Mitochondrial Phosphoproteome Revealed by an Improved IMAC Method and MS/MS/MS

Abstract

IMAC in combination with mass spectrometry is a promising approach for global analysis of protein phosphorylation. Nevertheless this approach suffers from two shortcomings: inadequate efficiency of IMAC and poor fragmentation of phosphopeptides in the mass spectrometer. Here we report optimization of the IMAC procedure using 32P-labeled tryptic peptides and development of MS/MS/MS (MS3) for identifying phosphopeptide sequences and phosphorylation sites. The improved IMAC method allowed recovery of phosphorylated tryptic peptides up to ∼77% with only minor retention of unphosphorylated peptides. MS3 led to efficient fragmentation of the peptide backbone in phosphopeptides for sequence assignment. Proteomics of mitochondrial phosphoproteins using the resulting IMAC protocol and MS3 revealed 84 phosphorylation sites in 62 proteins, most of which have not been reported before. These results revealed diverse phosphorylation pathways involved in the regulation of mitochondrial functions. Integration of the optimized batchwise IMAC protocol with MS3 offers a relatively simple and more efficient approach for proteomics of protein phosphorylation. IMAC in combination with mass spectrometry is a promising approach for global analysis of protein phosphorylation. Nevertheless this approach suffers from two shortcomings: inadequate efficiency of IMAC and poor fragmentation of phosphopeptides in the mass spectrometer. Here we report optimization of the IMAC procedure using 32P-labeled tryptic peptides and development of MS/MS/MS (MS3) for identifying phosphopeptide sequences and phosphorylation sites. The improved IMAC method allowed recovery of phosphorylated tryptic peptides up to ∼77% with only minor retention of unphosphorylated peptides. MS3 led to efficient fragmentation of the peptide backbone in phosphopeptides for sequence assignment. Proteomics of mitochondrial phosphoproteins using the resulting IMAC protocol and MS3 revealed 84 phosphorylation sites in 62 proteins, most of which have not been reported before. These results revealed diverse phosphorylation pathways involved in the regulation of mitochondrial functions. Integration of the optimized batchwise IMAC protocol with MS3 offers a relatively simple and more efficient approach for proteomics of protein phosphorylation. Global analysis of protein phosphorylation will provide insight into mechanisms by which this dynamic post-translational modification modulates diverse cellular processes. Mass spectrometry-based phosphoproteomics is a potentially powerful approach for global profiling and quantification of protein phosphorylation. Such studies usually involve selective isolation of phosphorylated peptides and their subsequent fragmentation in a mass spectrometer to assign the sequence and localize phosphorylation sites. Three strategies have been described for enriching phosphopeptides based on antibodies (1Hinsby A.M. Olsen J.V. Mann M. Tyrosine phosphoproteomics of fibroblast growth factor signaling: a role for insulin receptor substrate-4.J. Biol. Chem. 2004; 279: 46438-46447Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 2Rush J. Moritz A. Lee K.A. Guo A. Goss V.L. Spek E.J. Zhang H. Zha X.M. Polakiewicz R.D. Comb M.J. Immunoaffinity profiling of tyrosine phosphorylation in cancer cells.Nat. Biotechnol. 2005; 23: 94-101Crossref PubMed Scopus (964) Google Scholar), chemical derivatization (3McLachlin D.T. Chait B.T. Improved β-elimination-based affinity purification strategy for enrichment of phosphopeptides.Anal. Chem. 2003; 75: 6826-6836Crossref PubMed Scopus (224) Google Scholar, 4Tao W.A. Wollscheid B. O'Brien R. Eng J.K. Li X.J. Bodenmiller B. Watts J.D. Hood L. Aebersold R. Quantitative phosphoproteome analysis using a dendrimer conjugation chemistry and tandem mass spectrometry.Nat. Methods. 2005; 2: 591-598Crossref PubMed Scopus (282) Google Scholar), or ionic interactions (e.g. IMAC and strong ion exchange chromatography) (5Ficarro S.B. McCleland M.L. Stukenberg P.T. Burke D.J. Ross M.M. Shabanowitz J. Hunt D.F. White F.M. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae.Nat. Biotechnol. 2002; 20: 301-305Crossref PubMed Scopus (1499) Google Scholar, 6Gruhler A. Olsen J.V. Mohammed S. Mortensen P. Faergeman N.J. Mann M. Jensen O.N. Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway.Mol. Cell. Proteomics. 2005; 4: 310-327Abstract Full Text Full Text PDF PubMed Scopus (698) Google Scholar, 7Kokubu M. Ishihama Y. Sato T. Nagasu T. Oda Y. Specificity of immobilized metal affinity-based IMAC/C18 tip enrichment of phosphopeptides for protein phosphorylation analysis.Anal. Chem. 2005; 77: 5144-5154Crossref PubMed Scopus (188) Google Scholar, 8Ballif B.A. Villen J. Beausoleil S.A. Schwartz D. Gygi S.P. Phosphoproteomic analysis of the developing mouse brain.Mol. Cell. Proteomics. 2004; 3: 1093-1101Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 9Trinidad J.C. Specht C.G. Thalhammer A. Schoepfer R. Burlingame A.L. Comprehensive identification of phosphorylation sites in postsynaptic density preparations.Mol. Cell. Proteomics. 2006; 5: 914-922Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). These methods have achieved limited success in enriching phosphopeptides for proteomics studies. Among these approaches, IMAC is the most convenient and holds much potential for the efficient isolation of phosphopeptides (10Cao P. Stults J.T. Rapid Commun. Mass Spectrom. 2000; 14: 1600-1606Crossref PubMed Scopus (116) Google Scholar, 11Posewitz M.C. Tempst P. Immobilized gallium(III) affinity chromatography of phosphopeptides.Anal. Chem. 1999; 71: 2883-2892Crossref PubMed Scopus (788) Google Scholar, 12Haydon C.E. Eyers P.A. Aveline-Wolf L.D. Resing K.A. Maller J.L. Ahn N.G. Identification of novel phosphorylation sites on Xenopus laevis Aurora A and analysis of phosphopeptide enrichment by immobilized metal-affinity chromatography.Mol. Cell. Proteomics. 2003; 2: 1055-1067Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 13Liu H. Stupak J. Zheng J. Keller B.O. Brix B.J. Fliegel L. Li L. Open tubular immobilized metal ion affinity chromatography combined with MALDI MS and MS/MS for identification of protein phosphorylation sites.Anal. Chem. 2004; 76: 4223-4232Crossref PubMed Scopus (72) Google Scholar). The method has been used for several global analyses of protein phosphorylation in model organisms and cellular organelles (5Ficarro S.B. McCleland M.L. Stukenberg P.T. Burke D.J. Ross M.M. Shabanowitz J. Hunt D.F. White F.M. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae.Nat. Biotechnol. 2002; 20: 301-305Crossref PubMed Scopus (1499) Google Scholar, 6Gruhler A. Olsen J.V. Mohammed S. Mortensen P. Faergeman N.J. Mann M. Jensen O.N. Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway.Mol. Cell. Proteomics. 2005; 4: 310-327Abstract Full Text Full Text PDF PubMed Scopus (698) Google Scholar, 7Kokubu M. Ishihama Y. Sato T. Nagasu T. Oda Y. Specificity of immobilized metal affinity-based IMAC/C18 tip enrichment of phosphopeptides for protein phosphorylation analysis.Anal. Chem. 2005; 77: 5144-5154Crossref PubMed Scopus (188) Google Scholar, 8Ballif B.A. Villen J. Beausoleil S.A. Schwartz D. Gygi S.P. Phosphoproteomic analysis of the developing mouse brain.Mol. Cell. Proteomics. 2004; 3: 1093-1101Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 9Trinidad J.C. Specht C.G. Thalhammer A. Schoepfer R. Burlingame A.L. Comprehensive identification of phosphorylation sites in postsynaptic density preparations.Mol. Cell. Proteomics. 2006; 5: 914-922Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). Nevertheless further refinement of extant IMAC protocols is required to achieve high reproducibility and efficiency (14Nuehse T.S. Stensballe A. Jensen O.N. Peck S.C. Large-scale analysis of in vivo phosphorylated membrane proteins by immobilized metal ion affinity chromatography and mass spectrometry.Mol. Cell. Proteomics. 2003; 2: 1234-1243Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar). Poor fragmentation of phosphopeptides in the mass spectrometer represents the second major challenge for phosphoproteomics. The availability of a relatively low energy fragmentation pathway via β-elimination of the phosphate moiety limits fragmentation at peptide bonds that would be informative for identifying the sequence and site(s) of phosphorylation of the peptide. Mass spectrometers under development, such as instruments using electron transfer dissociation (15Coon J.J. Ueberheide B. Syka J.E. Dryhurst D.D. Ausio J. Shabanowitz J. Hunt D.F. Protein identification using sequential ion/ion reactions and tandem mass spectrometry.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 9463-9468Crossref PubMed Scopus (340) Google Scholar), might address this problem. However, such mass spectrometers are not currently commercially available. In addition, fragmentation of doubly charged and singly charged peptides in electron transfer dissociation mass spectrometry is compromised. In summary, despite extensive effort in the past several years, efficient proteomics of protein phosphorylation remains a daunting challenge. Here we report analysis of the phosphoproteome of mitochondria using an improved method that integrates an optimized batchwise IMAC protocol for isolation of phosphopeptides and MS3 1The abbreviations used are: MS3, MS/MS/MS; MS2, MS/MS; MS1, MS; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; VDAC, voltage-dependent anion channel. for fragmentation of phosphopeptides. The improved IMAC procedure allowed recovery of ∼77% of phosphopeptides while retaining few unphosphorylated peptides. MS3 addressed the poor fragmentation of phosphopeptides by generating highly informative fragmentation patterns that allow peptide identification. Analysis of mitochondrial phosphorylation revealed 84 phosphorylation sites from 62 proteins. Most identified phosphorylation sites have not been reported before, providing novel information about mitochondrial regulatory mechanisms. The optimized batchwise IMAC protocol in combination with MS3 offers a relatively simple and more efficient approach for proteomics of protein phosphorylation. The reagents used in this work included SelfPack Poros 20 MC beads from Applied Biosystems (Foster City, CA), [γ-32P]ATP from Amersham Biosciences, TFA from Fluka (Buchs, Switzerland), sequencing-grade trypsin from Promega Corp. (Madison, WI), and iodoacetamide and 99.99% acetic acid from Sigma. Four 10-cm dishes of A431 cells (80% confluence) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The cells were washed with cold Dulbecco's PBS twice, and 200 μl of reaction buffer (50 mm HEPES, pH 7.2, 5 mm MgCl2, 100 mm KCl, 1 mm ATP, and protease inhibitor mixture) were added. The cells were harvested by scraping and transferred to 1.5-ml microcentrifuge tubes. The cells were drawn into a sterile 1-ml syringe through a 30-gauge needle and expelled by a steady pressure on the plunger. The process was repeated 12 times. [γ-32P]ATP (0.2 mCi/ml of cell lysate) was added to the lysate and incubated for 1 h at 30 °C. After labeling of the whole cell lysate proteins by autophosphorylation in vitro, remaining free [γ-32P]ATP was removed by two cycles of TCA precipitation as briefly described below. The cytosolic proteins were precipitated by adding 1/9 volume of 100% TCA followed by a 2-h incubation on ice. After centrifugation at 15,300 × g for 20 min, the supernatant was removed, and the protein pellet was washed twice with cold acetone. The protein pellet was redissolved in 8 m urea, and the TCA precipitation was repeated to remove remaining free [γ-32P]ATP. The mitochondrial fraction from mouse liver was prepared and digested as described in the supplemental information. Methylation of carboxylic groups in acidic amino acid residues (Asp and Glu) and at the C termini of peptides was performed as described previously (5Ficarro S.B. McCleland M.L. Stukenberg P.T. Burke D.J. Ross M.M. Shabanowitz J. Hunt D.F. White F.M. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae.Nat. Biotechnol. 2002; 20: 301-305Crossref PubMed Scopus (1499) Google Scholar). Briefly 100 μg of tryptic digest was dried in a SpeedVac (Thermo Savant, San Jose, CA) for 6 h. Then 50 μl of 2 m methanolic HCl was added to the dried peptides, and the reaction was carried out for 2 h on a shaker. Anhydrous used to methanolic HCl been in the of The peptide was and the reaction was repeated to of peptides to their Poros 20 MC beads were to the procedure in the supplemental of beads were with 200 μl of in acetic acid of mitochondrial tryptic peptides were in 20 μl of and the pH was to with 2 m The peptide was at × g for 20 to remove which might have peptides. The resulting supernatant was with IMAC beads and incubated at with for 30 The was in a microcentrifuge at × g for 1 min, and the supernatant was The resulting beads were washed with 200 μl of buffer acid 100 mm and twice with 200 μl of buffer acid phosphopeptides were from the IMAC beads with of mass spectrometry was carried out as described previously Y. S.C. Y. approach for of peptides identified by protein sequence with tandem mass 2005; 4: PubMed Scopus Google that a of buffer acetic in A buffer acetic at a of was The mass spectrometer was in a in which MS was followed by of energy was to and MS3 MS3 was a of or was the most in the The were used for peptide identification as described in the supplemental information. The IMAC of a chromatography IMAC beads and has been used for isolation of phosphopeptides for proteomics approach has the of low of batchwise such as and efficient convenient and high that phosphate groups have relatively low affinity for IMAC that are potentially and that phosphorylated peptides are in the at low we that the of batchwise isolation the efficiency of IMAC of phosphopeptides from a peptide through batchwise IMAC be into of the carboxylic acid groups of peptides to their of phosphopeptides to the IMAC beads of unphosphorylated peptides from the beads and of phosphopeptides from the beads of is to phosphopeptide recovery while from unphosphorylated peptides. phosphopeptides were used to the IMAC The of the IMAC procedure was by from unphosphorylated peptides was by of the fraction and peptide identification via sequence 32P-labeled peptides, proteins from cell lysate are usually in phosphorylated and digested with The resulting tryptic peptides are to or chromatography to remove using 32P-labeled revealed that beads and beads more 1% of the that was from 32P-labeled from the supplemental information for the that only a of in 32P-labeled will be transferred proteins in is that 1% of the 32P-labeled would be used for in phosphorylation. was to remove by using a method or beads IMAC the in the to proteins phosphorylated in were precipitated twice with TCA and digested with Such precipitation remove in 32P-labeled The tryptic peptides were further to purification to remove to used for IMAC of carboxylic acid groups in peptides to their of unphosphorylated peptides to IMAC beads through acidic The reaction is and of the reaction is by the of used to the reaction to 100 μg of tryptic were dried in a SpeedVac for 6 h to of from the the used in the reaction was to remove in the the reaction was performed After tryptic peptides from A431 cells with we not tryptic peptides by a reaction not the IMAC method on the charged groups and the immobilized the chemical modification of acidic groups to the and of the its proteins and is acidic amino acid the more acidic from carboxylic acid groups would to a of for the analysis of peptides that the reaction was to groups as we not to the of the would be for in the These that to of the peptides be by a buffer After the the dried peptide was for is to the of peptides on and a of acidic with was was by was achieved in with acetic acid buffer is for IMAC as the high of ionic interactions immobilized and phosphate pH the affinity phosphate and by the of the phosphate the of phosphopeptides in the IMAC and on the pH of the buffer The recovery was achieved a relatively high pH was used phosphate a more more to the IMAC However, such a high pH is to of peptides, which might be in to the IMAC a recovery and a pH of was used enrichment of on we would not the buffer pH as a of The recovery from using a buffer pH of was at pH at pH the carboxylic have a of the phosphate has a of is that the of recovery using a buffer of pH would be by a of The of the is to of phosphopeptides to the IMAC beads while unphosphorylated peptides. phosphate groups and while interactions and the IMAC will interactions the IMAC beads and groups in the amino The of in the of and in the of phosphopeptides by in the was to as in the was to from ionic or 100 mm was included in the which led to an of not A procedure was that phosphopeptide recovery while from unphosphorylated peptides. The procedure included the using acid 100 mm and the second and using 50 mm and TFA were for efficiency with 32P-labeled peptide These reagents the interactions and the phosphate or and the acid and and of phosphopeptides to the IMAC beads these were used was included in the the be only of the in the beads phosphopeptides from to the IMAC beads through on these a and was as the for not in the buffer a high would phosphopeptides from to the this 32P-labeled phosphopeptides were allowed to to beads with of the supplemental information for the an the of the beads and the of in the buffer IMAC and in a of a of phosphopeptides to the to in the In summary, to IMAC efficiency we optimized at of the and The optimized procedure a phosphopeptide recovery of which into combined at the and and the Protein identification and modification sites tryptic peptides are performed using MS/MS to protein sequence of the MS/MS a role in such MS/MS results from fragmentation of peptide to of diverse that are by the peptide sequence and of post-translational to phosphopeptides are by the of via a relatively low energy fragmentation pathway that MS/MS of phosphopeptides usually limited mass information that is of the peptide sequence address this we of mass spectrometry for phosphopeptides. In this the the mass of the and the second the phosphate using low energy energy to or acid The (MS3) is fragmentation by of the resulting peptide which to unphosphorylated peptides in the mass spectrometer. The information from MS3 be used to peptides and localize phosphorylation sites The mass which has a high such a strategy is that phosphorylated peptides identified through using MS3 be the of two not two sites in the peptide as of the be from a the is described further in the supplemental information. The is a in for its role in phosphorylation and the cellular cell of and and and has been in such as and from the of and phosphoproteins identified in that phosphorylation an role in mitochondrial D.J. J.E. phosphorylation Sci. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). Nevertheless the of protein phosphorylation in mitochondria and the through which protein phosphorylation diverse mitochondrial applied the optimized IMAC protocol and MS3 to the phosphoproteome of mitochondria from mouse proteins were digested with trypsin and for phosphopeptides using The resulting peptide was by mass spectrometry using a MS3 The MS3 were used to the for mouse using the of in identification of 84 phosphorylation sites from 62 phosphoproteins 1 for the peptide and for the MS3 with The MS/MS for peptide is unphosphorylated peptides were identified using the combined and MS3 from The 62 phosphoproteins identified were into cell cell and the 84 identified phosphorylation were at and were at a phosphorylation these of of amino at the phosphorylation sites were and on a density was at the and acid was at the The phosphorylation sites and proteins identified in this analysis of the mitochondrial phosphoproteome further of the role of phosphorylation for mitochondrial proteins. the 62 proteins identified in this only were previously reported as mitochondrial The remaining novel and that phosphorylation has a on mitochondrial is currently the regulatory of few mitochondrial phosphorylation have been the of phosphorylation has been for two of the proteins identified in this and the of the of residues has been were identified in this at of the sites is to the of the M.J. M.C. of by 2003; PubMed Google Scholar). of the of the to of mitochondrial proteins, which to the phosphorylated and protein is a of the mitochondrial and Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). the of these phosphorylation phosphorylation the of mitochondrial proteins. phosphorylation of an of phosphorylation 1 has been to signaling in the a of or Biol. 2005; PubMed Scopus Google Scholar). the role of phosphorylation in the regulation of most of the proteins identified in this further of the phosphorylation sites with sequences reported for protein that the of mitochondrial proteins identified in this are potential of the kinase of sequences as for protein and protein Biol. Chem. Full Text PDF PubMed Google Scholar). is the role of in signaling and in mitochondria and the were the identified phosphopeptides is in mitochondria and a role in signaling in to a role for in the cellular and signaling in the a of or Biol. 2005; PubMed Scopus Google Scholar). of to mitochondria has been to be and mitochondrial signaling in the a of or Biol. 2005; PubMed Scopus Google Scholar). A of potential were identified in proteins involved in and pathways and proteins in to these potential is a of PKC, its phosphorylation in M. R. T. T. M.C. M. of protein kinase C to in and 2003; PubMed Scopus Google Scholar). is a protein kinase as a of and signaling from to insulin signaling and the cell of to its to and mitochondrial proteins have been to be signaling in the a of or Biol. 2005; PubMed Scopus Google Scholar, Rapid of in mitochondria 2003; PubMed Scopus Google Scholar). identified peptides that to the the of the kinase for a modification these based on sequences further a regulatory of phosphorylation in J.C. of cell signaling interactions using sequence 2003; PubMed Scopus Google of kinase phosphorylation and at high and medium identified phosphorylation kinase kinase kinase kinase Protein Open in a A protein be modified by post-translational in to these work in to protein identified proteins in this analysis that were identified in a proteomics analysis of proteins S.C. R. Y. Y. H. J. T. Y. H. L. White M. X.J. Y. and of revealed by a proteomics Cell. 2006; 23: Full Text Full Text PDF PubMed Scopus Google Scholar). identified in studies and protein might proteins these to be by signaling of mitochondrial to cellular Such of are of the and of to the of of and of the and of the its role in cellular and the of signaling on the regulation of its proteins through of the potential of the phosphorylation identified in this protein sequence was using the The protein sequences were for and of the identified phosphorylation proteins was the modified to be of the identified sites. that phosphorylation of mitochondrial proteins a regulatory pathway mouse and In this we addressed two major of proteomics of protein efficient isolation of phosphopeptides and informative optimized batchwise IMAC protocol allowed a phosphopeptide recovery of The high of singly phosphorylated peptides and low of unphosphorylated peptides identified mitochondrial proteins were the of IMAC offers the the IMAC beads be washed of phosphopeptides from the buffer is in the batchwise to be included in the buffer to interactions phosphopeptides and the IMAC recovery of the phosphopeptides. Such high be used in the method would a of phosphopeptides from to the the batchwise IMAC protocol high the potential to phosphopeptides in low optimized procedure is not with the as IMAC buffer a high of that the of phosphopeptides to in the of tryptic peptides a we of 1% acetic 100 mm buffer from S.B. McCleland M.L. Stukenberg P.T. Burke D.J. Ross M.M. Shabanowitz J. Hunt D.F. White F.M. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae.Nat. Biotechnol. 2002; 20: 301-305Crossref PubMed Scopus (1499) Google 50 mm buffer from S.B. McCleland M.L. Stukenberg P.T. Burke D.J. Ross M.M. Shabanowitz J. Hunt D.F. White F.M. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae.Nat. Biotechnol. 2002; 20: 301-305Crossref PubMed Scopus (1499) Google or TFA buffer from an and phosphopeptide The only of the phosphopeptides In by in IMAC we the efficiency of phosphopeptides from IMAC be that were used for optimization of the IMAC applied the procedure in to phosphorylated peptides from tryptic digest not In this only μl of IMAC beads was used protocol in the supplemental The of this was to a method of the of for phosphopeptide was through the of the isolation procedure via an optimized batchwise of the of the phosphate of phosphopeptides usually sequence to the peptides and to localize phosphorylation sites. is in MS3 in which the a of is used as the ion for further The that have the phosphate was on or acid the phosphate was on and to unphosphorylated peptides. The resulting fragmentation patterns are in information for peptide identification and of the previously phosphorylated sites. the mitochondrial phosphoproteome using optimized batchwise IMAC in combination with the MS3 we novel phosphorylation sites. The that approach is much more efficient previously reported methods for proteomics of protein phosphorylation. for with