Article Title: Data-Independent Acquisition and Quantification of Extracellular Matrix from Human Lung in Chronic Inflammation-Associated Carcinomas
Figure Lengend Snippet: Study Design and Proteomic Pipeline for ECM Analysis of Lung Squamous Cell Carcinoma. ‘Tumor’ and ‘Matched Normal’ fresh tissue specimens were collected from 10 patients with lung squamous cell carcinoma by the Ferri team at McGill University Health Centre (QC, Canada) or through the Cooperative Human Tissue Network Western Division (TN, USA). Fresh samples in UW Cold Storage Solution were sent to the Tlsty team at UCSF (CA, USA) for enrichment for insoluble extracellular matrix (ECM) proteins. ECM-enriched samples were further processed by the Schilling team at the Buck Institute (Novato, CA); proteins were solubilized, in-gel digested with Lys-C and trypsin, and extracted proteolytic peptides were de-glycosylated with PNGase F. All resulting samples were analyzed in duplicate on a nanoLC-TripleTOF 6600 system (QqTOF) operated in data-independent acquisition (DIA) mode, and data were processed with Spectronaut (Biognosys). Finally, candidates were validated on independent cohorts by immunofluorescence-based immunohistochemistry by the Tlsty team.
Article Snippet: Glycerol-free PNGase F was purchased from New England BioLabs (Ipswich, MA).
Techniques: Western Blot, Immunofluorescence, Immunohistochemistry