Mass Spectrometry‐Based Bottom‐Up Proteomics: Sample Preparation, LC‐MS/MS Analysis, and Database Query Strategies

Matthew J. Wither1, Kirk C. Hansen1, Julie A. Reisz1

1 Biological Mass Spectrometry Core, Department of Biochemistry and Molecular Genetics, University of Colorado Denver, Aurora
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 16.4
DOI:  10.1002/cpps.18
Online Posting Date:  November, 2016
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Recent technological advances in mass spectrometry (MS) have made possible the investigation and quantification of complex mixtures of biomolecules. The exceptional sensitivity and resolving power of today's mass spectrometers allow for the detection of proteins and peptides at low femtomole quantities; however, these attributes demand high sample purity to minimize artifacts and achieve the highest degree of biomolecule identification. Tissue preparation for proteomic studies is particularly challenging due to their heterogeneity in cell type, presence of insoluble biomaterials, and wide diversity of biomolecules. The workflow described herein details sample preparation from tissues through protein extraction, proteolysis, and purification to generate peptides for MS analysis. Increased peptide resolution and a corresponding increase in protein identification is accomplished using polarity‐based fractionation (C18 resin) at the peptide level. Additionally, approaches to instrument set up, including the use of nanoscale liquid chromatography and quadrupole Orbitrap MS, along with database searching, are described. © 2016 by John Wiley & Sons, Inc.

Keywords: LC‐MS/MS; mass spectrometry; peptide sequencing; proteomics; fractionation

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Sample Preparation and nanoLC‐MS/MS Analysis
  • Alternate Protocol 1: nanoLC‐MS Parameters for Thermo LTQ Orbitrap Velos Pro AND Eksigent nanoLC Ultra 2D
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Sample Preparation and nanoLC‐MS/MS Analysis

  Materials
  • Samples/tissues of interest: 3 to 10 mg of dry organ or tissue is generally sufficient; use 2 to 3 times this amount when using wet weight
  • Liquid nitrogen
  • Extraction buffer (see recipe)
  • Protease Inhibitor (e.g., ProteaseArrest; G‐Biosciences, cat. no. 786‐108 or Halt Protease Inhibitor Cocktail, Thermo Scientific, cat. no. 87786]
  • Pierce BCA Protein Assay kit (ThermoFisher Scientific, cat. no. 23225) or Sigma‐Aldrich BCA kit (Sigma‐Aldrich, cat. no. BCA‐1)
  • Urea buffer (see recipe)
  • Dithiothreitol (DTT) solution (see recipe)
  • Iodoacetamide (IAM) solution (see recipe)
  • Ammonium bicarbonate (NH 4HCO 3)
  • Trypsin solution (see recipe)
  • Double‐distilled water
  • Ammonium hydroxide (NH 4OH)
  • Acetonitrile, Optima LC‐MS (Fisher Scientific, cat. no. A955‐4)
  • High‐purity water, such as double‐distilled or commercial (HPLC grade)
  • Acetonitrile with 0.1% formic acid (Optima LC‐MS; Fisher Scientific, cat. no. LS120‐4)
  • Water with 0.1% formic acid (Optima LC‐MS; Fisher Scientific)
  • Ceramic mortar and pestle
  • Analytical Balance (Sartorius, Entris 64‐1 S)
  • 1‐mm glass beads (Next Advance)
  • Bead Beater (Next Advance, Bullet Blender Storm 24)
  • Benchtop centrifuge capable of 14,000 × g (Eppendorf 5424 w/24 place rotor)
  • 10 kDa molecular‐weight cut‐off (MWCO) filters (Sartorius, Vivacon 500, cat. no. VN01H02)
  • Temperature‐controlled incubator
  • Nano‐LC system
  • C18 Spin Tips (Pierce, Thermo Scientific, cat. no. 84850)
  • Autosampler vials (Phenomenex, cat. no. AR0‐9992‐13)
  • Vacuum centrifuge
IMPORTANT NOTE: Sample contamination by ambient material (particularly keratins) is challenging to avoid in proteomic workflows. Keratins are introduced to samples through skin cells, hair, fingerprints, and dust or lint in the laboratory. To avoid introduction of keratin and other contaminants, wear clean gloves and keep samples, pipet tips, and all reagents covered/capped when not in use.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Aebersold, R. and Mann, M. 2003. Mass spectrometry‐based proteomics. Nature 422:198‐207. doi: 10.1038/nature01511.
  Ahn, Y.H., Kim, J.Y., and Yoo, J.S. 2015. Quantitative mass spectrometric analysis of glycoproteins combined with enrichment methods. Mass Spectrom. Rev. 34:148‐165. doi: 10.1002/mas.21428.
  Baez, N.O.D., Reisz, J.A., and Furdui, C.M. 2015. Mass spectrometry in studies of protein thiol chemistry and signaling: Opportunities and caveats. Free Radic. Biol. Med. 80:191‐211. doi: 10.1016/j.freeradbiomed.2014.09.016.
  Baker, P.R., Trinidad, J.C., and Chalkley, R.J. 2011. Modification site localization scoring integrated into a search engine. Mol. Cell Proteomics 10:M111.008078. doi: 10.1074/mcp.M111.008078.
  Bern, M., Kil, Y.J., and Becker, C. 2012. Byonic: Advanced peptide and protein identification software. Curr. Protoc. Bioinform. 40:13.20.1‐13.20.14. doi: 10.1002/0471250953.bi1320s40.
  Cagney, G., Amiri, S., Premawaradena, T., Lindo, M., and Emili, A. 2003. In silico proteome analysis to facilitate proteomics experiments using mass spectrometry. Proteome Sci. 1:5. doi: 10.1186/1477‐5956‐1‐5.
  Camerini, S. and Mauri, P. 2015. The role of protein and peptide separation before mass spectrometry analysis in clinical proteomics. J. Chromatogr. A 1381:1‐12. doi: 10.1016/j.chroma.2014.12.035.
  Chahrour, O., Cobice, D., and Malone, J. 2015. Stable isotope labelling methods in mass spectrometry‐based quantitative proteomics. J. Pharm. Biomed. Anal. 113:2‐20. doi: 10.1016/j.jpba.2015.04.013.
  Chambers, M.C., Maclean, B., Burke, R., Amodei, D., Ruderman, D.L., Neumann, S., Gatto, L., Fischer, B., Pratt, B., Egertson, J., Hoff, K., Kessner, D., Tasman, N., Shulman, N., Frewen, B., Baker, T.A., Brusniak, M.Y., Paulse, C., Creasy, D., Flashner, L., Kani, K., Moulding, C., Seymour, S.L., Nuwaysir, L.M., Lefebvre, B., Kuhlmann, F., Roark, J., Rainer, P., Detlev, S., Hemenway, T., Huhmer, A., Langridge, J., Connolly, B., Chadick, T., Holly, K., Eckels, J., Deutsch, E.W., Moritz, R.L., Katz, J.E., Agus, D.B., MacCoss, M., Tabb, D.L., and Mallick, P. 2012. A cross‐platform toolkit for mass spectrometry and proteomics. Nat. Biotechnol. 30:918‐920. doi: 10.1038/nbt.2377.
  Clark, D.J., Fondrie, W.E., Liao, Z., Hanson, P.I., Fulton, A., Mao, L., and Yang, A.J. 2015. Redefining the breast cancer exosome proteome by tandem mass tag quantitative proteomics and multivariate cluster analysis. Anal. Chem. 87:10462‐10469. doi: 10.1021/acs.analchem.5b02586. doi: 10.1021/acs.analchem.5b02586.
  Craig, R., Cortens, J.P., and Beavis, R.C. 2004. Open source system for analyzing, validating, and storing protein identification data. J. Proteome Res. 3:1234‐1242. doi: 10.1021/pr049882h.
  D'Alessandro, A., D'Amici, G.M., Vaglio, S., and Zolla, L. 2012. Time‐course investigation of SAGM‐stored leukocyte‐filtered red bood cell concentrates: From metabolism to proteomics. Haematologica 97:107‐115. doi: 10.3324/haematol.2011.051789.
  Deutsch, E.W., Lam, H., and Aebersold, R. 2008. PeptideAtlas: A resource for target selection for emerging targeted proteomics workflows. EMBO Rep. 9:429‐434. doi: 10.1038/embor.2008.56.
  Dzieciatkowska, M., Hill, R., and Hansen, K.C. 2014. GeLC‐MS/MS analysis of complex protein mixtures. Methods Mol. Biol. 1156:53‐66. doi: 10.1007/978‐1‐4939‐0685‐7_4.
  Dzieciatkowska, M., Wohlauer, M.V., Moore, E.E., Damle, S., Peltz, E., Campsen, J., Kelher, M., Silliman, C., Banerjee, A., and Hansen, K.C. 2011. Proteomic analysis of human mesenteric lymph. Shock 35:331‐338. doi: 10.1097/SHK.0b013e318206f654.
  Eliuk, S. and Makarov, A. 2015. Evolution of orbitrap mass spectrometry instrumentation. Annu. Rev. Anal. Chem. 8:61‐80. doi: 10.1146/annurev‐anchem‐071114‐040325.
  Ford, K.L., Zeng, W., Heazlewood, J.L., and Bacic, A. 2015. Characterization of protein N‐glycosylation by tandem mass spectrometry using complementary fragmentation techniques. Front. Plant Sci. 6:674. doi: 10.3389/fpls.2015.00674.
  Gallien, S., Kim, S.Y., and Domon, B. 2015. Large‐scale targeted proteomics using internal standard triggered‐parallel reaction monitoring (IS‐PRM). Mol. Cell. Proteomics 14:1630‐1644. doi: 10.1074/mcp.O114.043968.
  Grundner‐Culemann, K., Dybowski, J.N., Klammer, M., Tebbe, A., Schaab, C., and Daub, H. 2016. Comparative proteome analysis across non‐small cell lung cancer cell lines. J. Proteomics 130:1‐10. doi: 10.1016/j.jprot.2015.09.003.
  Hill, R.C., Calle, E.A., Dzieciatkowska, M., Niklason, L.E., and Hansen, K.C. 2015a. Quantification of extracellular matrix proteins from a rat lung scaffold to provide a molecular readout for tissue engineering. Mol. Cell. Proteomics 14:961‐973. doi: 10.1074/mcp.M114.045260.
  Hill, R.C., Wither, M.J., Nemkov, T., Barrett, A., D'Alessandro, A., Dzieciatkowska, M., and Hansen, K.C. 2015b. Preserved proteins from extinct bison latifrons identified by tandem mass spectrometry; Hydroxylysine glycosides are a common feature of ancient collagen. Mol. Cell. Proteomics 14:1946‐1958. doi: 10.1074/mcp.M114.047787.
  Isasa, M., Rose, C.M., Elsasser, S., Navarrete‐Perea, J., Paulo, J.A., Finley, D.J., and Gygi, S.P. 2015. Multiplexed, proteome‐wide protein expression profiling: Yeast deubiquitylating enzyme knockout strains. J. Proteome Res. 14:5306‐5317. doi: 10.1021/acs.jproteome.5b00802.
  Lennicke, C., Rahn, J., Heimer, N., Lichtenfels, R., Wessjohann, L.A., and Seliger, B. 2016. Redox proteomics: Methods for the identification and enrichment of redox‐modified proteins and their applications. Proteomics 16:197‐213. doi: 10.1002/pmic.201500268.
  Mallet, C.R., Lu, Z., and Mazzeo, J.R. 2004. A study of ion suppression effects in electrospray ionization from mobile phase additives and solid‐phase extracts. Rapid Commun. Mass Spectrom. 18:49‐58. doi: 10.1002/rcm.1276.
  McClatchy, D.B. and Yates, J.R. 2014. Stable isotope labeling in mammals (SILAM). Methods Mol. Biol. 1156:133‐146. doi: 10.1007/978‐1‐4939‐0685‐7_8.
  Nardelli, S.C., Che, F.‐Y., Silmon de Monerri, N.C., Xiao, H., Nieves, E., Madrid‐Aliste, C., Angel, S.O., Sullivan, W.J., Angeletti, R.H., Kim, K., and Weiss, L.M. 2013. The histone code of Toxoplasma gondii comprises conserved and unique posttranslational modifications. MBio 4:e00922‐00913. doi: 10.1128/mBio.00922‐13.
  Olson, B.J. and Markwell, J. 2007. Assays for determination of protein concentration. Curr. Protoc. Protein Sci. 48:3.4.1‐3.4.29. doi: 10.1002/0471140864.ps0304s48.
  Ong, S.‐E., Blagoev, B., Kratchmarova, I., Kristensen, D.B., Steen, H., Pandey, A., and Mann, M. 2002. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell. Proteomics 1:376‐386. doi: 10.1074/mcp.M200025‐MCP200.
  Picotti, P., Lam, H., Campbell, D., Deutsch, E.W., Mirzaei, H., Ranish, J., Domon, B., and Aebersold, R. 2008. A database of mass spectrometric assays for the yeast proteome. Nat. Methods 5:913‐914. doi: 10.1038/nmeth1108‐913.
  Porras‐Yakushi, T.R., Sweredoski, M.J., and Hess, S. 2015. ETD Outperforms CID and HCD in the analysis of the ubiquitylated proteome. J. Am. Soc. Mass Spectrom. 26:1580‐1587. doi: 10.1007/s13361‐015‐1168‐0.
  Rosenthal, F., Nanni, P., Barkow‐Oesterreicher, S., and Hottiger, M.O. 2015. Optimization of LTQ‐Orbitrap mass spectrometer parameters for the identification of ADP‐ribosylation sites. J. Proteome Res. 14:4072‐4079. doi: 10.1021/acs.jproteome.5b00432.
  Ross, P.L., Huang, Y.N., Marchese, J.N., Williamson, B., Parker, K., Hattan, S., Khainovski, N., Pillai, S., Dey, S., Daniels, S., Purkayastha, S., Juhasz, P., Martin, S., Bartlet‐Jones, M., He, F., Jacobson, A., and Pappin, D.J. 2004. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine‐reactive isobaric tagging reagents. Mol. Cell. Proteomics 3:1154‐1169. doi: 10.1074/mcp.M400129‐MCP200.
  Schilling, B., Rardin, M.J., MacLean, B.X., Zawadzka, A.M., Frewen, B.E., Cusack, M.P., Sorensen, D.J., Bereman, M.S., Jing, E., Wu, C.C., Verdin, E., Kahn, C.R., Maccoss, M.J., and Gibson, B.W. 2012. Platform‐independent and label‐free quantitation of proteomic data using MS1 extracted ion chromatograms in skyline: Application to protein acetylation and phosphorylation. Mol. Cell. Proteomics 11:202‐214. doi: 10.1074/mcp.M112.017707.
  Silliman, C.C., Dzieciatkowska, M., Moore, E.E., Kelher, M.R., Banerjee, A., Liang, X., Land, K.J., and Hansen, K.C. 2012. Proteomic analyses of human plasma: Venus versus Mars. Transfusion 52:417‐424. doi: 10.1111/j.1537‐2995.2011.03316.x.
  Ternent, T., Csordas, A., Qi, D., Gómez‐Baena, G., Beynon, R.J., Jones, A.R., Hermjakob, H., and Vizcaíno, J.A. 2014. How to submit MS proteomics data to ProteomeXchange via the PRIDE database. Proteomics 14:2233‐2241. doi: 10.1002/pmic.201400120.
  Thompson, A., Schäfer, J., Kuhn, K., Kienle, S., Schwarz, J., Schmidt, G., Neumann, T., Johnstone, R., Mohammed, A.K.A., and Hamon, C. 2003. Tandem mass tags: A novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Anal. Chem. 75:1895‐1904. doi: 10.1021/ac0262560.
  Trinkle‐Mulcahy, L. 2012. Resolving protein interactions and complexes by affinity purification followed by label‐based quantitative mass spectrometry. Proteomics 12:1623‐1638. doi: 10.1002/pmic.201100438.
  Tyanova, S., Mann, M., and Cox, J. 2014. MaxQuant for in‐depth analysis of large SILAC datasets. Methods Mol. Biol. 1188:351‐364. doi: 10.1007/978‐1‐4939‐1142‐4_24.
  Wang, H., Sun, S., Zhang, Y., Chen, S., Liu, P., and Liu, B. 2015. An off‐line high pH reversed‐phase fractionation and nano‐liquid chromatography‐mass spectrometry method for global proteomic profiling of cell lines. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 974:90‐95. doi: 10.1016/j.jchromb.2014.10.031.
  Wang, Y., Yang, F., Gritsenko, M.A., Wang, Y., Clauss, T., Liu, T., Shen, Y., Monroe, M.E., Lopez‐Ferrer, D., Reno, T., Moore, R.J., Klemke, R.L., Camp, D.G. 2nd, and Smith RD. 2011. Reversed‐phase chromatography with multiple fraction concatenation strategy for proteome profiling of human MCF10A cells. Proteomics 11:2019‐2026. doi: 10.1002/pmic.201000722.
  Williams, A. and Frasca, V. 2001. Ion‐Exchange Chromatography. Curr. Protoc. Protein Sci. 15:8.2:8.2.1‐8.2.30.
  Wiśniewski, J.R., Zougman, A., Nagaraj, N., and Mann, M. 2009. Universal sample preparation method for proteome analysis. Nat. Methods 6:359‐362. doi: 10.1038/nmeth.1322.
  Yang, C., Zhong, X., and Li, L. 2014. Recent advances in enrichment and separation strategies for mass spectrometry‐based phosphoproteomics. Electrophoresis 35:3418‐3429. doi: 10.1002/elps.201400017.
  Yin, H., Tan, Z., Wu, J., Zhu, J., Shedden, K.A., Marrero, J., and Lubman, D.M. 2015. Mass‐selected site‐specific core‐fucosylation of serum proteins in hepatocellular carcinoma. J. Proteome Res. 14:4876‐4884. doi: 10.1021/acs.jproteome.5b00718.
  Yue, Q., Feng, L., Cao, B., Liu, M., Zhang, D., Wu, W., Jiang, B., Yang, M., Liu, X., and Guo, D. 2016. Proteomic analysis revealed the important role of vimentin in human cervical carcinoma HeLa cells treated with gambogic acid. Mol. Cell. Proteomics 15:26‐44. doi: 10.1074/mcp.M115.053272.
  Zhang, G., Annan, R.S., Carr, S.A., and Neubert, T.A. 2014. Overview of Peptide and protein analysis by mass spectrometry. Curr. Protoc. Mol. Biol. 108:10.21.1‐10.21.30. doi: 10.1002/0471140864.ps1601s62.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library