Differential Protease Specificity by Collagenase as a Novel Approach to Serum Proteomics That Includes Identification of Extracellular Matrix Proteins without Enrichment

Abstract

Circulating extracellular matrix (ECM) proteins are serological biomarkers of interest due to their association with pathologies involving disease processes such as fibrosis and cancers. In this study, we investigate the potential for serum biomarker research using differential protease specificity (DPS), leveraging alternate protease specificity as a targeting mechanism to selectively digest circulating ECM protein serum proteins. A proof-of-concept study is presented using serum from patients with cirrhotic liver or hepatocellular carcinoma. The approach uses collagenase DPS for digestion of deglycosylated serum and liquidchromatography-trapped ion mobility-tandem mass spectrometry (LC-TIMS-MS/MS) to enhance the detection of ECM proteins in serum. It requires no sample enrichment and minimizes the albumin average precursor intensity readout to less than 1.2%. We further demonstrate the capabilities for using the method as a high-throughput matrix-assisted laser/desorption ionization mass spectrometry (MALDI-MS) assay coupled with reference library searching. A goal is to improve the depth and breadth of biofluid proteomics for noninvasive assays.

The Spatial Extracellular Proteomic Tumor Microenvironment Distinguishes Molecular Subtypes of Hepatocellular Carcinoma

“…Increased fibrinogen levels in serum and plasma have been associated with advanced tumor stage and poor survival in hepatocellular carcinoma. ,, Through LC-MS/MS sequencing, data showed higher proportions of fibrinogen sampled from S1 compared to S3 (S1 753 spectra, 15.7%; S3 99 spectra; 2.6%) (Figures A and S ). Two fibrinogen peptides that matched the image data showed significant elevation in peak intensity in S1 tumors compared with S3 tumors.…”

“…Following epitope retrieval, the tris-buffer was sequentially exchanged with HPLC-grade H 2 O by diluting three times prior to drying via a desiccator. Collagenase type III was used for localized digestion of the extracellular matrix as done previously. − In addition to the manufacturer’s characterization, collagenase type III was characterized by collagen activity assays (Abcam ab196999) prior to use. The absence of tryptic activity was validated by using a colorimetric trypsin activity assay kit (Abcam ab102531).…”