Advertisement

Preparation and Immunoaffinity Depletion of Fresh Frozen Tissue Homogenates for Mass Spectrometry-Based Proteomics in the Context of Drug Target/Biomarker Discovery

  • DaRue A. Prieto
  • King C. Chan
  • Donald J. JohannJr
  • Xiaoying Ye
  • Gordon Whitely
  • Josip BlonderEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1647)

Abstract

The discovery of novel drug targets and biomarkers via mass spectrometry (MS)-based proteomic analysis of clinical specimens has proven to be challenging. The wide dynamic range of protein concentration in clinical specimens and the high background/noise originating from highly abundant proteins in tissue homogenates and serum/plasma encompass two major analytical obstacles. Immunoaffinity depletion of highly abundant blood-derived proteins from serum/plasma is a well-established approach adopted by numerous researchers; however, the utilization of this technique for immunodepletion of tissue homogenates obtained from fresh frozen clinical specimens is lacking. We first developed immunoaffinity depletion of highly abundant blood-derived proteins from tissue homogenates, using renal cell carcinoma as a model disease, and followed this study by applying it to different tissue types. Tissue homogenate immunoaffinity depletion of highly abundant proteins may be equally important as is the recognized need for depletion of serum/plasma, enabling more sensitive MS-based discovery of novel drug targets, and/or clinical biomarkers from complex clinical samples. Provided is a detailed protocol designed to guide the researcher through the preparation and immunoaffinity depletion of fresh frozen tissue homogenates for two-dimensional liquid chromatography, tandem mass spectrometry (2D-LC-MS/MS)-based molecular profiling of tissue specimens in the context of drug target and/or biomarker discovery.

Key words

Immunoaffinity depletion Fresh frozen tissue Drug discovery Biomarker discovery Clinical proteomics Mass spectrometry (MS)- based proteomics 2D-LC-MS/MS 

Notes

Acknowledgment

“This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.”

References

  1. 1.
    Johann DJ Jr, Blonder J (2007) Biomarker discovery: tissues versus fluids versus both. Expert Rev Mol Diagn 7(5):473–475CrossRefPubMedGoogle Scholar
  2. 2.
    Puangpila C, Mayadunne E, Rassi SE (2015) Liquid-phase-based separation systems for depletion, pre-fractionation and enrichment of proteins in biological fluids and matrices for in-depth proteomics analysis–an update covering the period 2011–2014. Electrophoresis 36(1):238–252CrossRefPubMedGoogle Scholar
  3. 3.
    Bjorhall K, Miliotis T, Davidson P (2005) Comparison of different depletion strategies for improved resolution in proteomic analysis of human serum samples. Proteomics 5(1):307–317CrossRefPubMedGoogle Scholar
  4. 4.
    Faca V, Pitteri SJ, Newcomb L et al (2007) Contribution of protein fractionation to depth of analysis of the serum and plasma proteomes. J Proteome Res 6(9):3558–3565CrossRefPubMedGoogle Scholar
  5. 5.
    Yadav AK, Bhardwaj G, Basak T et al (2011) A systematic analysis of eluted fraction of plasma post immunoaffinity depletion: implications in biomarker discovery. PLoS One 6(9):e24442CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Huang Z, Yan G, Gao M et al (2016) Array-based online two dimensional liquid chromatography system applied to effective depletion of high-abundance proteins in human plasma. Anal Chem 88(4):2440–2445CrossRefPubMedGoogle Scholar
  7. 7.
    Borg J, Campos A, Diema C et al (2011) Spectral counting assessment of protein dynamic range in cerebrospinal fluid following depletion with plasma-designed immunoaffinity columns. Clin Proteomics 8:6–14CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Cheon DH, Nam EJ, Park KH et al (2016) Comprehensive analysis of low-molecular-weight human plasma proteome using top-down mass spectrometry. J Proteome Res 5(1):229–244CrossRefGoogle Scholar
  9. 9.
    Fountoulakis M, Juranville JF, Jiang L et al (2004) Depletion of the high-abundance plasma proteins. Amino Acids 27(3–4):249–259CrossRefPubMedGoogle Scholar
  10. 10.
    Martosella J, Zolotarjova N, Liu H et al (2005) Reversed-phase high-performance liquid chromatographic pre-fractionation of immunodepleted human serum proteins to enhance mass spectrometry identification of lower-abundant proteins. J Proteome Res 4(5):1522–1537CrossRefPubMedGoogle Scholar
  11. 11.
    Hyung SW, Piehowski PD, Moore RJ et al (2014) Microscale depletion of high abundance proteins in human biofluids using IgY14 immunoaffinity resin: analysis of human plasma and cerebrospinal fluid. Anal Bioanal Chem 406:7117–7125CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Galindo AN, Kussman M, Dayon L (2015) Proteomics of cerebrospinal fluid: throughput and robustness using a scalable automated analysis pipeline for biomarker discovery. Anal Chem 87(21):10755–10761CrossRefGoogle Scholar
  13. 13.
    Cellar NA, Karnoup AS, Albers DR (2009) Immunodepletion of high abundance proteins coupled on-line with reversed-phase liquid chromatography: a two-dimensional LC sample enrichment and fractionation technique for mammalian proteomics. J Chromatogr B Analyt Technol Biomed Life Sci 877(1–2):79–85CrossRefPubMedGoogle Scholar
  14. 14.
    Bellei E, Bergamini S, Monari E et al (2011) High-abundance protein depletion for serum proteomic analysis: concomitant removal of non-targeted proteins. Amino Acids 40:145–156CrossRefPubMedGoogle Scholar
  15. 15.
    Millioni R, Tolin S, Puricelli L et al (2011) High abundance proteins depletion vs low abundance proteins enrichment: comparison of methods to reduce the plasma proteome complexity. PLoS One 6(5):e19603CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Janecki DJ, Pomerantz SC, Beil EJ et al (2012) A fully integrated multi-column system for abundant protein depletion from serum/plasma. J Chromatogr B Analyt Technol Biomed Life Sci 902:35–41CrossRefPubMedGoogle Scholar
  17. 17.
    Kullolli M, Warren J, Arampatzidou M et al (2013) Performance evaluation of affinity ligands for depletion of abundant plasma proteins. J Chromatogr B Analyt Technol Biomed Life Sci 939:10–16CrossRefPubMedGoogle Scholar
  18. 18.
    Johann DJ, Wei BR, Prieto DA et al (2010) Combined blood/tissue analysis for cancer biomarker discovery: application to renal cell carcinoma. Anal Chem 82(5):1584–1588CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Prieto DA, Johann DJ Jr, Wei BR et al (2014) Mass spectrometry in cancer biomarker research: a case for immunodepletion of abundant blood-derived proteins from clinical tissue specimens. Biomark Med 8(2):269–286CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Blonder J, Chan KC, Issaq HJ et al (2006) Identification of membrane proteins from mammalian cell/tissue using methanol-facilitated solubilization and tryptic digestion coupled with 2D-LC-MS/MS. Nat Protoc 1(6):2784–2790CrossRefPubMedGoogle Scholar
  21. 21.
    Tran JC, Doucette A (2008) Gel-eluted liquid fraction entrapment electrophoresis: an electrophoretic method for broad molecular weight range proteome separation. Anal Chem 80:1568–1573CrossRefPubMedGoogle Scholar
  22. 22.
    Slebos RJ, Brock JW, Winters NF et al (2008) Evaluation of strong cation exchange versus isoelectric focusing of peptides for multidimensional liquid chromatography-tandem mass spectrometry. J Proteome Res 12:5286–5294CrossRefGoogle Scholar
  23. 23.
    Dzieciatkowska M, Hill R, Hansen KC (2014) GeLC-MS/MS analysis of complex protein mixtures. Methods Mol Biol 1156:53–66CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Gundry RL, White MY, Murray CI et al (2010) Preparation of proteins and peptides for mass spectrometry analysis in a bottom-up proteomics workflow. Curr Protoc Mol Biol Chapter 10: Unit 10.25Google Scholar
  25. 25.
    Shevchenko A, Tomas H, Havlis J et al (2006) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1(6):2856–2860CrossRefPubMedGoogle Scholar
  26. 26.
    Giansanti P, Tsiatsiani L, Low TY et al (2016) Six alternative proteases for mass spectrometry-based proteomics beyond trypsin. Nat Protoc 11(5):993–1006CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • DaRue A. Prieto
    • 1
  • King C. Chan
    • 1
  • Donald J. JohannJr
    • 2
  • Xiaoying Ye
    • 3
  • Gordon Whitely
    • 3
  • Josip Blonder
    • 3
    Email author
  1. 1.Cancer Research Technology Program, Protein Characterization LaboratoryLeidos Biomedical Research Inc., Frederick National Laboratory for Cancer ResearchFrederickUSA
  2. 2.Wintrop P Rockefeller Cancer Institute, University of Arkansas for Medical SciencesLittle RockUSA
  3. 3.Cancer Research Technology Program, Antibody Characterization LaboratoryLeidos Biomedical Research Inc., Frederick National Laboratory for Cancer ResearchFrederickUSA

Personalised recommendations