Quantitative proteomic profiling of extracellular matrix and site-specific collagen post-translational modifications in an in vitro model of lung fibrosis

Abstract

Lung fibrosis is characterized by excessive deposition of extracellular matrix (ECM), in particular collagens, by fibroblasts in the interstitium. Transforming growth factor-β1 (TGF-β1) alters the expression of many extracellular matrix (ECM) components produced by fibroblasts, but such changes in ECM composition as well as modulation of collagen post-translational modification (PTM) levels have not been comprehensively investigated. Here, we performed mass spectrometry (MS)-based proteomics analyses to assess changes in the ECM deposited by cultured lung fibroblasts from idiopathic pulmonary fibrosis (IPF) patients upon stimulation with transforming growth factor β1 (TGF-β1). In addition to the ECM changes commonly associated with lung fibrosis, MS-based label-free quantification revealed profound effects on enzymes involved in ECM crosslinking and turnover as well as multiple positive and negative feedback mechanisms of TGF-β1 signaling. Notably, the ECM changes observed in this in vitro model correlated significantly with ECM changes observed in patient samples. Because collagens are subject to multiple PTMs with major implications in disease, we implemented a new bioinformatic platform to analyze MS data that allows for the comprehensive mapping and site-specific quantitation of collagen PTMs in crude ECM preparations. These analyses yielded a comprehensive map of prolyl and lysyl hydroxylations as well as lysyl glycosylations for 15 collagen chains. In addition, site-specific PTM analysis revealed novel sites of prolyl-3-hydroxylation and lysyl glycosylation in type I collagen. Interestingly, the results show, for the first time, that TGF-β1 can modulate prolyl-3-hydroxylation and glycosylation in a site-specific manner. Taken together, this proof of concept study not only reveals unanticipated TGF-β1 mediated regulation of collagen PTMs and other ECM components but also lays the foundation for dissecting their key roles in health and disease. The proteomic data has been deposited to the ProteomeXchange Consortium via the MassIVE partner repository with the data set identifier MSV000082958.

Extracellular matrix alterations in low‐grade lung adenocarcinoma compared with normal lung tissue by imaging mass spectrometry

“…Bioinformatics done on noncollagen ECM‐type proteins reported main functions of collagen binding (eight proteins, 20%), collagen regulation (five proteins, 12%), and collagen fibril formation (four proteins, 10%) (Figure B; Table S2). By conventional proteomics and antibody staining, type 1 collagen, but not type III or IV, has been reported the major collagen produced in lung tumor cells, xenografts, and patient‐derived cancer cells . Our data supported this, demonstrating that COL1A1 and COL1A2 were the major collagens in the wedge resection (6.1% and 18.6% of total spectral counts, respectively).…”

“…A total of five peptides were confidently identified with hydroxylated lysine residues (Table S3). We attribute this to limited material and potential cross‐linking in formalin‐fixed tissue sections; studies on ECM secreted by fibrotic lung cells have identified 14 hydroxylated lysine residues on ECM . However, both the current study and previous study identified that lysine residue 277 on COL1A1 was hydroxylated.…”

“…Our data supported this, demonstrating that COL1A1 and COL1A2 were the major collagens in the wedge resection (6.1% and 18.6% of total spectral counts, respectively). Comparison of the FFPE collagenase digest with recent ECM proteomic papers using trypsin digestion on primary human lung cells from end‐stage idiopathic pulmonary fibrosis and a mouse model of lung fibrosis to LUAD demonstrated good overlap in protein identifications 60% (24/40 proteins) and 50% (20/40 proteins). The cited studies utilized trypsin digestion on four biological replicates of ECM from 25 000 cells (initial plating) or on 50 to 100 mg of fresh tissue as compared with our approach on a single section of FFPE tissue in duplicate.…”

“…However, both the current study and previous study identified that lysine residue 277 on COL1A1 was hydroxylated. Hydroxylation of this particular lysine is reduced by TGFβ1 stimulation, suggesting a role in the development of fibrosis . Investigation on hydroxylated proline modifications reported that 8.7% of all identified peptides were HYP modified (Figure A).…”

“…Comparison with conventional tryptic digestion proteomics on ECM from cell culture and fresh tissue demonstrated good overlap in protein identifications. This included identifying a common hydroxylated lysine residue (K 277 ) in COL1A1 sensitive to TGFβ1 stimulation . Proteins identified associated with cell‐collagen adhesion, which could be useful for understanding immune cell recruitment throughout the tumor and linked to oxidative stress, including the unfolded protein response, could be leveraged to understand mechanisms of ECM deposition in emergent cancer.…”