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Comprehensive and Reproducible Phosphopeptide Enrichment Using Iron Immobilized Metal Ion Affinity Chromatography (Fe-IMAC) Columns

2014; Elsevier BV; Volume: 14; Issue: 1 Linguagem: Inglês

10.1074/mcp.m114.043109

ISSN

1535-9484

Autores

Benjamin Ruprecht, Heiner Koch, Guillaume Médard, Max Mundt, Bernhard Küster, Simone Lemeer,

Tópico(s)

Monoclonal and Polyclonal Antibodies Research

Resumo

Advances in phosphopeptide enrichment methods enable the identification of thousands of phosphopeptides from complex samples. Current offline enrichment approaches using TiO2, Ti, and Fe immobilized metal ion affinity chromatography (IMAC) material in batch or microtip format are widely used, but they suffer from irreproducibility and compromised selectivity. To address these shortcomings, we revisited the merits of performing phosphopeptide enrichment in an HPLC column format. We found that Fe-IMAC columns enabled the selective, comprehensive, and reproducible enrichment of phosphopeptides out of complex lysates. Column enrichment did not suffer from bead-to-sample ratio issues and scaled linearly from 100 μg to 5 mg of digest. Direct measurements on an Orbitrap Velos mass spectrometer identified >7500 unique phosphopeptides with 90% selectivity and good quantitative reproducibility (median cv of 15%). The number of unique phosphopeptides could be increased to more than 14,000 when the IMAC eluate was subjected to a subsequent hydrophilic strong anion exchange separation. Fe-IMAC columns outperformed Ti-IMAC and TiO2 in batch or tip mode in terms of phosphopeptide identification and intensity. Permutation enrichments of flow-throughs showed that all materials largely bound the same phosphopeptide species, independent of physicochemical characteristics. However, binding capacity and elution efficiency did profoundly differ among the enrichment materials and formats. As a result, the often quoted orthogonality of the materials has to be called into question. Our results strongly suggest that insufficient capacity, inefficient elution, and the stochastic nature of data-dependent acquisition in mass spectrometry are the causes of the experimentally observed complementarity. The Fe-IMAC enrichment workflow using an HPLC format developed here enables rapid and comprehensive phosphoproteome analysis that can be applied to a wide range of biological systems. Advances in phosphopeptide enrichment methods enable the identification of thousands of phosphopeptides from complex samples. Current offline enrichment approaches using TiO2, Ti, and Fe immobilized metal ion affinity chromatography (IMAC) material in batch or microtip format are widely used, but they suffer from irreproducibility and compromised selectivity. To address these shortcomings, we revisited the merits of performing phosphopeptide enrichment in an HPLC column format. We found that Fe-IMAC columns enabled the selective, comprehensive, and reproducible enrichment of phosphopeptides out of complex lysates. Column enrichment did not suffer from bead-to-sample ratio issues and scaled linearly from 100 μg to 5 mg of digest. Direct measurements on an Orbitrap Velos mass spectrometer identified >7500 unique phosphopeptides with 90% selectivity and good quantitative reproducibility (median cv of 15%). The number of unique phosphopeptides could be increased to more than 14,000 when the IMAC eluate was subjected to a subsequent hydrophilic strong anion exchange separation. Fe-IMAC columns outperformed Ti-IMAC and TiO2 in batch or tip mode in terms of phosphopeptide identification and intensity. Permutation enrichments of flow-throughs showed that all materials largely bound the same phosphopeptide species, independent of physicochemical characteristics. However, binding capacity and elution efficiency did profoundly differ among the enrichment materials and formats. As a result, the often quoted orthogonality of the materials has to be called into question. Our results strongly suggest that insufficient capacity, inefficient elution, and the stochastic nature of data-dependent acquisition in mass spectrometry are the causes of the experimentally observed complementarity. The Fe-IMAC enrichment workflow using an HPLC format developed here enables rapid and comprehensive phosphoproteome analysis that can be applied to a wide range of biological systems. Protein phosphorylation is a reversible post-translational modification with pivotal roles in cellular signaling. It is implicated in many essential biological processes, and aberrant protein phosphorylation is causally linked to numerous diseases (1.Blume-Jensen P. Hunter T. Oncogenic kinase signalling.Nature. 2001; 411: 355-365Crossref PubMed Scopus (3133) Google Scholar). At least one-third to one-half of all human proteins are thought to be phosphorylated at some point (2.Cohen P. The regulation of protein function by multisite phosphorylation—a 25 year update.Trends Biochem. 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Because of this molecular complexity and the often substoichiometric extent of phosphorylation, specific enrichment prior to analysis via liquid chromatography–tandem mass spectrometry (LC-MS/MS) 1The abbreviations used are:LC-MS/MSliquid chromatography–tandem mass spectrometryFDRfalse discovery rateIMACimmobilized metal ion affinity chromatographyACNacetonitrileFAformic acidhSAXhydrophilic strong anion exchangeHPLChigh-performance liquid chromatographyTiO2titanium dioxide. 1The abbreviations used are:LC-MS/MSliquid chromatography–tandem mass spectrometryFDRfalse discovery rateIMACimmobilized metal ion affinity chromatographyACNacetonitrileFAformic acidhSAXhydrophilic strong anion exchangeHPLChigh-performance liquid chromatographyTiO2titanium dioxide. is generally required (4.Lemeer S. Heck A.J. The phosphoproteomics data explosion.Curr. Opin. Chem. Biol. 2009; 13: 414-420Crossref PubMed Scopus (140) Google Scholar, 5.Ruprecht B. Lemeer S. Proteomic analysis of phosphorylation in cancer.Expert Rev. Proteomics. 2014; 11: 259-267Crossref PubMed Scopus (35) Google Scholar). Most current such strategies exploit the affinity of phosphate groups for metals immobilized on carrier resins. These approaches include Fe3+ (6.Andersson L. Porath J. Isolation of phosphoproteins by immobilized metal (Fe3+) affinity chromatography.Anal. Biochem. 1986; 154: 250-254Crossref PubMed Scopus (644) Google Scholar), Ga3+ (7.Posewitz M.C. Tempst P. Immobilized gallium(III) affinity chromatography of phosphopeptides.Anal. Chem. 1999; 71: 2883-2892Crossref PubMed Scopus (786) Google Scholar), and Zr4+ (8.Zhou H. Xu S. Ye M. Feng S. Pan C. Jiang X. Li X. Han G. Fu Y. Zou H. Zirconium phosphonate-modified porous silicon for highly specific capture of phosphopeptides and MALDI-TOF MS analysis.J. Proteome Res. 2006; 5: 2431-2437Crossref PubMed Scopus (157) Google Scholar) immobilized metal ion affinity chromatography (IMAC); metal oxide affinity chromatography (TiO2 (9.Pinkse M.W. Uitto P.M. Hilhorst M.J. Ooms B. Heck A.J. Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-nanoLC-ESI-MS/MS and titanium oxide precolumns.Anal. Chem. 2004; 76: 3935-3943Crossref PubMed Scopus (825) Google Scholar), ZrO2 (10.Kweon H.K. Håkansson K. Selective zirconium dioxide-based enrichment of phosphorylated peptides for mass spectrometric analysis.Anal. Chem. 2006; 78: 1743-1749Crossref PubMed Scopus (470) Google Scholar), and others); and the recently introduced Ti-IMAC, which is a hybrid of the other two methods (11.Zhou H. Low T.Y. Hennrich M.L. Toorn H.v. d. Schwend T. Zou H. Mohammed S. Heck A.J.R. Enhancing the identification of phosphopeptides from putative basophilic kinase substrates using Ti (IV) based IMAC enrichment.Mol. Cell. Proteomics. 2011; 10Abstract Full Text Full Text PDF Scopus (77) Google Scholar, 12.Lai A.C.-Y. Tsai C.-F. Hsu C.-C. Sun Y.-N. Chen Y.-J. Complementary Fe3+- and Ti4+-immobilized metal ion affinity chromatography for purification of acidic and basic phosphopeptides.Rapid Commun. Mass Spectrom. 2012; 26: 2186-2194Crossref PubMed Scopus (45) Google Scholar). It is often stated, if not generally accepted, that these enrichment methods are capable of purifying complementary parts of the phosphoproteome with unique physicochemical characteristics (11.Zhou H. Low T.Y. Hennrich M.L. Toorn H.v. d. Schwend T. Zou H. Mohammed S. Heck A.J.R. Enhancing the identification of phosphopeptides from putative basophilic kinase substrates using Ti (IV) based IMAC enrichment.Mol. Cell. Proteomics. 2011; 10Abstract Full Text Full Text PDF Scopus (77) Google Scholar, 13.Bodenmiller B. Mueller L.N. Mueller M. Domon B. Aebersold R. Reproducible isolation of distinct, overlapping segments of the phosphoproteome.Nat. Methods. 2007; 4: 231-237Crossref PubMed Scopus (505) Google Scholar, 14.Tsai C.-F. Hsu C.-C. Hung J.-N. Wang Y.-T. Choong W.-K. Zeng M.-Y. Lin P.-Y. Hong R.-W. Sung T.-Y. Chen Y.-J. Sequential phosphoproteomic enrichment through complementary metal-directed immobilized metal ion affinity chromatography.Anal. Chem. 2014; 86: 685-693Crossref PubMed Scopus (81) Google Scholar). For example, Ti-IMAC is thought to be better for purifying basophilic phosphopeptides than TiO2 (11.Zhou H. Low T.Y. Hennrich M.L. Toorn H.v. d. Schwend T. Zou H. Mohammed S. Heck A.J.R. Enhancing the identification of phosphopeptides from putative basophilic kinase substrates using Ti (IV) based IMAC enrichment.Mol. Cell. Proteomics. 2011; 10Abstract Full Text Full Text PDF Scopus (77) Google Scholar), whereas Fe-IMAC is credited with the more efficient enrichment of multiply phosphorylated peptides (15.Thingholm T.E. Jensen O.N. Robinson P.J. Larsen M.R. SIMAC (sequential elution from IMAC), a phosphoproteomics strategy for the rapid separation of monophosphorylated from multiply phosphorylated peptides.Mol. Cell. Proteomics. 2008; 7: 661-671Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar). The current consensus in the field is that no method is superior over others and none alone is sufficient for the comprehensive purification of the phosphoproteome, a view that we challenge in this study. Most phosphopeptide enrichments are conducted either in a batch format or in self-constructed microcolumns packed into pipette tips. Both formats have been found to suffer from considerable variability introduced by various manual steps in the process. Moreover, differences in loading conditions including additives (16.Larsen M.R. Thingholm T.E. Jensen O.N. Roepstorff P. Jorgensen T.J. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns.Mol. Cell. Proteomics. 2005; 4: 873-886Abstract Full Text Full Text PDF PubMed Scopus (1333) Google Scholar, 17.Kettenbach A.N. Gerber S.A. Rapid and reproducible single-stage phosphopeptide enrichment of complex peptide mixtures: application to general and phosphotyrosine-specific phosphoproteomics experiments.Anal. Chem. 2011; 83: 7635-7644Crossref PubMed Scopus (129) Google Scholar), acid concentration (17.Kettenbach A.N. Gerber S.A. Rapid and reproducible single-stage phosphopeptide enrichment of complex peptide mixtures: application to general and phosphotyrosine-specific phosphoproteomics experiments.Anal. Chem. 2011; 83: 7635-7644Crossref PubMed Scopus (129) Google Scholar), incubation time (17.Kettenbach A.N. Gerber S.A. Rapid and reproducible single-stage phosphopeptide enrichment of complex peptide mixtures: application to general and phosphotyrosine-specific phosphoproteomics experiments.Anal. Chem. 2011; 83: 7635-7644Crossref PubMed Scopus (129) Google Scholar, 18.Montoya A. Beltran L. Casado P. Rodríguez-Prados J.-C. Cutillas P.R. Characterization of a TiO2 enrichment method for label-free quantitative phosphoproteomics.Methods. 2011; 54: 370-378Crossref PubMed Scopus (94) Google Scholar), and wash volume (18.Montoya A. Beltran L. Casado P. Rodríguez-Prados J.-C. Cutillas P.R. Characterization of a TiO2 enrichment method for label-free quantitative phosphoproteomics.Methods. 2011; 54: 370-378Crossref PubMed Scopus (94) Google Scholar) can lead to different results. One prominent parameter that was found to have a considerable effect on the enrichment quality is the ratio of bead amount to protein digest quantity, and finding the optimal ratio is often a tradeoff between comprehensiveness and selectivity (13.Bodenmiller B. Mueller L.N. Mueller M. Domon B. Aebersold R. Reproducible isolation of distinct, overlapping segments of the phosphoproteome.Nat. Methods. 2007; 4: 231-237Crossref PubMed Scopus (505) Google Scholar, 18.Montoya A. Beltran L. Casado P. Rodríguez-Prados J.-C. Cutillas P.R. Characterization of a TiO2 enrichment method for label-free quantitative phosphoproteomics.Methods. 2011; 54: 370-378Crossref PubMed Scopus (94) Google Scholar, 19.Li Q.R. Ning Z.B. Tang J.S. Nie S. Zeng R. Effect of peptide-to-TiO2 beads ratio on phosphopeptide enrichment selectivity.J. Proteome Res. 2009; 8: 5375-5381Crossref PubMed Scopus (108) Google Scholar, 20.Yue X.-S. Hummon A.B. Combination of multistep IMAC enrichment with high-pH reverse phase separation for in-depth phosphoproteomic profiling.J. Proteome Res. 2013; 12: 4176-4186Crossref PubMed Scopus (40) Google Scholar, 21.Zhou H. Di Palma S. Preisinger C. Peng M. Polat A.N. Heck A.J.R. Mohammed S. Toward a comprehensive characterization of a human cancer cell phosphoproteome.J. Proteome Res. 2013; 12: 260-271Crossref PubMed Scopus (297) Google Scholar). Consequently, careful a priori evaluation and optimization of enrichment conditions is generally required on a case-by-case basis for every experimental system. Even then, the use of such formats often comes at the expense of intra-experimental and, even more so, inter-experimental accuracy. As a means to overcome these issues, direct coupling of the chromatographic enrichment step with the LC-MS/MS system has been applied. Although these online systems increase reproducibility, sensitivity, and robustness, they suffer from limited capacity, which is why they have been primarily used for the analysis of limited sample amounts or samples of rather low complexity (9.Pinkse M.W. Uitto P.M. Hilhorst M.J. Ooms B. Heck A.J. Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-nanoLC-ESI-MS/MS and titanium oxide precolumns.Anal. Chem. 2004; 76: 3935-3943Crossref PubMed Scopus (825) Google Scholar, 22.Ficarro S.B. Salomon A.R. Brill L.M. Mason D.E. Stettler-Gill M. Brock A. Peters E.C. Automated immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry platform for profiling protein phosphorylation sites.Rapid Commun. Mass Spectrom. 2005; 19: 57-71Crossref PubMed Scopus (89) Google Scholar, 23.Wang J. Zhang Y. Jiang H. Cai Y. Qian X. Phosphopeptide detection using automated online IMAC-capillary LC-ESI-MS/MS.Proteomics. 2006; 6: 404-411Crossref PubMed Scopus (46) Google Scholar, 24.Pinkse M.W.H. Mohammed S. Gouw J.W. van Breukelen B. Vos H.R. Heck A.J.R. Highly robust, automated, and sensitive online TiO2-based phosphoproteomics applied to study endogenous phosphorylation in Drosophila melanogaster.J. Proteome Res. 2008; 7: 687-697Crossref PubMed Scopus (163) Google Scholar, 25.Ndassa Y.M. Orsi C. Marto J.A. Chen S. Ross M.M. Improved immobilized metal affinity chromatography for large-scale phosphoproteomics applications.J. Proteome Res. 2006; 5: 2789-2799Crossref PubMed Scopus (106) Google Scholar, 26.Zhang Y. Wolf-Yadlin A. Ross P.L. Pappin D.J. Rush J. Lauffenburger D.A. White F.M. Time-resolved mass spectrometry of tyrosine phosphorylation sites in the epidermal growth factor receptor signaling network reveals dynamic modules.Mol. Cell. Proteomics. 2005; 4: 1240-1250Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar, 27.Zarling A.L. Ficarro S.B. White F.M. Shabanowitz J. Hunt D.F. Engelhard V.H. Phosphorylated peptides are naturally processed and presented by Major Histocompatibility Complex Class I molecules in vivo.J. Exp. Med. 2000; 192: 1755-1762Crossref PubMed Scopus (167) Google Scholar). liquid chromatography–tandem mass spectrometry false discovery rate immobilized metal ion affinity chromatography acetonitrile formic acid hydrophilic strong anion exchange high-performance liquid chromatography titanium dioxide. liquid chromatography–tandem mass spectrometry false discovery rate immobilized metal ion affinity chromatography acetonitrile formic acid hydrophilic strong anion exchange high-performance liquid chromatography titanium dioxide. In order to address this, phosphopeptide enrichment workflows often employ an upstream peptide separation step followed by phosphopeptide enrichment from each fraction. Examples of first-dimension separations include hydrophilic interaction liquid chromatography (28.Zarei M. Sprenger A. Gretzmeier C. Dengjel J. Combinatorial use of electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) and strong cation exchange (SCX) chromatography for in-depth phosphoproteome analysis.J. Proteome Res. 2012; 11: 4269-4276Crossref PubMed Scopus (32) Google Scholar), electrostatic repulsion hydrophilic interaction chromatography (29.Zarei M. Sprenger A. Metzger F. Gretzmeier C. Dengjel J. Comparison of ERLIC–TiO2, HILIC–TiO2, and SCX–TiO2 for global phosphoproteomics approaches.J. Proteome Res. 2011; 10: 3474-3483Crossref PubMed Scopus (81) Google Scholar), strong anion exchange (30.Dai J. Wang L.-S. Wu Y.-B. Sheng Q.-H. Wu J.-R. Shieh C.-H. Zeng R. Fully automatic separation and identification of phosphopeptides by continuous pH-gradient anion exchange online coupled with reversed-phase liquid chromatography mass spectrometry.J. Proteome Res. 2009; 8: 133-141Crossref PubMed Scopus (56) Google Scholar), and strong cation exchange (11.Zhou H. Low T.Y. Hennrich M.L. Toorn H.v. d. Schwend T. Zou H. Mohammed S. Heck A.J.R. Enhancing the identification of phosphopeptides from putative basophilic kinase substrates using Ti (IV) based IMAC enrichment.Mol. Cell. Proteomics. 2011; 10Abstract Full Text Full Text PDF Scopus (77) Google Scholar, 31.Villén J. Gygi S.P. The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry.Nat. Protoc. 2008; 3: 1630-1638Crossref PubMed Scopus (498) Google Scholar). Although powerful and frequently used, enrichment after fractionation can be irreproducible and time consuming. As a consequence, reversing the order (i.e. enrichment of phosphopeptides first, followed by fractionation of the phosphopeptide pool) has recently become popular (17.Kettenbach A.N. Gerber S.A. Rapid and reproducible single-stage phosphopeptide enrichment of complex peptide mixtures: application to general and phosphotyrosine-specific phosphoproteomics experiments.Anal. Chem. 2011; 83: 7635-7644Crossref PubMed Scopus (129) Google Scholar, 18.Montoya A. Beltran L. Casado P. Rodríguez-Prados J.-C. Cutillas P.R. Characterization of a TiO2 enrichment method for label-free quantitative phosphoproteomics.Methods. 2011; 54: 370-378Crossref PubMed Scopus (94) Google Scholar, 32.Engholm-Keller K. Birck P. Størling J. Pociot F. Mandrup-Poulsen T. Larsen M.R. TiSH—a robust and sensitive global phosphoproteomics strategy employing a combination of TiO2, SIMAC, and HILIC.J. Proteomics. 2012; 75: 5749-5761Crossref PubMed Scopus (149) Google Scholar). This, however, requires that the phosphopeptide enrichment step be of exquisite selectivity, have sufficient capacity, and allow complete elution (17.Kettenbach A.N. Gerber S.A. Rapid and reproducible single-stage phosphopeptide enrichment of complex peptide mixtures: application to general and phosphotyrosine-specific phosphoproteomics experiments.Anal. Chem. 2011; 83: 7635-7644Crossref PubMed Scopus (129) Google Scholar, 18.Montoya A. Beltran L. Casado P. Rodríguez-Prados J.-C. Cutillas P.R. Characterization of a TiO2 enrichment method for label-free quantitative phosphoproteomics.Methods. 2011; 54: 370-378Crossref PubMed Scopus (94) Google Scholar). Phosphopeptides purified in this way can be directly analyzed via LC-MS/MS (33.Thakur S.S. Geiger T. Chatterjee B. Bandilla P. Fröhlich F. Cox J. Mann M. Deep and highly sensitive proteome coverage by LC-MS/MS without prefractionation.Mol. Cell. Proteomics. 2011; 10Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 34.Pirmoradian M. Budamgunta H. Chingin K. Zhang B. Astorga-Wells J. Zubarev R.A. Rapid and deep human proteome analysis by single-dimension shotgun proteomics.Mol. Cell. Proteomics. 2013; 12: 3330-3338Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), typically using shallow reversed phase LC gradients (35.Köcher T. Pichler P. Swart R. Mechtler K. Analysis of protein mixtures from whole-cell extracts by single-run nanoLC-MS/MS using ultralong gradients.Nat. Protoc. 2012; 7: 882-890Crossref PubMed Scopus (90) Google Scholar). However, it turns out that current mass spectrometers still lack the scan speed and dynamic range required to reach complete (phospho)peptide sampling in direct LC-MS/MS measurements (36.Michalski A. Cox J. Mann M. More than 100,000 detectable peptide species elute in single shotgun proteomics runs but the majority is inaccessible to data-dependent LC-MS/MS.J. Proteome Res. 2011; 10: 1785-1793Crossref PubMed Scopus (476) Google Scholar, 37.Hebert A.S. Richards A.L. Bailey D.J. Ulbrich A. Coughlin E.E. Westphall M.S. Coon J.J. The one hour yeast proteome.Mol. Cell. Proteomics. 2014; 13: 339-347Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar). In this article, we describe a robust and flexible workflow based on offline chromatographic enrichment of phosphopeptides using a commercially available Fe-IMAC column. This approach offers selective, comprehensive, and reproducible enrichment and scales over a wide range of sample quantities. We show that this method outperformed all Ti-IMAC and TiO2 methods tested, and our data argue that the apparent orthogonality of all three methods is caused by a combination of format inadequacies, inefficient elution, and insufficient data acquisition speed, rather than the different physicochemical characteristics of phosphopeptides. Human epidermoid A431 cells were grown in Iscove's modified Dulbecco's medium supplemented with 10% (v/v) fetal bovine serum and 1% antibiotic/antimycotic solution. For all phosphopeptide enrichment optimization experiments, cells were treated with 1 mm pervanadate for 5 min prior to lysis. After harvesting, cells were washed two times with ice-cold PBS and lysed in 8 m urea, 40 mm Tris/HCl (pH 7.6), 1× EDTA-free protease inhibitor mixture (Complete Mini, Roche), and 1× phosphatase inhibitor mixture (Sigma). The lysate was centrifuged at 20,000 rpm for 1 h at 4 °C. The protein concentration was determined using the Bradford method (Coomassie (Bradford) Protein Assay Kit, Thermo Scientific). The supernatant was reduced with 10 mm DTT at 56 °C for 1 h and alkylated with 25 mm iodoacetamide for 45 min at room temperature in the dark. The protein mixture was diluted with 40 mm Tris/HCl to a final urea concentration of 1.6 m. We induced digestion by adding sequencing-grade trypsin (Promega, Mannheim, Germany; 1:100 enzyme:substrate ratio) and allowing samples to incubate at 37 °C for 4 h. Subsequently another 1:100 quantity of trypsin was added for overnight digestion at 37 °C. Samples were acidified with TFA to pH 2 to stop the trypsin activity. SepPack columns (C18 cartridges, Sep-Pak Vac, 1 cc (50 mg), Waters Corp., Eschborn, Germany; solvent A, 0.07% TFA; solvent B, 0.07% TFA, 50% ACN) were used for peptide desalting according to the manufacturer's instructions, and eluates were dried down and stored at −80 °C. Smaller sample amounts of up to 100 μg were desalted as described elsewhere (38.Rappsilber J. Mann M. Ishihama Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips.Nat. Protoc. 2007; 2: 1896-1906Crossref PubMed Scopus (2570) Google Scholar). Fe-IMAC batch enrichments were essentially performed as described in Ref. 31.Villén J. Gygi S.P. The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry.Nat. Protoc. 2008; 3: 1630-1638Crossref PubMed Scopus (498) Google Scholar. Briefly, 100 μl of Fe-IMAC beads (PhosSelect iron affinity gel, Sigma) were washed four times with 1 ml of binding solvent (25 mm FA, 40% ACN), and a 50% slurry in binding solvent was prepared. Dried-down peptides were resuspended in binding solvent at a concentration of 1 μg/μl. After the beads and dissolved peptides had been combined, samples were incubated for 1 h at room temperature with vigorous shaking. Subsequently, Fe-IMAC beads were transferred on top of previously equilibrated C18 StageTips and washed by being sequentially passed through 50 μl of Fe-IMAC binding solvent (twice) and 40 μl of 1% FA. Elution was achieved via the application of 70 μl of 500 mm K2HPO4, pH 7 (twice). Peptides that were retained on the C18 material were washed with 40 μl of 1% FA and eluted directly into an MS plate using 40 μl of 60% ACN, 0.1% FA. Eluates were dried down and stored at −80 °C. The desalted digest was reconstituted in 0.5 ml of Fe-IMAC solvent A (30% ACN, 0.07% (v/v) TFA) and loaded onto an analytical Fe-IMAC column (4 × 50 mm or 9 × 50 mm ProPac IMAC-10, Thermo Fisher Scientific) connected to an HPLC system (ÄKTA Explorer FPLC system, Amersham Biosciences Pharmacia) with a 1-ml sample loop. The column was charged with iron according to the manufacturer's instructions. Briefly, the column was rinsed with 3 column volumes of 20 mm formic acid and charged using 3 column volumes of 25 mm FeCl3, 100 mm acetic acid. To wash out unbound iron ions, we flushed the column with 20 column volumes of 20 mm formic acid and subsequently removed it from the HPLC system. The HPLC lines were flushed first with 20 ml of double-deionized H2O and then with 20 ml of 50 mm EDTA and 10 ml of double-deionized H2O to remove remaining iron ions. After column reconnection and baseline equilibration, the gradient was started. Sample was loaded (0.1 ml/min over 10 min), and unbound peptides were washed out with Fe-IMAC solvent A (0.3 ml/min over 16 min). Subsequent phosphopeptide elution was achieved with a linear gradient from 0% to 45% Fe-IMAC solvent B (0.5% (v/v) NH4OH) (0.2 ml/min over 60 min). After an increase to 100% solvent B and a 5-min holding step, the column was re-equilibrated with Fe-IMAC solvent A (0.5 ml/min over 30 min). Flow-through and a phosphopeptide fraction were collected according to the UV signal (280 nm), dried down, and stored at −80 °C. The column was recharged after a maximum of three enrichments. Ti-IMAC beads were synthesized as previously described by Zhou et al. (39.Zhou H. Ye M. Dong J. Corradini E. Cristobal A. Heck A.J. Zou H. Mohammed S. Robust phosphoproteome enrichment using monodisperse microsphere-based immobilized titanium (IV) ion affinity chromatography.Nat. Protoc. 2013; 8: 461-480Crossref PubMed Scopus (243) Google Scholar), with some modifications and additional quality control steps. A detailed description can be found in the supplemental "Materials and Methods" section. Enrichment of up to 250 μg of sample was performed as described in Ref. 39.Zhou H. Ye M. Dong J. Corradini E. Cristobal A. Heck A.J. Zou H. Mohammed S. Robust phosphoproteome enrichment using monodisperse microsphere-based immobilized titanium (IV) ion affinity chromatography.Nat. Protoc. 2013; 8: 461-480Crossref PubMed Scopus (243) Google Scholar; the phosphopeptide enrichment protocol was modified for sample amounts exceeding 250 μg. Sep-Pak cartridges (C18 cartridges, Sep-Pak Vac, 1 cc (50 mg), Waters Corp.) were attached to a vacuum manifold and flushed with 1 ml of Ti-IMAC loading solvent (80% ACN, 6% TFA). Subsequently, 50 mg of Ti-IMAC beads were loaded on top of the C18 material and equilibrated with 5 × 1 ml loading solvent. Dried-down digests were dissolved in 1 ml of binding solvent and slowly passed through the cartridges. After reapplication of the flow-through, columns were washed with 10 ml of washing solution 1 (50% (v/v) ACN, 0.5% (v/v) TFA, 200 mm NaCl) and 10 ml of washing solution 2 (50% (v/v) ACN, 0.1% (v/v) TFA). Bound peptides were eluted sequentially with 0.8 ml of 10% (v/v) NH4OH and 0.2 ml of 80% (v/v) ACN, 2% (v/v) FA. Flow-through and elution fractions were dried down and stored at −80 °C. TiO2 batch enrichment was performed as described by Kettenbach and Gerber (17.Kettenbach A.N. Gerber S.A. Rapid and reproducible single-stage phosphopeptide enrichment of complex peptide mixtures: application to general and phosphotyrosine-specific phosphoproteomics experiments.Anal. Chem. 2011; 83: 7635-7644Crossref PubMed Scopus (129) Google Scholar), with some modifications. TiO2 beads (5 μm, GL Sciences Inc., Mainz, Germany) were washed twice with 1 ml of washing solvent (50% ACN, 0.1% TFA) and four times with 1 ml of binding solvent (2 m lactic acid, 50% ACN, 0.1% TFA). In between, beads were spun down and the supernatant was discarded. Peptides were dissolved in 0.5 ml of binding solvent, and after the addition of 0.25 ml of equilibrated bead slurry, the mixture was incubated for 1 h at room temperature with vigorous shaking. Subsequently, the beads were washed four times with 0.2 ml of binding solvent and five times with 1 ml of washing solution. Bound peptides were eluted in two 10-min incubation steps with 200 μl of elution solvent (50 mm KH2PO4, 0.5% (v/v) NH4OH, pH 11.3). The supernatant was quenched by the addition of 30 μl of 100% FA, dried down, and stored at −80 °C. For chromatographic separation

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