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

Macdonald, Jade K.; Clift, Cassandra L.; Saunders, Janet; Zambrzycki, Stephen C.; Mehta, Anand S.; Drake, Richard R.; Angel, Peggi M.

doi:10.1021/jasms.3c00366

Cited by 3 publications

(2 citation statements)

References 63 publications

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“…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.…”

Section: Resultsmentioning

confidence: 97%

“…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).…”

Section: Experimental Proceduresmentioning

confidence: 99%

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

Macdonald,

Taylor,

Wang

et al. 2024

J. Proteome Res.

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Hepatocellular carcinoma (HCC) mortality rates continue to increase faster than those of other cancer types due to high heterogeneity, which limits diagnosis and treatment. Pathological and molecular subtyping have identified that HCC tumors with poor outcomes are characterized by intratumoral collagenous accumulation. However, the translational and post-translational regulation of tumor collagen, which is critical to the outcome, remains largely unknown. Here, we investigate the spatial extracellular proteome to understand the differences associated with HCC tumors defined by Hoshida transcriptomic subtypes of poor outcome (Subtype 1; S1; n = 12) and better outcome (Subtype 3; S3; n = 24) that show differential stroma-regulated pathways. Collagen-targeted mass spectrometry imaging (MSI) with the same-tissue reference libraries, built from untargeted and targeted LC-MS/MS was used to spatially define the extracellular microenvironment from clinically-characterized, formalin-fixed, paraffin-embedded tissue sections. Collagen α-1(I) chain domains for discoidindomain receptor and integrin binding showed distinctive spatial distribution within the tumor microenvironment. Hydroxylated proline (HYP)-containing peptides from the triple helical regions of fibrillar collagens distinguished S1 from S3 tumors. Exploratory machine learning on multiple peptides extracted from the tumor regions could distinguish S1 and S3 tumors (with an area under the receiver operating curve of ≥0.98; 95% confidence intervals between 0.976 and 1.00; and accuracies above 94%). An overall finding was that the extracellular microenvironment has a high potential to predict clinically relevant outcomes in HCC.

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Section: Resultsmentioning

confidence: 97%

Section: Experimental Proceduresmentioning

confidence: 99%

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

Macdonald,

Taylor,

Wang

et al. 2024

J. Proteome Res.

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Molecular analysis of the extracellular microenvironment: from form to function

Macdonald,

Mehta,

Drake

et al. 2024

FEBS Letters

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The extracellular matrix (ECM) proteome represents an important component of the tissue microenvironment that controls chemical flux and induces cell signaling through encoded structure. The analysis of the ECM represents an analytical challenge through high levels of post‐translational modifications, protease‐resistant structures, and crosslinked, insoluble proteins. This review provides a comprehensive overview of the analytical challenges involved in addressing the complexities of spatially profiling the extracellular matrix proteome. A synopsis of the process of synthesizing the ECM structure, detailing inherent chemical complexity, is included to present the scope of the analytical challenge. Current chromatographic and spatial techniques addressing these challenges are detailed. Capabilities for multimodal multiplexing with cellular populations are discussed with a perspective on developing a holistic view of disease processes that includes both the cellular and extracellular microenvironment.

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Determining the N-Glycan and Collagen/Extracellular Matrix Protein Compositions in a Novel Outcome Cohort of Prostate Cancer Tissue Microarrays Using MALDI-MSI

Hartig,

Bejar,

Young

et al. 2024

Cancer Research Communications

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Prostate cancer is a significant health concern, with metastasis posing major clinical challenges and resulting in poor patient outcome. Despite screening and treatment advances, a critical need for novel biomarkers to predict prostate cancer progression at the time of prostatectomy persists. Here, we assessed aberrant N-glycosylation patterns and alterations in extracellular matrix proteins as potential biomarkers of predicting prostate cancer severity in a unique patient outcome cohort. Tissue microarray slides were assembled from primary prostatectomy specimens that were categorized into “no evidence of disease (NED)” and “metastasis (MET)” designations based on >5-year disease progression outcomes. Serial mass spectrometry imaging techniques were performed to analyze N-glycans and extracellular matrix (ECM) components in formalin-fixed paraffin-embedded cores. The results revealed a significant upregulation of bisecting and multi-antennary core fucosylated N-glycans in MET tissues when compared to NED tissues. Alterations in ECM composition in both NED and MET cohorts were observed, particularly in collagen species and the amount of hydroxyproline content. Results suggest a coordinated alteration of ECM protein and glycosylation content in prostate cancer tissues can be predictive for post-prostatectomy disease progression.

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

Cited by 3 publications

References 63 publications

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

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

Molecular analysis of the extracellular microenvironment: from form to function

Determining the N-Glycan and Collagen/Extracellular Matrix Protein Compositions in a Novel Outcome Cohort of Prostate Cancer Tissue Microarrays Using MALDI-MSI

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