Human Serum and Plasma Proteomics

Ian Musselman1, David W. Speicher1

1 The Wistar Institute, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 24.1
DOI:  10.1002/0471140864.ps2401s42
Online Posting Date:  December, 2005
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Abstract

This unit focuses on several related methods for profiling and identifying proteins in human serum or plasma. While the methods are specifically described for human serum and plasma, with the possible exception of the major protein depletion steps, they can be readily adapted to serum and plasma from other species as well as to other biological fluids. A very powerful multidimensional protein‐profiling method is described that utilizes major protein depletion coupled to two other protein separations, MicroSol IEF fractionation and SDS‐PAGE, followed by LC‐MS/MS. Other protein‐profiling strategies include the depletion of abundant proteins followed by analysis either via 2‐D gels or using offline Multidimensional Protein Identification Technology (MudPIT) or LC/LC‐MS/MS. Additional protein profiling methods and strategies are also discussed.

Keywords: proteomics; fractionation; serum; plasma; protein profiling; MudPIT; protein depletion

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Multidimensional Protein and Peptide Separation
  • Alternate Protocol 1: Major Protein Depletion Followed by 2‐D Gel Analysis
  • Alternate Protocol 2: Major Protein Depletion Followed by Offline Multidimensional Protein Identification Technology (MudPIT)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Multidimensional Protein and Peptide Separation

  Materials
  • Human serum or plasma sample
  • 1× Agilent MARS Buffer A
  • 1× Agilent MARS Buffer B
  • Human serum or plasma sample
  • Isopropanol
  • 1 M NaOH
  • 10 mM sodium phosphate buffer, pH 7.0 ( appendix 2E)
  • 1 M Tris⋅Cl, pH 8.3 ( appendix 2E)
  • Urea
  • Dithiothreitol (DTT)
  • Dimethylacrylamide
  • Thiourea
  • 3‐[(3‐cholamidopropyl)‐dimethylammonio]‐1‐propanesulfonate (CHAPS)
  • IPG buffer (carrier amphoytes, pH 3 to 10 L; Amersham Biosciences)
  • 200 mM ammonium bicarbonate in 50% (v/v) acetonitrile
  • 0.02 µg/µl modified trypsin (Promega) in 40 mM ammonium bicarbonate/10% acetonitrile, pH 8.1
  • Argon or nitrogen source
  • 40 mM ammonium bicarbonate in 10% (v/v) acetonitrile, pH 8.1
  • Concentrated acetic acid
  • Solvent A: 0.58% (v/v) acetic acid in Milli‐Q or HPLC‐grade H 2O
  • Solvent B: 0.58% acetic acid in acetonitrile
  • HPLC system (unit 8.7)
  • 1× Agilent MARS column, 4.6 × 50–mm or 4.6 × 100–mm
  • 0.22‐µm spin filters
  • Microcentrifuge
  • 5‐kDa MWCO spin separators (Amicon Ultra 4; Millipore)
  • Gel‐cutting device (e.g., MEF‐1.5 Gel Cutter; The Gel Company, http://www.gelcompany.com/)
  • Digestion tubes: microcentrifuge tubes precleaned by rinsing several times in 0.1% trifluoroacetic acid/50% acetonitrile
  • 37°C shaking water bath
  • 37°C thermostatically controlled oven
  • Reversed‐phase 75 µm i.d. × 15 cm length PicoFrit column (New Objective, Inc.), packed with MAGIC C18 resin, 5 µm particle size (Michrom BioResources)
  • Linear ion‐trap mass spectrometer with microscale autosampler and injection loop (also see Chapter 16)
  • MS/MS data analysis software (e.g., Sequest; http://fields.scripps.edu/sequest/).
  • Additional reagents and equipment for HPLC (unit 8.7), SDS‐PAGE (unit 10.1), protein assay (unit 3.4), Zoom IEF fractionation (unit 22.6), running Zoom IEF fractions on 1‐D minigels (unit 22.6), staining with colloidal Coomassie blue (unit 10.5), and RP‐HPLC (unit 8.7), and mass spectrometry (Chapter 16)

Alternate Protocol 1: Major Protein Depletion Followed by 2‐D Gel Analysis

  • 1.1× IEF sample buffer (see recipe)
  • Equilibration buffer I: add 463 mg, dithiothreitol (DTT) to 100 ml equilibration buffer base solution (see recipe) immediately before use
  • Equilibration buffer II: add 4.37 g iodoacetamide to 100 ml equilibration buffer base solution (see recipe) immediately before use
  • 24‐cm pH 3‐10 L IPG strips (Amersham Biosciences)
  • 25 × 20 cm, 1 mm thick 10% Tris⋅glycine polyacrylamide gels (unit 10.4)
  • Gel‐image analysis software (e.g., Melanie 4; GeneBio)
  • Additional reagents and equipment for 2‐D electrophoresis using immobilized pH gradient gel strips (unit 10.4, Basic Protocol 2) and staining of gels (unit 10.5).

Alternate Protocol 2: Major Protein Depletion Followed by Offline Multidimensional Protein Identification Technology (MudPIT)

  • 50 mM ammonium bicarbonate in 10% (v/v) acetonitrile, pH 8.1
  • Modified trypsin (Promega)
  • Additional reagents and equipment for SCX and RP‐LC‐MS/MS (unit 23.1, Basic Protocol 1)
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Figures

Videos

Literature Cited

   Anderson, N.L. and Anderson, N.G. 2002. The human plasma proteome: History, character, and diagnostic prospects. Mol. Cell. Proteomics 1:845‐867.
   Anderson, N.L., Polanski, M., Pieper, R., Gatlin, T., Tirumalai, R.S., Conrads, T.P., Veenstra, T.D., Adkins, J.N., Pounds, J.G., Fagan, R., and Lobley, A. 2004. The human plasma proteome: A nonredundant list developed by combination of four separate sources. Mol. Cell. Proteomics 3:311‐326.
   Chan, K.C., Lucas, D.A., Hise, D., Schaefer, C.F., Xiao, Z., Janini, G.M., Buetow, K.H., Issaq, H.J., Veenstra, T.D., and Conrads, T.P. 2004. Analysis of the human serum proteome. Clinical Proteomics 1:101‐226.
   Check, E. 2004. Proteomics and cancer: Running before we can walk? Nature 429:496‐497.
   Diamandis, E.P. 2004. Mass spectrometry as a diagnostic and a cancer biomarker discovery tool: Opportunities and potential limitations. Mol. Cell. Proteomics 3:367‐378.
   Echan, L.A., Tang, H.Y., Ali‐Khan, N., Lee, K., and Speicher, D.W. 2005. Depletion of multiple high abundance proteins improves protein profiling capacities of human serum and plasma. Proteomics 5:3292‐3303.
   Link, A.J., Eng, J., Schieltz, D.M., Carmack, E., Mize, G.J., Morris, D.R., Garvik, B.M., and Yates, J.R. 3rd. 1999. Direct analysis of protein complexes using mass spectrometry. Nat. Biotechnol. 17:676‐682.
   Marshall, J., Kupchak, P., Zhu, W., Yantha, J., Vrees, T., Furesz, S., Jacks, K., Smith, C., Kireeva, I., Zhang, R., Takahashi, M., Stanton, E., and Jackowski, G. 2003. Processing of serum proteins underlies the mass spectral fingerprinting of myocardial infarction. J. Proteome. Res. 2:361‐372.
   Misek, D.E., Kuick, R., Wang, H., Galchev, V., Deng, B., Zhao, R., Tra, J., Pisano, M.R., Amunugama, R., Allen, D., Walker, A.K., Strahler, J.R., Andrews, P., Omenn, G.S., and Hanash, S.M. 2005. A wide range of protein isoforms in serum and plasma uncovered by a quantitative intact protein analysis system. Proteomics 5:3343‐3352.
   Omenn, G.S., States, D.J., Adamski, M., Blackwell, T.W., Menon, R., Hermjakob, H., Apweiler, R., Haab, B.B., Simpson, R.J., Eddes, J.S., Kapp, E.A., Moritz, R.L., Chan, D.W., Rai, A.J., Admon, A., Aebersold, R., Eng, J., Hancock, W.S., Hefta, S.A., Meyer, H., Paik, Y.K., Yoo, J.S., Ping, P., Pounds, J., Adkins, J., Qian, X., Wang, R., Wasinger, V., Wu, C.Y., Zhao, X., Zeng, R., Archakov, A., Tsugita, A., Beer, I., Pandey, A., Pisano, M., Andrews, P., Tammen, H., Speicher, D.W., and Hanash, S.M. 2005. Overview of the HUPO Proteome Project: Results from the pilot phase with 35 collaborating laboratories and multiple analytical groups, generating a core dataset of 3020 proteins and a publically available database. Proteomics 5:3226‐3245.
   Petricoin, E.F., Ardekani, A.M., Hitt, B.A., Levine, P.J., Fusaro, V.A., Steinberg, S.M., Mills, G.B., Simone, C., Fishman, D.A., Kohn, E.C., and Liotta, L.A. 2002. Use of proteomic patterns in serum to identify ovarian cancer. Lancet 359:572‐577.
   Rai, A.J., Gelfand, C.A., Haywood, B.C., Warunek, D.J., Yi, J., Schuchard, M.D., Mehigh, R.J., Cockrill, S.L., Scott, G.B., Tammen, H., Schulz‐Knappe, P., Speicher, D.W., Vitzthum, F., Haab, B.B., Siest, G., and Chan, D.W. 2005. HUPO plasma proteome project specimen collection and handling: Towards the standardization of parameters for plasma proteome samples. Proteomics. 5:3262‐3277.
   Schuchard, M.D., Mehigh, R.J., Cockrill, S.L., Lipscomb, G.T., Stephan, J.D., Wildsmith, J., Valdes‐Camin, R., Kappel, W.K., Rai, A.J., and Scott, G.B. 2005. Artifactual isoform profile modification following treatment of human plasma or serum with protease inhibitor, monitored by 2‐dimensional electrophoresis and mass spectrometry. Biotechniques. 39:239‐247.
   Shen, Y., Jacobs, J.M., Camp, D.G. 2nd, Fang, R., Moore, R.J., Smith, R.D., Xiao, W., Davis, R.W., and Tompkins, R.G. 2004. Ultra‐high‐efficiency strong cation exchange LC/RPLC/MS/MS for high dynamic range characterization of the human plasma proteome. Anal. Chem. 76:1134‐1144.
   Tammen, H., Schulte, I., Hess, R., Menzel, C., Kellmann, M., Mohring, T., and Schulz‐Knappe, P. 2005. Peptidomic analysis of human blood specimens: Comparison between plasma specimens and serum by differential peptide display. Proteomics 5:3414‐3422.
   Tang, H.Y., Ali‐Khan, N., Echan, L.A., Levenkova, N., Rux, J.J., and Speicher, D.W. 2005. A novel four‐dimensional strategy combing protein and peptide separation methods enables detection of low‐abundance proteins in human plasma and serum proteomes. Proteomics. In press.
   Tirumalai, R.S., Chan, K.C., Prieto, D.A., Issaq, H.J., Conrads, T.P., and Veenstra, T.D. 2003. Characterization of the low molecular weight human serum proteome. Mol Cell. Proteomics. 2:1096‐1103.
   Villanueva, J., Philip, J., Entenberg, D., Chaparro, C.A., Tanwar, M.K., Holland, E.C., and Tempst, P. 2004. Serum peptide profiling by magnetic particle‐assisted, automated sample processing and MALDI‐TOF mass spectrometry. Anal. Chem. 76:1560‐1570.
Internet Resources
   http://www.specialtylabs.com
  Specialty Laboratories publications, Santa Monica, Ca. August 10, 2005. This web site provided the information shown in Figure . Searching the site for specific proteins will provide information on abundance ranges.
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