Immunohistochemistry Enhances Proteomic Profiling from Paraffin-Embedded Tissue Sections by Mass-Spectrometry: A Novel Approach with Wide-Ranging Potential Applications in Pathology
JA Vrana, JD Gamez, JD Theis, A Dogan. Mayo Clinic, Rochester, MN
Background: We have recently shown that the proteome of amyloid extracellular deposits can be reproducibly identified in paraffin-embedded tissue sections with great sensitivity and specificity using nano-flow liquid chromatography electrospray tandem mass spectrometry (LC-MS/MS) based proteomic analysis (Lab Invest, 2008 Aug 18 Epub). In this study, we illustrate the principle of utilizing immunohistochemistry (IHC) to successfully identify differentially expressed proteins in 2 physiological subsets of skeletal muscle.
Design: Tissue sections of normal skeletal muscle were stained with a monoclonal antibody (MY32) to fast skeletal muscle myosin. Individual muscle cells that stained positive and negative were laser microdissected and collected into separate tubes. The dissected cells were processed and trypsin digested into peptides. The peptides were analyzed by LC-MS/MS, and the resulting data were correlated to theoretical fragmentation patterns of tryptic peptide sequences from the Swissprot database using Scaffold. Peptide identifications were accepted if they could be established at greater than 90.0% probability and protein identifications were accepted if they could be established at greater than 90.0% probability and contain at least 2 identified spectra. The identified proteins were subsequently examined for the presence or absence of muscle related peptides.
Results: LC-MS/MS gave peptide profiles containing 60 proteins consistent with a variety of fast and slow twitch muscle proteins as well as numerous general muscle protein markers. 12 of 37 proteins identified by IHC were specific to fast twitch muscle (most abundant proteins being beta-enolase and fast skeletal myosin light chain 2) and 6 of 26 proteins identified by negative IHC were specific to slow twitch muscle (most abundant proteins being myosin light chain 3 and myosin regulatory light chain 2). General muscle markers, such as myoglobin, were observed in both stained and unstained cells.
Conclusions: Our study shows that detailed proteomic analysis can be successfully performed on IHC stained tissue sections. Using this methodology, the proteome of single muscle cells can be identified and subtle differences in protein expression between physiological subsets of skeletal muscle can be demonstrated. As this technology is readily applicable to routine clinical biopsy specimens, it is likely that LC MS/MS- based proteomic analysis will have wide ranging applications in biomarker discovery and clinical diagnosis.
Category: Special Category for 2009 - Pan-genomic/Pan-proteomic approaches to Cancer
Tuesday, March 10, 2009 9:30 AM
Poster Session III # 250, Tuesday Morning