Proteomic Profiling of Breast Tissue Collagens and Site-specific Characterization of Hydroxyproline Residues of Collagen Alpha-1-(I)

In a quantitative proteomics-based breast cancer study
of complementary
normal and tumor biopsies, 22 collagen isoforms were detected by LC–MALDI
TOF/TOF MS. By applying proline oxidation, representing hydroxyproline,
in database search parameters a substantial increase in assigned MS/MS
was achieved, boosting the average (three experiments) number of peptides
from 306 to 8126 for collagen alpha-1­(I). The plethora of peptide
identities for alpha-1­(I) was disproportionate with full length protein
sequence coverage which only increased from 28.3 to 64.4%. The peptides,
in fact, constituted an extensive two-dimensional array of isomers
exhibiting heterogeneity in degree and location of hydroxyproline
residues. A total of 3433 peptides, scores >36 (p < 0.01), constituting 94% of the triple helix region of collagen
alpha-1­(I) provided a census of proline hydroxylation levels defined
as the rate of site occupancy for each peptide isomer (r) and the total site occupancy for each proline residue (t). MS/MS and MS/MS/MS analysis, by MALDI-QIT-TOF MS, was
used to corroborate site-specific proline hydroxylation of the original
data. In addition, iTRAQ data for each collagen isoform in each of
10 patients (grouped by disease) was determined and indicated an increase
in fibrillar collagens in invasive carcinoma but little change in
fibroadenoma or DCIS.