Abstract
The extracellular matrix (ECM) is composed of a dynamically regulated collagenous scaffold that provides structure, conveys cellular and environmental communication, and contributes to disease progression. Collagen proteins derived from clinically archived formalin-fixed, paraffin-embedded (FFPE) tissues are analytically challenging due to dense post-translational modifications, high proline content, and insolubility. A recent advancement in ECM proteomics is the use of collagenase type III, an ECM-specific bacterial protease, to target native collagenous structures on-tissue for peptide imaging. The resulting collagenase-generated peptides have biochemical differences compared to tryptic peptides, creating analytical challenges in elucidating peptide sequence information. In this study, we characterize collagenase as a proteomic enzyme for ECM-targeted liquid chromatography trapped ion mobility spectrometry tandem mass spectrometry (LC-TIMS-MS/MS) and matrix-assisted laser/desorption ionization mass spectrometry imaging (MALDI-MSI) proteomic workflows. We then optimized collagenase-generated peptide sequencing for MALDI-MSI peptide identification from clinically archived FFPE tissue sections. Soluble rat tail collagen solution is used as a collagen standard to elucidate tryptic and collagenase cleavage sites within collagen. Proteomic readouts of FFPE tissue are compared across trypsin and collagenase digests to assess for ECM enrichment by collagenase in biologically complex samples. Optimized methods for MALDI-MSI peptide identification are comprehensively detailed from sample preparation to MS data acquisition and MS data analysis for reproducible implementation. On-tissue digestion followed by liquid surface extraction (LSE), inclusion of singly charged peptides during data acquisition, and implementation of nonspecific cleavage during database searching resulted in the most collagenase-generated peptide spectrum matches as well as MALDI-MSI peptide identifications. This research establishes parameters for the optimal identification of peptides from collagenase-directed ECM proteomic workflows for targeted spatial analysis of the ECM.
| Original language | English |
|---|---|
| Pages (from-to) | 7672-7681 |
| Number of pages | 10 |
| Journal | Analytical Chemistry |
| Volume | 97 |
| Issue number | 14 |
| DOIs | |
| State | Published - Apr 15 2025 |
Funding
J.K.M. was supported by NIH/NIGMS 5T32GM132055. S.C.Z. was supported by NIH/NCI T32 CA193201 and PF-23-1029103-01-MM. P.M.A. was supported by NIH/NCI R21CA263464, R21CA286287, and R01CA253460; R21AR084303 and in part by P30CA138313, 5P20GM130457, the Biorepository and Tissue Analysis Shared Resource, and the Translational Science Laboratory, Hollings Cancer Center, MUSC. The Mass Spectrometry Facility is supported by NIH/NIGMS P20GM103542, with shared instrumentation NIH/OD S10OD010731, S10OD025126, and S10OD030212. The contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.