title: A quantitative proteomic workflow for characterization of frozen clinical biopsies: laser capture microdissection coupled with label-free mass spectrometry.
abstract: This paper describes a simple, highly efficient and robust proteomic workflow for routine liquid-chromatography tandem mass spectrometry analysis of Laser Microdissection Pressure Catapulting (LMPC) isolates. Highly efficient protein recovery was achieved by optimization of a "one-pot" protein extraction and digestion allowing for reproducible proteomic analysis on as few as 500 LMPC isolated cells. The method was combined with label-free spectral count quantitation to characterize proteomic differences from 3000-10,000 LMPC isolated cells. Significance analysis of spectral count data was accomplished using the edgeR tag-count R package combined with hierarchical cluster analysis. To illustrate the capability of this robust workflow, two examples are presented: 1) analysis of keratinocytes from human punch biopsies of normal skin and a chronic diabetic wound and 2) comparison of glomeruli from needle biopsies of patients with kidney disease. Differentially expressed proteins were validated by use of immunohistochemistry. These examples illustrate that tissue proteomics carried out on limited clinical material can obtain informative proteomic signatures for disease pathogenesis and demonstrate the suitability of this approach for biomarker discovery.
authors.affiliation: Department of Molecular Virology, Immunology and Medical Genetics, College of Medicine, Columbus, OH 43210, USA.
authors.forename: John P
authors.initials: JP
authors.lastname: Shapiro
authors.forename: Sabyasachi
authors.initials: S
authors.lastname: Biswas
authors.forename: Anand S
authors.initials: AS
authors.lastname: Merchant
authors.forename: Anjali
authors.initials: A
authors.lastname: Satoskar
authors.forename: Cenny
authors.initials: C
authors.lastname: Taslim
authors.forename: Shili
authors.initials: S
authors.lastname: Lin
authors.forename: Brad H
authors.initials: BH
authors.lastname: Rovin
authors.forename: Chandan K
authors.initials: CK
authors.lastname: Sen
authors.forename: Sashwati
authors.initials: S
authors.lastname: Roy
authors.forename: Michael A
authors.initials: MA
authors.lastname: Freitas
mesh_terms: D015415:Biomarkers / Q000032:analysis / Q000378:metabolism; D001706:Biopsy; D015925:Cryopreservation / Q000379:methods*; D005260:Female; D006801:Humans; D061346:Laser Capture Microdissection / Q000379:methods*; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D040901:Proteomics / Q000379:methods*
chemical_list: D015415:Biomarkers