title: Proteomic profiling of breast tissue collagens and site-specific characterization of hydroxyproline residues of collagen alpha-1-(I).
abstract: 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.
authors.affiliation: Koichi Tanaka Research Laboratory, Shimadzu Corporation, Manchester, United Kingdom.
authors.forename: Helen
authors.initials: H
authors.lastname: Montgomery
authors.forename: Nitin
authors.initials: N
authors.lastname: Rustogi
authors.forename: Andreas
authors.initials: A
authors.lastname: Hadjisavvas
authors.forename: Koichi
authors.initials: K
authors.lastname: Tanaka
authors.forename: Kyriacos
authors.initials: K
authors.lastname: Kyriacou
authors.forename: Chris W
authors.initials: CW
authors.lastname: Sutton
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000595:Amino Acid Sequence; D001706:Biopsy; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D016022:Case-Control Studies; D024042:Collagen Type I / Q000032:analysis* / Q000378:metabolism; D000090602:Collagen Type I, alpha 1 Chain; D030562:Databases, Protein; D005260:Female; D006801:Humans; D006900:Hydroxylation; D006909:Hydroxyproline / Q000032:analysis*; D008875:Middle Aged; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010084:Oxidation-Reduction; D011392:Proline / Q000378:metabolism; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D055815:Young Adult
chemical_list: D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D020543:Proteome; D011392:Proline; D006909:Hydroxyproline