title: Optimized nLC-MS workflow for laser capture microdissected breast cancer tissue.
abstract: Reliable sample preparation is of utmost importance for comparative proteome analysis, particularly when investigating minute amounts of clinical specimens, such as laser capture microdissected tumor tissue. In this study, we present an optimized nanoLC-MS workflow specifically for the analysis of laser capture microdissected breast cancer tissue. Analytical performance of different laser capture microdissection (LCM) functions available on the PALM system, time dependent trypsin digestion efficiency, effect of sample preparation and digestion time on peptide modification, semi-tryptic peptides and missed cleavages were evaluated. Our results show that microdissection from uncoated glass slides results in protein degradation; that protease and phosphatase inhibitors do not result in detectable improvement in number of peptides or semi-tryptic peptides; and that digestion time longer than four hours drastically reduces the number of missed cleavages, but also increases the number of unexpectedly modified peptides. Overalkylation was the most dominant side-reaction, which significantly increased overnight (P=0.05). The latter effect could almost completely be reverted by the use of a quenching agent (P=0.001). Taken together, our results show that it is of importance to carefully control sample handling steps so that reliable protein identification and quantitation can be performed within comparative proteomics studies using LCM. This article is part of a Special Issue entitled: Proteomics: The clinical link.
authors.affiliation: Department of Medical Oncology, Erasmus Medical Center, Rotterdam, The Netherlands. r.braakman@erasmusmc.nl
authors.forename: René B H
authors.initials: RB
authors.lastname: Braakman
authors.forename: Madeleine M A
authors.initials: MM
authors.lastname: Tilanus-Linthorst
authors.forename: Ning Qing
authors.initials: NQ
authors.lastname: Liu
authors.forename: Christoph
authors.initials: C
authors.lastname: Stingl
authors.forename: Lennard J M
authors.initials: LJ
authors.lastname: Dekker
authors.forename: Theo M
authors.initials: TM
authors.lastname: Luider
authors.forename: John W M
authors.initials: JW
authors.lastname: Martens
authors.forename: John A
authors.initials: JA
authors.lastname: Foekens
authors.forename: Arzu
authors.initials: A
authors.lastname: Umar
mesh_terms: D014408:Biomarkers, Tumor / Q000032:analysis / Q000378:metabolism; D001943:Breast Neoplasms / Q000737:chemistry / Q000175:diagnosis* / Q000378:metabolism / Q000473:pathology; D002138:Calibration; D002277:Carcinoma / Q000737:chemistry / Q000175:diagnosis* / Q000378:metabolism / Q000473:pathology; D002853:Chromatography, Liquid / Q000379:methods / Q000592:standards; D016000:Cluster Analysis; D005260:Female; D005629:Frozen Sections; D006801:Humans; D061346:Laser Capture Microdissection / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods / Q000592:standards*; D009363:Neoplasm Proteins / Q000032:analysis / Q000378:metabolism; D040901:Proteomics / Q000379:methods / Q000592:standards; D013048:Specimen Handling / Q000379:methods; D057188:Workflow
chemical_list: D014408:Biomarkers, Tumor; D009363:Neoplasm Proteins