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Proteomic Analysis of Frozen Tissue Samples Using Laser Capture Microdissection

  • Sumana Mukherjee
  • Jaime Rodriguez-Canales
  • Jeffrey Hanson
  • Michael R. Emmert-Buck
  • Michael A. Tangrea
  • DaRue A. Prieto
  • Josip Blonder
  • Donald J. JohannJr.
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1002)

Abstract

The discovery of effective cancer biomarkers is essential for the development of both advanced molecular diagnostics and new therapies/medications. Finding and exploiting useful clinical biomarkers for cancer patients is fundamentally linked to improving outcomes. Towards these aims, the heterogeneous nature of tumors represents a significant problem. Thus, methods establishing an effective functional linkage between laser capture microdissection (LCM) and mass spectrometry (MS) provides for an enhanced molecular profiling of homogenous, specifically targeted cell populations from solid tumors. Utilizing frozen tissue avoids molecular degradation and bias that can be induced by other preservation techniques. Since clinical samples are often of a small quantity, tissue losses must be minimized. Therefore, all steps are carried out in the same single tube. Proteins are identified through peptide sequencing and subsequent matching against a specific proteomic database. Using such an approach enhances clinical biomarker discovery in the following ways. First, LCM allows for the complexity of a solid tumor to be reduced. Second, MS provides for the profiling of proteins, which are the ultimate bio-effectors. Third, by selecting for tumor proper or microenvironment-specific cells from clinical samples, the heterogeneity of individual solid tumors is directly addressed. Finally, since proteins are the targets of most pharmaceuticals, the enriched protein data streams can then be further analyzed for potential biomarkers, drug targets, pathway elucidation, as well as an enhanced understanding of the various pathologic processes under study. Within this context, the following method illustrates in detail a synergy between LCM and MS for an enhanced molecular profiling of solid tumors and clinical biomarker discovery.

Key words

Biomarker Cancer Laser capture microdissection (LCM) Liquid chromatography-mass spectrometry (LC-MS) Solid tumor heterogeneity Frozen tissue 

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Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Sumana Mukherjee
    • 1
  • Jaime Rodriguez-Canales
    • 1
  • Jeffrey Hanson
    • 1
  • Michael R. Emmert-Buck
    • 1
  • Michael A. Tangrea
    • 1
  • DaRue A. Prieto
    • 2
  • Josip Blonder
    • 2
  • Donald J. JohannJr.
    • 1
  1. 1.National Cancer InstituteBethesdaUSA
  2. 2.Frederick National Laboratory for Cancer ResearchFrederickUSA

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