A Combined Shotgun and Targeted Mass Spectrometry Strategy for Breast Cancer Biomarker Discovery

It is of highest
importance to find proteins responsible for breast
cancer dissemination, for use as biomarkers or treatment targets.
We established and performed a combined nontargeted LC–MS/MS
and a targeted LC–SRM workflow for discovery and validation
of protein biomarkers. Eighty breast tumors, stratified for estrogen
receptor status and development of distant recurrence (DR ± ),
were collected. After enrichment of N-glycosylated peptides, label-free
LC–MS/MS was performed on each individual tumor in triplicate.
In total, 1515 glycopeptides from 778 proteins were identified and
used to create a map of the breast cancer N-glycosylated proteome.
Based on this specific proteome map, we constructed a 92-plex targeted
label-free LC–SRM panel. These proteins were quantified across
samples by LC–SRM, resulting in 10 proteins consistently differentially
regulated between DR+/DR– tumors. Five proteins were further
validated in a separate cohort as prognostic biomarkers at the gene
expression level. We also compared the LC–SRM results to clinically
reported HER2 status, demonstrating its clinical accuracy. In conclusion,
we demonstrate a combined mass spectrometry strategy, at large scale
on clinical samples, leading to the identification and validation
of five proteins as potential biomarkers for breast cancer recurrence.
All MS data are available via ProteomeXchange and PASSEL with identifiers
PXD001685 and PASS00643.