Proteomic View of Basement Membranes from Human Retinal Blood Vessels, Inner Limiting Membranes, and Lens Capsules

Abstract

Basement membranes (BMs) are extracellular matrix sheets comprising the laminins, type-IV collagens, nidogens, and the heparan sulfate proteoglycans, perlecan, collagen XVIII, and agrin. In intact BMs, BM proteins are physiologically insoluble and partially resistant to proteolytic digestion, making BMs a challenge to study. Here three types of BMs from adult human eyes, the inner limiting membrane (ILM), the retinal vascular BMs, and the lens capsule, were isolated for analysis by 1D-SDS-PAGE and LC-MS/MS. Peptide and protein identifications were done using MaxQuant. 1129 proteins were identified with a 1% false discovery rate. Data showed that BMs are composed of multiple laminins, collagen IVs, nidogens, and proteoglycans. The dominant laminin family member in all BMs was laminin α5β2γ1. The dominant collagen IV trimer in lens capsule (LC) and blood vessel (BV) BMs had a chain composition of α1(IV), α2 (IV), whereas the dominant collagen IV in the ILM had the α3(IV), α4(IV), α5(IV) chain composition. The data also showed that the ratio of laminin and collagen IVs varied among different BM types: the ratio of collagen IV to the other BM proteins is highest in LC, followed by BV and lowest for the ILM. The data have been deposited to the ProteomeXchange with identifier PXD001025.

Comprehensive Characterization of Glycosylation and Hydroxylation of Basement Membrane Collagen IV by High-Resolution Mass Spectrometry

“…To test the capabilities of our improved bioinformatics workflow for the mapping of hydroxylation and O-glycosylation of lysine residues and hydroxylation of proline residues, we downloaded the lens capsule LC–MS/MS data generated by Uechi et al, which provided a proteomic overview of human eye BM. 52 Although the data sets were generated by trypsin digestion using conventional CID fragmentation, an extensive 1D-gel LC fractionation strategy was undertaken for each sample analyzed. Analyses of the multiple .raw files generated for these sample sets with our bioinformatics approach identified 66, 68, and 58 distinct peptides corresponding to 1207, 1364, and 767 spectra, resulting in an increased sequence coverage of the entire col4a1 molecule (excluding signal peptide) from 29% to about 85% (Figure 6, Supporting Figure S3, and Table 2).…”

“…These problems are evident in many proteomic analyses of BM proteins, in which collagen IV is reported with low abundance and low sequence coverage. 52–56 In fact, when we analyzed collagen IV by using a standardized proteomics approach, we obtained extremely poor coverage (Supporting Figure S1) likely due to the extensive presence of PTMs that hampered identification of modified peptides. Interestingly, the majority of peptides identified when searching with standard database strategies mapped to the NC1 domain sequence.…”

“…49–51 Indeed, although collagen IV is very abundant, a surprising underrepresentation of sequence coverage has been observed in proteomics inventories of highly enriched BM preparations. 52–56 This may be explained by the inherent properties of collagen IV such as repetitive nature of primary sequence, high insolubility, cross-linking, and resistance to cleavage due to extensive PTM, which could lead to low peptide abundance, poor primary sequence coverage, and poor identification of the location of PTMs. Alternative approaches such as use of complementary ionization techniques, 57–59 glycosylated peptide enrichment, 60 and multiple enzyme digestions 61 have been used for MS characterization of other collagens but not tried with the BM collagen IV.…”

An active role for basement membrane assembly and modification in tissue sculpting

“…matrixome.com/bm/Home/home/home.asp and the human protein atlas, http://www.proteinatlas.org) (Hynes and Naba, 2012;Pontén et al, 2011;Uhlen et al, 2010). As proteomic studies of isolated basement membranes have revealed over 200 core matrix and matrix-associated proteins, the composition or structure of basement membranes can likely be modified in many ways to create specialized or context-specific assemblies (Uechi et al, 2014).…”

Applying Proteomics to Investigate Extracellular Matrix in Health and Disease

“…Peptide and protein identifications were performed using MaxQuant, and 1129 proteins were identified (Uechi, Sun, Schreiber, Halfter, & Balasubramani, 2014). According to MatrixDB, the in silico matrisome and the Uniprot database 202 proteins were identified as ECM.…”