent was from Waters, calf intestinal phosphatase (CIP) was bought from Fermentas (reference EF0341; 1 unit/μl), and shrimp alkaline phosphatase (SAP) was from Promega (reference 9PIM820; 1 unit/μl). The amino-reactive bifunctional cross-linker dithiobis(succinimidyl propionate) (DSP) was purchased from Pierce (reference 22585). Six samples consisting of human angiotensin II (designated “A”) and Tyr-phosphorylated angiotensin II (designated “pA”) (references 05-23-0101 and 05-23-0111, Merck/Calbiochem) were prepared in proportions (A/pA) 0:100, 20:80, 40:60, 60:40, 80:20, and 100:0, respectively. Each sample, containing a total of 600 pmol of peptide in water, was split in two halves, which were completely dried by SpeedVac and then resuspended in 3 μl of Dissolution Buffer. Two iTRAQ reagent tubes (116 and 117) were dissolved in 70 μl of ethanol, and 11 μl of these reagent solutions were used to perform a differential labeling reaction of the two halves of each mixture (A/pA). After 1-h incubation on the bench, samples were dried by SpeedVac and then resuspended in 20 μl of CIP buffer (Fermentas; provided with the enzyme: 10 mm Tris-HCl (pH 7.5 at 37 °C), 10 mm MgCl2). After 30 min allowing complete hydrolysis of the excess iTRAQ reagents, 1 unit of CIP was added to every 117-labeled sample. All samples (116- and 117-labeled) were incubated at 37 °C for 2.5 h. After heating at 85 °C for 15 min to inactivate the phosphatase, samples were finally pooled by 116-117 pairs to reconstitute the whole (A/pA) samples. Fifteen picomoles of each treated peptide pair A/pA were cleaned on a ZipTipC18 (Millipore), following the manufacturer's instructions to remove buffers and excess iTRAQ reagents. The eluates were dried by SpeedVac, and peptides were resuspended in 50 μl of 0.1% (v/v) TFA in water. From each sample A/pA, 0.5 μl was spotted on a MALDI plate and mixed with 0.5 μl of matrix solution containing 4 mg/ml of α-cyano-4-hydroxycinnamic acid (Fluka, reference 28480; >99% pure) in (ACN/water/TFA, 70:30:0.1, v/v/v). Samples were analyzed on a MALDI-TOF/TOF 4800 instrument (Applied Biosystems) by accumulating 1500 laser shots in MS analysis mode. In MS/MS analysis, 1500 shots were accumulated for fragmentation of angiotensin, and 4500 shots were summed for its phosphorylated counterpart. To test the relevance of applying a correction factor to measured iTRAQ ratios A(117)/A(116) and A(115)/A(114), a sample consisting of pure angiotensin (600 pmol) and 4 μg of β-lactoglobulin A (Sigma, reference L7880) was submitted to the previous protocol described above for A/pA mixtures but using iTRAQ labels 114 and 115. A yeast strain expressing TAP-tagged Kin28 was obtained from Open Biosystems. Cells were grown at 30 °C until the culture reached an OD of 1.15. Cells were harvested and pelleted at 8000 × g for 10 min. Pellets were washed (20% glycerol, 1 mm EDTA, 50 mm Tris-HCl, pH 7.8, 150 mm KCl) and distributed into 50-ml tubes. Cells were centrifuged again at 4000 rpm in a Microfuge T, and the pellets (∼4–5-ml pellet were obtained from 3 liters of culture) were quick frozen in liquid nitrogen to be stored at −80 °C until use. Two such pellets were used for tandem affinity purification. The procedure followed was close to that described by Rigaut et al. (25Rigaut G. Shevchenko A. Rutz B. Wilm M. Mann M. Seraphin B. A generic protein purification method for protein complex characterization and proteome exploration.Nat. Biotechnol. 1999; 17: 1030-1032Crossref PubMed Scopus (2287) Google Scholar) and is detailed in Gingras et al. (26Gingras A.C. Caballero M. Zarske M. Sanchez A. Hazbun T.R. Fields S. Sonenberg N. Hafen E. Raught B. Aebersold R. A novel, evolutionarily conserved protein phosphatase complex involved in cisplatin sensitivity.Mol. Cell. Proteomics. 2005; 4: 1725-1740Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Specifically the following modifications were implemented to accommodate the higher cell amount used. Five milliliters of ice-cold lysis buffer were added to each cell pellet, and cells were resuspended and distributed as 500-μl aliquots into 2-ml tubes. After adding 500 μl of 0.5-mm glass beads, cells were lysed by repeating the following cycle three times: 10-min agitation of the tubes on a vortexer (Vortex-Genie 2, Scientific Industries, Inc., reference SI-0276; equipped with a 1.5-ml Snap-top Microtube Holder, reference 0A-0563-010) at maximum speed and 10-min cooling on ice. After discarding the cell debris by centrifugation at 13,000 rpm (Centrifuge 5415 R, Eppendorf), approximately 10 ml of protein extract (350 mg of protein, determined by protein assay (Bio-Rad)) were obtained. This lysate was precleared on 400 μl of packed glutathione 4B beads (Amersham Biosciences, reference 17-0756-01) for 2 h. The extract was then incubated on 300 μl of packed IgG beads (Amersham Biosciences, reference 17-0969-01) for 4.5 h. IgG beads were rinsed with 3 ml of lysis buffer and then three times with 3 ml of TEV buffer containing PMSF. The tagged protein was released by adding 600 μl of TEV buffer containing 30 μl of TEV protease (Invitrogen, reference 10127017). After overnight incubation on a rotating wheel at 4 °C, eluted proteins were collected by pooling the supernatant and three washes of the IgG beads with 300 μl of calmodulin binding buffer (10-min incubation of the IgG beads in that buffer each time). The resulting 1.5 ml of protein sample were incubated with 100 μl of packed calmodulin beads (50 μl from each manufacturer: Amersham Biosciences reference 17-0529-01 and Stratagene reference 214303) for 3.75 h. After rinsing the beads, the retained proteins were eluted with 3 × 100 μl of elution buffer supplemented with 0.2 mg/ml Rapigest. Each time, the 100 μl of eluting buffer were pipetted up and down for 1 min to thoroughly resuspend the beads and improve protein elution. This purified sample was heated at 95 °C to denature proteins and then split in two halves to mimic two independent protein samples. The resulting samples were dried by SpeedVac to eliminate the ammonium bicarbonate present in the final eluting buffer. Samples were sonicated for 10 min in 40 μl of Dissolution Buffer to help resolubilize proteins. Both samples were reduced, alkylated, and digested by addition of 0.3 μg of trypsin and overnight incubation at 37 °C. Each digest was split in two halves (around 20 μl each), and the four resulting tubes were labeled with the four different iTRAQ reagents (114 and 115 for one digest and 116 and 117 for the other) resuspended in 70 μl of ethanol. After 1-h reaction at ambient temperature, samples were concentrated to dryness and resuspended in 40 μl of CIP buffer. Tubes were further left on the bench for 60 min to allow hydrolysis of excess iTRAQ reagents. After addition of 1 m MgCl2 to reach a final concentration of 100 mm and fully react with EGTA (used in the elution from calmodulin beads and now at around 95 mm in the four tubes), 2 μl of CIP were added to the 115- and 117-labeled samples. The four tubes were placed at 37 °C for 3 h, and then CIP was inactivated by heating at 85 °C for 15 min. The four samples were pooled and redigested by addition of 1 μg of trypsin and incubation at 37 °C for 1.5 h. The detergent Rapigest was cleaved by adding a solution of 1 m HCl until reaching a pH of approximately 2. After incubation at 37 °C for 45 min, the tube was centrifuged at 13,000 rpm for 10 min (Eppendorf Centrifuge 5415 R) to discard the bottom 10 μl containing the insoluble part from Rapigest. The sample was finally cleaned by using consecutively three ZipTipC18 pipette tips to enhance peptide recovery. A sample consisting of 100 μg of β-casein (reference C6905 from Sigma; >90% pure) in 50 μl of Dissolution Buffer was treated following the protocol in Fig. 1 using the combination of CIP and SAP. The sample was supplemented with Rapigest at a final concentration of 0.3% (w/v) and then heated at 95 °C for 5 min. After reduction and alkylation of the sample, 50 μl of Dissolution Buffer and 2 μg of trypsin were added to the sample before overnight incubation at 37 °C. The obtained peptide mixture was split in two halves, which were labeled with iTRAQ reagents 114 and 115. The resulting samples were dried by SpeedVac and resuspended in 100 μl of CIP buffer. Sample tubes were left on the bench for 30 min to allow complete hydrolysis of residual iTRAQ reagents. Then 2 units of CIP and 2 units of SAP were added to the 115-labeled sample. Both tubes were incubated at 37 °C for 2 h before being heated at 85 °C for 15 min to inactivate the phosphatases. The pooled sample was finally redigested by addition of 0.8 μg of trypsin and incubation at 37 °C for 2 h. After cleavage of Rapigest performed as for the TAP sample, the sample was cleaned on a ZipTipC18 and analyzed by MALDI-TOF/TOF MS (deposition of 350 fmol/spot) in the same conditions as the angiotensin samples. In an alternative experiment, casein peptides were treated with 2 units of λ-phosphatase (Sigma-Aldrich reference P9614) using the buffer delivered with the enzyme. To finally inactivate phosphatase, the tubes were heated at 85 °C for 30 min and supplemented with 6 mm orthovanadate. After pooling the two tube contents, the sample was redigested by addition of 0.8 μg of trypsin and incubation at 37 °C for 2 h. Kc167 cells were transfected with either empty pMHW-Blast (hemagglutinin (HA) control) or pMHW-chico-Blast, which allowed CuSO4-inducible expression of HA-tagged Chico under control of the metallothionein promoter. Stably transfected cell pools were obtained by selection on 25 μg/ml blasticidin for 2 weeks. After selection cells were grown in six 175-cm2 flasks containing 32 ml of full medium (Schneider's Drosophila medium (Invitrogen)) supplemented with 10% (v/v) fetal calf serum, 100 units of penicillin (Invitrogen), 100 μg/ml streptomycin (Invitrogen), and 10 μg/ml blasticidin (Invitrogen) per flask. When a concentration around 5–6·106 cells/ml was reached, cells were supplemented with 7 ml of fresh full medium per flask and with 600 μm CuSO4 for overnight induction of HA-Chico or HA-control expression (induction lasted typically 14 h). Cell suspensions were then collected in four 500-ml beakers, to obtain two comparable pairs of Chico/control samples, and harvested by centrifugation at 1500 rpm for 5 min (using a Sorvall GS3 rotor precooled at 4 °C). The resulting pellets were washed with 40 ml of ice-cold PBS, transferred into four 50-ml tubes, and centrifuged again at 400 × g for 5 min. One Chico/control pair of cell pellets was quickly frozen in liquid nitrogen to be stored at −80 °C until use. The pair of Chico/control immunoprecipitations (IPs) obtained from these pellets is later called sample “Chico2.” The other pair of cell pellets was immediately processed for immunopurification. The resulting pair of IPs is later called sample “Chico1.” Cell pellets in each tube were lysed in 5 ml of lysis buffer (40 mm HEPES-KOH, pH 7.5, 120 mm NaCl, 1 mm EDTA, and 1% (v/v) Triton X-100 freshly supplemented with 1 mm PMSF, 50 mm NaF, 10 mm pyrophosphate, 10 mm glycerophosphate, 2 mm orthovanadate, EDTA-free protease inhibitor mixture (one “Complete” tablet (Roche Applied Science)/20 ml of buffer), and 2.4 mg/ml DSP). After homogenizing with 10 strokes using a tight fitting pistil of a Dounce homogenizer, the cell lysates were left on ice for 40 min. DSP was used to try to increase recovery of Chico interactors by forming covalent intermolecular linkages. The cross-linking reaction was quenched by addition of 1.25 ml of 1 m Tris-HCl, pH 7.4, and incubation on ice for 30 min. Cell debris were centrifuged at 13,000 rpm for 20 min using a Sorvall SS-34 rotor equilibrated at 4 °C. Cell extracts were then precleared by incubation with 100 μl of packed Protein A-Sepharose (Amersham Biosciences) beads for 1 h on a rocker at 4 °C. Beads were removed by centrifugation at 800 rpm (130 × g) at 4 °C (Eppendorf 5810 R). Protein concentration was estimated to be at 6 μg/μl in the HA-Chico lysate and 5.2 μg/μl for the control lysate (6 and 5 μg/μl, respectively, in the HA-Chico and control lysates providing sample Chico2). Lysates were then incubated with rotation at 4 °C for 3.75 h in a 15-ml tube with 75 μl of packed Protein A-Sepharose beads packed with supernatant of the hybridoma cell line 12CA5. After centrifuging the beads at 800 rpm for 2 min (130 × g), beads were washed three times with lysis buffer (not containing DSP) and then three times with lysis buffer containing neither detergent nor inhibitors (40 mm Hepes-KOH, pH 7.5, 120 mm NaCl, 1 mm EDTA). Proteins interacting with the beads were eluted with 3 × 200 μl of 2% (v/v) acetic acid in water. The eluates were concentrated by SpeedVac down to 50 μl, rediluted with 150 μl of iTRAQ Dissolution Buffer to reach a close to neutral pH, and supplemented with 0.1% (w/v) Rapigest. Samples were again concentrated by SpeedVac down to a volume below 40 μl. Both protein samples were reduced, alkylated, and received additional Dissolution Buffer to reach 150 μl. Protein digestion was performed by addition of 1.2 μg of trypsin to both samples and overnight incubation at 37 °C. Samples were then split in two halves and concentrated by SpeedVac down to 30 μl. Four vials of iTRAQ reagents, 114 to 117, were resuspended in 70 μl of ethanol and added to the samples as follows: reagents 114 and 115 were used for the digested HA-Chico IP, and reagents 116 and 117 were used for the control IP. After 1-h incubation on the bench, samples were dried by SpeedVac and resuspended in 100 μl of CIP buffer. They were further left for at least 30 min on the bench to allow complete hydrolysis of the excess iTRAQ reagents. Samples labeled with iTRAQ reagents 115 and 117 were then treated by addition of 2 units of CIP and 2 units of SAP and incubation at 37 °C for 2 h. The four samples were finally heated at 85 °C for 15 min to inactivate phosphatases and pooled to be redigested by 1 μg of trypsin (1.5–2-h incubation at 37 °C). After cl
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