Koichi Tanaka Research Laboratory, Shimadzu Corporation, Manchester, United Kingdom.
J Proteome Res. 2012 Dec 7;11(12):5890-902. doi: 10.1021/pr300656r. Epub 2012 Nov 14.
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.
在一项基于定量蛋白质组学的乳腺癌互补正常和肿瘤活检研究中，通过 LC-MALDI TOF/TOF MS 检测到 22 种胶原蛋白同工型。通过在数据库搜索参数中应用脯氨酸氧化，代表羟脯氨酸，实现了分配的 MS/MS 的大量增加，从而将胶原蛋白 alpha-1(I)的平均（三个实验）肽数从 306 个增加到 8126 个。alpha-1(I)的肽同一性数量与全长蛋白质序列覆盖率不成比例，仅从 28.3%增加到 64.4%。事实上，这些肽构成了一个广泛的异构二肽的二维阵列，表现出羟脯氨酸残基的程度和位置的异质性。共有 3433 个肽，分数>36（p<0.01），构成胶原蛋白 alpha-1(I)的三螺旋区的 94%，提供了脯氨酸羟化水平的普查，定义为每个肽异构体（r）的位点占有率率和每个脯氨酸残基（t）的总位点占有率。通过 MALDI-QIT-TOF MS 进行的 MS/MS 和 MS/MS/MS 分析用于证实原始数据中特定位点的脯氨酸羟化。此外，对 10 名患者（按疾病分组）的每种胶原蛋白同工型的 iTRAQ 数据进行了测定，并表明在浸润性癌中纤维胶原蛋白增加，但在纤维腺瘤或 DCIS 中变化不大。
