Mass spectrometry (MS) is a powerful and sensitive method often used for the identification of phosphoproteins. However, in phosphoproteomics, there is an identified need to compensate for the low abundance, insufficient ionization, and suppression effects of non-phosphorylated peptides. These may hamper the subsequent liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis, resulting in incomplete phosphoproteome characterization, even when using high-resolution instruments. To overcome these drawbacks, we present here an effective microgradient chromatographic technique that yields specific fractions of enriched phosphopeptides compatible with LC-MS/MS analysis. The purpose of our study was to increase the number of identified phosphopeptides, and thus, the coverage of the sample phosphoproteome using the reproducible and straightforward fractionation method. This protocol includes a phosphopeptide enrichment step followed by the optimized microgradient fractionation of enriched phosphopeptides and final LC-MS/MS analysis of the obtained fractions. The simple fractionation system consists of a gas-tight microsyringe delivering the optimized gradient mobile phase to reversed-phase microcolumn. Our data indicate that combining the phosphopeptide enrichment with the microgradient separation is a promising technique for in-depth phosphoproteomic analysis due to moderate input material requirements and more than 3-fold enhanced protein identification.

Department of Biological and Biochemical Sciences Faculty of Chemical Technology University of Pardubice 532 10 Pardubice Czech Republic

Department of Molecular Biology and Pathology Faculty of Military Health Sciences University of Defense in Brno 500 01 Hradec Kralove Czech Republic

Department of Radiobiology Faculty of Military Health Sciences University of Defense in Brno 500 01 Hradec Kralove Czech Republic

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Cohen P. The origins of protein phosphorylation. Nat. Cell Biol. 2002;4:E127–E130. doi: 10.1038/ncb0502-e127. PubMed DOI

Ardito F., Giuliani M., Perrone D., Troiano G., Lo Muzio L. The crucial role of protein phosphorylation in cell signaling and its use as targeted therapy (Review) Int. J. Mol. Med. 2017;40:271–280. doi: 10.3892/ijmm.2017.3036. PubMed DOI PMC

Vlastaridis P., Kyriakidou P., Chaliotis A., Van de Peer Y., Oliver S.G., Amoutzias G.D. Estimating the total number of phosphoproteins and phosphorylation sites in eukaryotic proteomes. Gigascience. 2017;6:1–11. doi: 10.1093/gigascience/giw015. PubMed DOI PMC

Chan C.Y.X., Gritsenko M.A., Smith R.D., Qian W.-J. The current state of the art of quantitative phosphoproteomics and its applications to diabetes research. Expert Rev. Proteom. 2016;13:421–433. doi: 10.1586/14789450.2016.1164604. PubMed DOI PMC

Mayya V., Han D.K. Phosphoproteomics by Mass Spectrometry: insights, implications, applications, and limitations. Expert Rev. Proteom. 2009;6:605–618. doi: 10.1586/epr.09.84. PubMed DOI PMC

Tichy A., Salovska B., Rehulka P., Klimentova J., Vavrova J., Stulik J., Hernychova L. Phosphoproteomics: searching for a needle in a haystack. J. Proteom. 2011;74:2786–2797. doi: 10.1016/j.jprot.2011.07.018. PubMed DOI

Fíla J., Honys D. Enrichment techniques employed in phosphoproteomics. Amino Acids. 2012;43:1025–1047. doi: 10.1007/s00726-011-1111-z. PubMed DOI PMC

Dunn J.D., Reid G.E., Bruening M.L. Techniques for phosphopeptide enrichment prior to analysis by mass spectrometry. Mass Spectrom. Rev. 2010;29:29–54. doi: 10.1002/mas.20219. PubMed DOI

Thingholm T.E., Larsen M.R. The use of titanium dioxide for selective enrichment of phosphorylated peptides. Methods Mol. Biol. 2016;1355:135–146. doi: 10.1007/978-1-4939-3049-4_9. PubMed DOI

Pinkse M.W.H., Uitto P.M., Hilhorst M.J., Ooms B., Heck A.J.R. Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns. Anal. Chem. 2004;76:3935–3943. doi: 10.1021/ac0498617. PubMed DOI

Ruprecht B., Koch H., Medard G., Mundt M., Kuster B., Lemeer S. Comprehensive and reproducible phosphopeptide enrichment using iron immobilized metal ion affinity chromatography (Fe-IMAC) columns. Mol. Cell. Proteom. 2015;14:205–215. doi: 10.1074/mcp.M114.043109. PubMed DOI PMC

Fenselau C. A review of quantitative methods for proteomic studies. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2007;855:14–20. doi: 10.1016/j.jchromb.2006.10.071. PubMed DOI

Nita-Lazar A., Saito-Benz H., White F.M. Quantitative phosphoproteomics by mass spectrometry: past, present, and future. Proteomics. 2008;8:4433–4443. doi: 10.1002/pmic.200800231. PubMed DOI PMC

Deracinois B., Flahaut C., Duban-Deweer S., Karamanos Y. Comparative and quantitative global proteomics approaches: an overview. Proteomes. 2013;1:180–218. doi: 10.3390/proteomes1030180. PubMed DOI PMC

Manadas B., Mendes V.M., English J., Dunn M.J. Peptide fractionation in proteomics approaches. Expert Rev. Proteom. 2010;7:655–663. doi: 10.1586/epr.10.46. PubMed DOI

Ly L., Wasinger V.C. Protein and peptide fractionation, enrichment and depletion: tools for the complex proteome. Proteomics. 2011;11:513–534. doi: 10.1002/pmic.201000394. PubMed DOI

Cao Z., Tang H.-Y., Wang H., Liu Q., Speicher D.W. Systematic comparison of fractionation methods for in-depth analysis of plasma proteomes. J. Proteome Res. 2012;11:3090–3100. doi: 10.1021/pr201068b. PubMed DOI PMC

Gilar M., Olivova P., Daly A.E., Gebler J.C. Two-dimensional separation of peptides using RP-RP-HPLC system with different pH in first and second separation dimensions. J. Sep. Sci. 2005;28:1694–1703. doi: 10.1002/jssc.200500116. PubMed DOI

Que A.H., Kahle V., Novotny M.V. A microgradient elution system for capillary electrochromatography. J. Microcolumn Sep. 2000;12:1–5. doi: 10.1002/(SICI)1520-667X(2000)12:1<1::AID-MCS1>3.0.CO;2-W. DOI

Moravcová D., Kahle V., Rehulková H., Chmelík J., Rehulka P. Short monolithic columns for purification and fractionation of peptide samples for matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry analysis in proteomics. J. Chromatogr. A. 2009;1216:3629–3636. doi: 10.1016/j.chroma.2009.01.075. PubMed DOI

Franc V., Řehulka P., Medda R., Padiglia A., Floris G., Šebela M. Analysis of the glycosylation pattern of plant copper amine oxidases by MALDI-TOF/TOF MS coupled to a manual chromatographic separation of glycans and glycopeptides. Electrophoresis. 2013;34:2357–2367. doi: 10.1002/elps.201200622. PubMed DOI

Franc V., Řehulka P., Raus M., Stulík J., Novak J., Renfrow M.B., Šebela M. Elucidating heterogeneity of IgA1 hinge-region O-glycosylation by use of MALDI-TOF/TOF mass spectrometry: role of cysteine alkylation during sample processing. J. Proteom. 2013;92:299–312. doi: 10.1016/j.jprot.2013.07.013. PubMed DOI PMC

Franc V., Sebela M., Rehulka P., Končitíková R., Lenobel R., Madzak C., Kopečný D. Analysis of N-glycosylation in maize cytokinin oxidase/dehydrogenase 1 using a manual microgradient chromatographic separation coupled offline to MALDI-TOF/TOF mass spectrometry. J. Proteom. 2012;75:4027–4037. doi: 10.1016/j.jprot.2012.05.013. PubMed DOI

Rehulka P., Zahradnikova M., Rehulkova H., Dvorakova P., Nenutil R., Valik D., Vojtesek B., Hernychova L., Novotny M.V. Microgradient separation technique for purification and fractionation of permethylated N-glycans before mass spectrometric analyses. J. Sep. Sci. 2018;41:1973–1982. doi: 10.1002/jssc.201701339. PubMed DOI

Lenobel R., Rehulkova H., Sebela M., Franc V., Kahle V., Moravcova D., Rehulka P. Analysis of peptide mixtures for proteomics research using LC–ESI-MS with a simple microgradient device. LC GC N. Am. 2015;33:420–428.

Yang F., Shen Y., Camp D.G., Smith R.D. High-pH reversed-phase chromatography with fraction concatenation for 2D proteomic analysis. Expert Rev. Proteom. 2012;9:129–134. doi: 10.1586/epr.12.15. PubMed DOI PMC

Batth T.S., Francavilla C., Olsen J.V. Off-line high-pH reversed-phase fractionation for in-depth phosphoproteomics. J. Proteome Res. 2014;13:6176–6186. doi: 10.1021/pr500893m. PubMed DOI

Matsuda H., Nakamura H., Nakajima T. New ceramic titania: selective adsorbent for organic phosphates. Anal. Sci. 1990;6:911–912. doi: 10.2116/analsci.6.911. DOI

Wang H., Chang-Wong T., Tang H.-Y., Speicher D.W. Comparison of extensive protein fractionation and repetitive LC-MS/MS analyses on depth of analysis for complex proteomes. J. Proteome Res. 2010;9:1032–1040. doi: 10.1021/pr900927y. PubMed DOI PMC

Yeh T.-T., Ho M.-Y., Chen W.-Y., Hsu Y.-C., Ku W.-C., Tseng H.-W., Chen S.-T., Chen S.-F. Comparison of different fractionation strategies for in-depth phosphoproteomics by liquid chromatography tandem mass spectrometry. Anal. Bioanal. Chem. 2019;411:3417–3424. doi: 10.1007/s00216-019-01823-0. PubMed DOI

McNulty D.E., Annan R.S. Hydrophilic interaction chromatography reduces the complexity of the phosphoproteome and improves global phosphopeptide isolation and detection. Mol. Cell. Proteom. 2008;7:971–980. doi: 10.1074/mcp.M700543-MCP200. PubMed DOI

Chien K.-Y., Liu H.-C., Goshe M.B. Development and application of a phosphoproteomic method using electrostatic repulsion-hydrophilic interaction chromatography (ERLIC), IMAC, and LC-MS/MS analysis to study Marek’s Disease Virus infection. J. Proteome Res. 2011;10:4041–4053. doi: 10.1021/pr2002403. PubMed DOI

Ritorto M.S., Cook K., Tyagi K., Pedrioli P.G.A., Trost M. Hydrophilic strong anion exchange (hSAX) chromatography for highly orthogonal peptide separation of complex proteomes. J. Proteome Res. 2013;12:2449–2457. doi: 10.1021/pr301011r. PubMed DOI PMC

Mertins P., Yang F., Liu T., Mani D.R., Petyuk V.A., Gillette M.A., Clauser K.R., Qiao J.W., Gritsenko M.A., Moore R.J., et al. Ischemia in tumors induces early and sustained phosphorylation changes in stress kinase pathways but does not affect global protein levels. Mol. Cell. Proteom. 2014;13:1690–1704. doi: 10.1074/mcp.M113.036392. PubMed DOI PMC

Mertins P., Qiao J.W., Patel J., Udeshi N.D., Clauser K.R., Mani D.R., Burgess M.W., Gillette M.A., Jaffe J.D., Carr S.A. Integrated proteomic analysis of post-translational modifications by serial enrichment. Nat. Methods. 2013;10:634–637. doi: 10.1038/nmeth.2518. PubMed DOI PMC

Batth T.S., Olsen J.V. Offline high pH reversed-phase peptide fractionation for deep phosphoproteome coverage. Methods Mol. Biol. 2016;1355:179–192. doi: 10.1007/978-1-4939-3049-4_12. PubMed DOI

Snyder L.R., Kirkland J.J., Dolan J.W. Introduction to Modern Liquid Chromatography. John Wiley & Sons, Ltd.; Hoboken, NJ, USA: 2010. Ionic samples: Reversed-phase, ion-pair, and ion-exchange chromatography; pp. 303–360.

Kemp B.E. Relative alkali stability of some peptide o-phosphoserine and o-phosphothreonine esters. FEBS Lett. 1980;110:308–312. doi: 10.1016/0014-5793(80)80099-8. PubMed DOI

Sickmann A., Meyer H.E. Phosphoamino acid analysis. Proteomics. 2001;1:200–206. doi: 10.1002/1615-9861(200102)1:2<200::AID-PROT200>3.0.CO;2-V. PubMed DOI

Kahle V., Vázlerová M., Welsch T. Automated microgradient system for capillary electrochromatography. J. Chromatogr. A. 2003;990:3–9. doi: 10.1016/S0021-9673(02)01806-X. PubMed DOI

Potel C.M., Lin M.-H., Heck A.J.R., Lemeer S. Defeating major contaminants in Fe3+- immobilized metal ion affinity chromatography (IMAC) phosphopeptide enrichment. Mol. Cell. Proteom. 2018;17:1028–1034. doi: 10.1074/mcp.TIR117.000518. PubMed DOI PMC

Yeung Y.-G., Stanley E.R. Rapid detergent removal from peptide samples with ethyl acetate for mass spectrometry analysis. Curr. Protoc. Protein Sci. 2010;59 doi: 10.1002/0471140864.ps1612s59. PubMed DOI PMC

Larsen M.R., Thingholm T.E., Jensen O.N., Roepstorff P., Jørgensen T.J.D. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol. Cell. Proteom. 2005;4:873–886. doi: 10.1074/mcp.T500007-MCP200. PubMed DOI

R Development Core Team . R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing; Vienna, Austria: 2009. [(accessed on 25 May 2020)]. Available online: https://www.R-project.org/