scholarly journals Methodology to Quantify Collagen Subtypes and Crosslinks: Application in Minipig Cartilages

Cartilage ◽  
2021 ◽  
pp. 194760352110605
Author(s):  
Benjamin J. Bielajew ◽  
Jerry C. Hu ◽  
Kyriacos A. Athanasiou
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tissue Characterization ◽  
Collagen Type ◽  
Facet Joint ◽  
Type I ◽  
Collagen Types ◽  
Collagen Crosslinks ◽  
Bottom Up ◽  
Ms Analysis

Introduction This study develops assays to quantify collagen subtypes and crosslinks with liquid chromatography-mass spectrometry (LC-MS) and characterizes the cartilages in the Yucatan minipig. Methods For collagen subtyping, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was performed on tissues digested in trypsin. For collagen crosslinks, LC-MS analysis was performed on hydrolysates. Samples were also examined histologically and with bottom-up proteomics. Ten cartilages (femoral condyle, femoral head, facet joint, floating rib, true rib, auricular cartilage, annulus fibrosus, 2 meniscus locations, and temporomandibular joint disc) were analyzed. Results The collagen subtyping assay quantified collagen types I and II. The collagen crosslinks assay quantified mature and immature crosslinks. Collagen subtyping revealed that collagen type I predominates in fibrocartilages and collagen type II in hyaline cartilages, as expected. Elastic cartilage and fibrocartilages had more mature collagen crosslink profiles than hyaline cartilages. Bottom-up proteomics revealed a spectrum of ratios between collagen types I and II, and quantified 42 proteins, including 24 collagen alpha-chains and 12 minor collagen types. Discussion The novel assays developed in this work are sensitive, inexpensive, and use a low operator time relative to other collagen analysis methods. Unlike the current collagen assays, these assays quantify collagen subtypes and crosslinks without an antibody-based approach or lengthy chromatography. They apply to any collagenous tissue, with broad applications in tissue characterization and tissue engineering. For example, a novel finding of this work was the presence of a large quantity of collagen type III in the white-white knee meniscus and a spectrum of hyaline and fibrous cartilages.

Conventional-Flow Liquid Chromatography–Mass Spectrometry for Exploratory Bottom-Up Proteomic Analyses

2018 ◽  
Vol 90 (8) ◽  
pp. 5381-5389 ◽  
Author(s):  
Juraj Lenčo ◽  
Marie Vajrychová ◽  
Kristýna Pimková ◽  
Magdaléna Prokšová ◽  
Markéta Benková ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Liquid Chromatography Mass Spectrometry ◽  
Bottom Up ◽  
Chromatography Mass Spectrometry ◽  
Proteomic Analyses

P0650URINE PEPTIDOME ANALYSIS IN HEART FAILURE, CHRONIC KIDNEY DISEASE AND CARDIORENAL SYNDROME TOWARDS THE DEFINITION OF DISEASE-SPECIFIC MARKERS

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Eleni Petra ◽  
Tianlin He ◽  
Agnieszka Latosinska ◽  
Rafael Stroggilos ◽  
Harald Mischak ◽  
...  
Keyword(s):  
Heart Failure ◽  
Chronic Kidney Disease ◽  
Kidney Disease ◽  
Collagen Type ◽  
Cardiorenal Syndrome ◽  
Collagen Type I ◽  
Type I ◽  
Collagen Types ◽  
Alpha 1 Antitrypsin ◽  
Urinary Peptides

Abstract Background and Aims The cardiorenal syndrome (CRS) reflects the complex interplay between kidney and heart diseases, but its molecular basis remains poorly understood. Multiple studies have demonstrated the association of urinary biomarkers with both heart and kidney diseases. However, their relevance and involvement in CRS have not been investigated yet. To address this gap, a study was designed with the aim to compare urinary biomarkers specific for heart failure (HF) and chronic kidney disease (CKD) with peptides representing CRS, with the ultimate target to connect these findings towards a better understanding of CRS pathophysiology. Method A total of 3.463 urinary peptidomic datasets from patients with HF, CKD, or with both HF and CKD (CRS) as well as patients with no apparent diseases (controls) were retrieved and analyzed from the urinary peptidomics database (Latosinska A et al., Electrophoresis 2019; 40: 2294-2308). Following the matching for age, gender, heart and kidney function, differences in the abundance of urinary peptides were investigated in a cohort comprised of 390 patients with HF, 257 patients with CKD, 392 patients with CRS and 356 controls. The non-parametric Mann-Whitney U test was applied, followed by correction for multiple testing using the Benjamini-Hochberg method. To map the peptides to the protein precursor, the alignment tool Geneious (www. geneious.com) was applied, while the PeptideRanker (http://distilldeep.ucd.ie/PeptideRanker/) was used to predict probability of peptide being bioactive. Results The multiple pair-wise comparisons resulted in the identification of numerous differentially abundant peptides (p<0.05) between the studied conditions, including among others 176 HF-specific, 146 CKD-specific and 35 CRS-specific peptides. Among the HF-specific peptides, the majority (n=94, 53.4%) originated from collagen type I, II and III. In the case of CKD-specific peptides, 24 (16.43%) originated from alpha-1-antitrypsin, 19 (13.0%) from b2-microglobulin and 15 (10.27%) from collagen type I. For the CRS specific peptides, fragments of Ig lambda-2 chain C regions (n=4, 11.42%), collagen type III (n=4, 11.42%), secreted and transmembrane protein 1 (n=3, 8.57%) and gelsolin (n=1, 2.85%) were identified (figure: 1). Of the 176 HF-specific peptides, 94 (53.40%) were predicted as bioactive, including, among others, fragments of collagen types I (n=43, 45.74%) and III (n=21, 22.34%). In the former, peptides with the higher bioactivity scores were aligned close to the N terminus of the precursor protein, whereas in the latter, peptides were in close proximity to both N and C termini. Along the same lines, 32 (21.91%) of the 146 CKD-specific peptides were predicted as bioactive, including peptides from collagen types I and III with the highest score, as well as fragments from collagen type V and the C terminus of the b2-microglobulin and alpha-1-antitrypsin proteins. No CRS-specific peptides could be predicted as bioactive. Conclusion Specific urinary peptides significantly associated with CRS, but not with HF or CKD, could be identified. These data indicate that on a molecular level, CRS is not merely the result of a combination of HF and CKD, but may represent a distinct pathology, defined via specific proteomic changes. It is expected that interpretation of these findings in the context of existing literature as well as in vitro activity assays will help to understand their biological relevance in CRS.

scholarly journals PATH-19. TUMOR HETEROGENEITY IN GLIOMAS: A PILOT STUDY OF HISTOPATHOLOGY-ASSOCIATED PROTEOME PROFILES ASSESSED BY LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY OF FFPE SAMPLES

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi147-vi147
Author(s):  
Aline Paixao Becker ◽  
Erica Hlavin Bell ◽  
S Jaharul Haque ◽  
Joseph McElroy ◽  
Jessica Fleming ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Tumor Heterogeneity ◽  
High Expression ◽  
Tandem Mass ◽  
Chromatography Tandem Mass Spectrometry ◽  
Ms Analysis ◽  
Ffpe Samples ◽  
Liquid Chromatography Tandem Mass

Abstract Herein, we aimed to scrutinize tumor heterogeneity of infiltrative gliomas based on histopathological phenotypes, through proteomic profiling of formalin-fixed, paraffin embedded (FFPE) tissue. FFPE tissues are promising samples for proteomic studies, which can support the elucidation of glioma evolution and identify therapeutically vulnerable proteins and signaling pathways that drive recurrence and resistance mechanisms. We represented 2–3 adjacent, phenotypically distinct areas from 12 grade II-IV gliomas diagnosed according to the 2016 WHO classification, in a total of 35 samples (1.0mm cores), that were analyzed employing liquid chromatography tandem mass spectrometry (LC-MS/MS) for label-free expression proteomics. The statistical analysis was performed using R and Qlucore™ omics explorer software. Overall, 9222 peptides were mapped to 1758 non-redundant proteins, 320 of which had a significant (p< 0.05) differential expression in glioblastomas versus lower grade gliomas (Wilcoxon test comparing average expression). Principal component analysis (PCA) of the whole set of proteins showed clustering of the samples by tumor grade and IDH status. Unsupervised hierarchical analysis of the most significantly expressed proteins (p= 0.01, FDR= 0.05) showed that IDHwt gliomas had high expression of proteins related to cell movement, DNA structure, and fatty acid metabolism throughout the samples. IDHmut gliomas largely displayed high expression of mitochondrial enzymes related to energy production and neurotransmitter metabolism, with subsets closely related to 1p19q status and histological grade. Importantly, we demonstrated that LC-MS/MS analysis of FFPE core samples is feasible and enables recognition of different proteome signatures across histopathological phenotypes within a single tumor. This is the first study, to our knowledge, exploring proteome profiles addressing histopathological heterogeneity in gliomas by LC-MS/MS analysis of FFPE samples, which warrants further validation in independent datasets including ones that utilize frozen specimens. FUNDING: R01CA108633, R01CA169368, RC2CA148190, U10CA180850-01 (NCI), Brain Tumor Funders Collaborative Grant, and the Ohio State University CCC (all to AC).

scholarly journals Lysyl hydroxylation in collagens from hyperplastic callus and embryonic bones

1992 ◽  
Vol 282 (2) ◽  
pp. 313-318 ◽  
Author(s):  
H W Lehmann ◽  
M Bodo ◽  
C Frohn ◽  
A Nerlich ◽  
D Rimek ◽  
...  
Keyword(s):  
Fetal Development ◽  
Bone Repair ◽  
Callus Tissue ◽  
Collagen Type ◽  
Compact Bone ◽  
Collagen Type I ◽  
Postnatal Period ◽  
Type I ◽  
Collagen Types ◽  
Hyperplastic Callus

Tissue from two patients with osteogenesis imperfecta suffering from a hyperplastic callus was studied. Although collagen type I from the compact bone and the skin and fibroblast cultures of these patients showed normal lysyl hydroxylation, collagen types I, II, III and V from the callus tissue were markedly overhydroxylated. Furthermore, the overhydroxylation of lysine residues covered almost equally the entire alpha 1 (I) collagen chain, as demonstrated by the analysis of individual CNBr-derived peptides. In addition, collagen type I was isolated from femoral compact bone of 33 individuals who died between the 16th week of gestational age and 22 years. Lysyl hydroxylation rapidly decreased in both collagen alpha 1 (I) and alpha 2 (I) chains during fetal development, and only little in the postnatal period. The transient increase in lysyl hydroxylation and the involvement of various collagen types in callus tissue argue for a regulatory mechanism that may operate in bone repair and during fetal development.

Immunohistochemical Localization of Collagen Types I, III, and IV, Laminin, Fibronectin, and Keratin in the Endolymphatic Sac

2000 ◽  
Vol 109 (2) ◽  
pp. 180-186 ◽  
Author(s):  
Teruhiko Harada ◽  
Youngki Kim ◽  
Steven K. Juhn ◽  
Yasuo Sakakura
Keyword(s):  
Collagen Type ◽  
Collagen Type I ◽  
Immunohistochemical Localization ◽  
Basement Membranes ◽  
Endolymphatic Sac ◽  
Type I ◽  
Collagen Types ◽  
Type Iv ◽  
Baseline Information ◽  
Subepithelial Layer

We have employed immunohistochemistry to obtain baseline information on the molecular constituents of the extracellular matrix (ECM) of the endolymphatic duct (ED) and endolymphatic sac (ES) of the chinchilla. The results demonstrated that collagen types I and III were distributed in the subepithelial layer in the ED and ES, type IV collagen and laminin in the basement membranes, and fibronectin in the subepithelial layer and partly in the conglomerated cells in the ES. Collagen type III was diffusely distributed in the whole subepithelial layer of the ES, whereas collagen type I was concentrated densely in the deep layer of the interstitium, although gradually, the cuboidal epithelium in the ES was transformed into a flatter type in the ED. The epithelial cells of the ED and ES were clearly positive for keratin. This study deals, in particular, with the normal distribution of ECM components of the ED and ES of the chinchilla.

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