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
Part of the Methods in Molecular Biology book series (MIMB, volume 1647)


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 



“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.”


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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

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