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Enrichment of Collagen Fragments Using Dimeric Collagen Hybridizing Peptide for Urinary Collagenomics.

ABSTRACT: Collagen remodeling in normal and pathologic conditions releases numerous collagen fragments into biological fluids. Although a few collagen fragments have been tested as biomarkers for disease indication, most occur at trace levels, making them nearly impossible to detect even with modern analytical tools. Here we report a new way to enrich collagen fragments that allows complete peptidomic analysis of collagen fragments in urine. Enrichment is made possible by dimeric collagen hybridizing peptides (CHPs) that bind collagen fragments originating from the triple helical regions of all collagen types with minimal sequence bias. LC-MS/MS analysis of enriched mouse urine revealed an average of 383 collagenous peptide fragments per sample (compared to 34 for unenriched sample), which could be mapped to all types of mouse collagens in the SwissProt database including FACITs and MACITs. Hierarchical clustering of a selected panel of the detected fragments separated osteopenic mice from healthy mice. The results demonstrate dimeric CHP's ability to enrich collagen fragments from biological fluid and its potential to aid peptidomics-based disease detection and biomarker discovery.

SUBMITTER: Kessler JL

PROVIDER: S-EPMC8204671 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Enrichment of Collagen Fragments Using Dimeric Collagen Hybridizing Peptide for Urinary Collagenomics.

Kessler Julian L JL Li Yang Y Fornetti Jaime J Welm Alana L AL Yu S Michael SM

Journal of proteome research 20200616 8

Collagen remodeling in normal and pathologic conditions releases numerous collagen fragments into biological fluids. Although a few collagen fragments have been tested as biomarkers for disease indication, most occur at trace levels, making them nearly impossible to detect even with modern analytical tools. Here we report a new way to enrich collagen fragments that allows complete peptidomic analysis of collagen fragments in urine. Enrichment is made possible by dimeric collagen hybridizing pept ...[more]

PMID: 32500704

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Project description:IntroductionDegenerative disc disease (DDD) is accompanied by structural changes in the intervertebral discs (IVD). Extra-cellular matrix degradation of the annulus fibrosus (AF) has been linked with degeneration of the IVD. Collagen is a vital component of the IVD. Collagen hybridizing peptide (CHP) is an engineered protein that binds to degraded collagen, which we used to quantify collagen damage in AF. This method was used to compare AF samples obtained from donors with no DDD to AF samples from patients undergoing surgery for symptomatic DDD.MethodsFresh AF tissue was embedded in an optimal cutting temperature compound and cryosectioned at a thickness of 8 μm. Hematoxylin and Eosin staining was performed on sections for general histomorphological assessment. Serial sections were stained with Cy3-conjugated CHP and the mean fluorescence intensity and areal fraction of Cy3-positive staining were averaged for three regions of interest (ROI) on each CHP-stained section.ResultsIncreases in mean fluorescence intensity (p = 0.0004) and percentage of positively stained area (p = 0.00008) with CHP were detected in DDD samples compared to the non-DDD samples. Significant correlations were observed between mean fluorescence intensity and percentage of positively stained area for both non-DDD (R = 0.98, p = 5E-8) and DDD (R = 0.79, p = 0.0012) samples. No significant differences were detected between sex and the lumbar disc level subgroups of the non-DDD and DDD groups. Only tissue pathology (non-DDD versus DDD) influenced the measured parameters. No three-way interactions between tissue pathology, sex, and lumbar disc level were observed.Discussion and conclusionsThese findings suggest that AF collagen degradation is greater in DDD samples compared to non-DDD samples, as evidenced by the increased CHP staining. Strong positive correlations between the two measured parameters suggest that when collagen degradation occurs, it is detected by this technique and is widespread throughout the tissue. This study provides new insights into the structural alterations associated with collagen degradation in the AF that occur during DDD.

Project description:The pathogenesis of diabetes mellitus (DM) is variable, comprising different inflammatory and immune responses. Proteome analysis holds the promise of delivering insight into the pathophysiological changes associated with diabetes. Recently, we identified and validated urinary proteomics biomarkers for diabetes. Based on these initial findings, we aimed to further validate urinary proteomics biomarkers specific for diabetes in general, and particularity associated with either type 1 (T1D) or type 2 diabetes (T2D).Therefore, the low-molecular-weight urinary proteome of 902 subjects from 10 different centers, 315 controls and 587 patients with T1D (n = 299) or T2D (n = 288), was analyzed using capillary-electrophoresis mass-spectrometry. The 261 urinary biomarkers (100 were sequenced) previously discovered in 205 subjects were validated in an additional 697 subjects to distinguish DM subjects (n = 382) from control subjects (n = 315) with 94% (95% CI: 92-95) accuracy in this study. To identify biomarkers that differentiate T1D from T2D, a subset of normoalbuminuric patients with T1D (n = 68) and T2D (n = 42) was employed, enabling identification of 131 biomarker candidates (40 were sequenced) differentially regulated between T1D and T2D. These biomarkers distinguished T1D from T2D in an independent validation set of normoalbuminuric patients (n = 108) with 88% (95% CI: 81-94%) accuracy, and in patients with impaired renal function (n = 369) with 85% (95% CI: 81-88%) accuracy. Specific collagen fragments were associated with diabetes and type of diabetes indicating changes in collagen turnover and extracellular matrix as one hallmark of the molecular pathophysiology of diabetes. Additional biomarkers including inflammatory processes and pro-thrombotic alterations were observed.These findings, based on the largest proteomic study performed to date on subjects with DM, validate the previously described biomarkers for DM, and pinpoint differences in the urinary proteome of T1D and T2D, indicating significant differences in extracellular matrix remodeling.

Dataset Information

Enrichment of Collagen Fragments Using Dimeric Collagen Hybridizing Peptide for Urinary Collagenomics.

Publications

Enrichment of Collagen Fragments Using Dimeric Collagen Hybridizing Peptide for Urinary Collagenomics.

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