title: Workflow comparison for label-free, quantitative secretome proteomics for cancer biomarker discovery: method evaluation, differential analysis, and verification in serum.
abstract: The cancer cell secretome has emerged as an attractive subproteome for discovery of candidate blood-based biomarkers. To choose the best performing workflow, we assessed the performance of three first-dimension separation strategies prior to nanoLC-MS/MS analysis: (1) 1D gel electrophoresis (1DGE), (2) peptide SCX chromatography, and (3) tC2 protein reversed phase chromatography. 1DGE using 4-12% gradient gels outperformed the SCX and tC2 methods with respect to number of identified proteins (1092 vs 979 and 580, respectively), reproducibility of protein identification (80% vs 70% and 72%, respectively, assessed in biological N = 3). Reproducibility of protein quantitation based on spectral counting was similar for all 3 methods (CV: 26% vs 24% and 24%, respectively). As a proof-of-concept of secretome proteomics for blood-based biomarker discovery, the gradient 1DGE workflow was subsequently applied to identify IGF1R-signaling related proteins in the secretome of mouse embryonic fibroblasts transformed with human IGF1R (MEF/Toff/IGF1R). VEGF and osteopontin were differentially detected by LC-MS/MS and verified in secretomes by ELISA. Follow-up in serum of mice bearing MEF/Toff/IGF1R-induced tumors showed an increase of osteopontin levels paralleling tumor growth, and reduction in the serum of mice in which IGF1R expression was shut off and tumor regressed.
authors.affiliation: OncoProteomics Laboratory, Department of Medical Oncology, VUmc-Cancer Center Amsterdam, VU University Medical Center, Amsterdam, The Netherlands. s.piersma@vumc.nl
authors.forename: Sander R
authors.initials: SR
authors.lastname: Piersma
authors.forename: Ulrike
authors.initials: U
authors.lastname: Fiedler
authors.forename: Simone
authors.initials: S
authors.lastname: Span
authors.forename: Andreas
authors.initials: A
authors.lastname: Lingnau
authors.forename: Thang V
authors.initials: TV
authors.lastname: Pham
authors.forename: Steffen
authors.initials: S
authors.lastname: Hoffmann
authors.forename: Michael H G
authors.initials: MH
authors.lastname: Kubbutat
authors.forename: Connie R
authors.initials: CR
authors.lastname: Jiménez
mesh_terms: D000818:Animals; D014408:Biomarkers, Tumor / Q000097:blood*; D002460:Cell Line; D002850:Chromatography, Gel / Q000379:methods*; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D051379:Mice; D009368:Neoplasm Transplantation; D053495:Osteopontin / Q000378:metabolism; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D017526:Receptor, IGF Type 1 / Q000378:metabolism; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
chemical_list: D014408:Biomarkers, Tumor; D020543:Proteome; C506896:Spp1 protein, mouse; D042461:Vascular Endothelial Growth Factor A; C467480:vascular endothelial growth factor A, mouse; D053495:Osteopontin; D017526:Receptor, IGF Type 1