title: Quantitative proteomic analysis of microdissected breast cancer tissues: comparison of label-free and SILAC-based quantification with shotgun, directed, and targeted MS approaches.
abstract: Quantitative proteomics plays an important role in validation of breast-cancer-related biomarkers. In this study, we systematically compared the performance of label-free quantification (LFQ) and SILAC with shotgun and directed methods for quantifying breast-cancer-related markers in microdissected tissues. We show that LFQ leads to slightly higher coefficient of variation (CV) for protein quantification (median CV = 16.3%) than SILAC quantification (median CV = 13.7%) (P < 0.0001), but LFQ method enables ∼60% more protein quantification and is also more reproducible (∼20% more proteins were quantified in all replicate samples). Furthermore, we describe a method to accurately quantify multiple proteins within one pathway, that is, "focal adhesion pathway", in trace amounts of breast cancer tissues using a SILAC-based SRM assay. Using this SILAC-based SRM assay, we precisely quantified five "focal adhesion" proteins with good quantitative precision (CV range: 2.4-5.9%) in replicate whole tissue lysate samples and replicate microdissected samples (CV range: 5.8-16.1%). Our results show that in microdissected breast cancer tissues LFQ in combination with shotgun proteomics performed the best overall and is therefore suitable for both biomarker discovery and validation in these types of specimens. The SILAC-based SRM method can be used for the development of clinically relevant protein assays in tumor biopsies.
authors: Department of Medical Oncology, Erasmus MC Cancer Institute and ‡Department of Neurology, Erasmus University Medical Center , Dr. Molewaterplein 50, Be-401, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands.
authors: Ning Qing
authors: NQ
authors: Liu
authors: Lennard J M
authors: LJ
authors: Dekker
authors: Christoph
authors: C
authors: Stingl
authors: Coşkun
authors: C
authors: Güzel
authors: Tommaso
authors: T
authors: De Marchi
authors: John W M
authors: JW
authors: Martens
authors: John A
authors: JA
authors: Foekens
authors: Theo M
authors: TM
authors: Luider
authors: Arzu
authors: A
authors: Umar
mesh_terms: D000595:Amino Acid Sequence; D014408:Biomarkers, Tumor / Q000737:chemistry / Q000378:metabolism*; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000378:metabolism; D045744:Cell Line, Tumor; D005260:Female; D022001:Focal Adhesions / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling; D061346:Laser Capture Microdissection; D008969:Molecular Sequence Data; D020543:Proteome / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics; D012015:Reference Standards; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods* / Q000592:standards
chemical_list: D014408:Biomarkers, Tumor; D015815:Cell Adhesion Molecules; D020543:Proteome