Comparison between chaotropic and detergent‐based sample preparation workflow in tendon for mass spectrometry analysis

Kharaz, Yalda Ashraf; Zamboulis, Danae E.; Sanders, Karen; Comerford, Eithne; Clegg, Peter; Peffers, Mandy J.

doi:10.1002/pmic.201700018

Cited by 26 publications

(33 citation statements)

References 39 publications

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“…The measured rate constants of protein turnover will also be dependent on the protein extraction methods used. We chose GuHCl extraction for whole tendon as this has previously been shown to result in best proteome coverage for tendon (Ashraf Kharaz et al, 2017). It was not possible to use the same extraction method for laser-captured samples due to the small amount of starting material, therefore we used a method previously optimised for this sample type (Thorpe et al, 2016b).…”

Section: Discussionmentioning

confidence: 99%

“…Tendons were finely chopped, flash frozen in liquid nitrogen and pulverised in a dismembrator (Sartorius, mikro-dismembrator U, 1800rpm, 2 mins.). Proteins were extracted using previously optimised methodologies (Ashraf Kharaz et al, 2017). Briefly, following deglycosylation in chondroitinase ABC, proteins were extracted in guanidine hydrocholoride (GuHCl) as described previously (Kharaz et al, 2016).…”

Section: Methodsmentioning

confidence: 99%

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Heterogeneity of proteome dynamics between connective tissue phases of adult tendon

Simpson

Choi²,

Wang³

et al. 2020

Preprint

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1Maintenance of connective tissue integrity is fundamental to sustain function, requiring 2 protein turnover to repair damaged tissue. However, connective tissue proteome dynamics 3 remain largely undefined, as do differences in turnover rates of individual proteins in the 4 collagen and glycoprotein phases of connective tissue extracellular matrix (ECM). Here, we 5 investigate proteome dynamics in the collagen and glycoprotein phases of connective tissues 6 by exploiting the spatially distinct fascicular (collagen-rich) and interfascicular (glycoprotein-7 rich) ECM phases of tendon. Using isotope labelling, mass spectrometry and bioinformatics, 8 we calculate turnover rates of individual proteins within rat Achilles tendon and its ECM 9 phases. Our results demonstrate complex proteome dynamics in tendon, with ~1000-fold 10 differences in protein turnover rates, and overall faster protein turnover within the 11 glycoprotein-rich interfascicular matrix compared to the collagen-rich fascicular matrix. 12These data provide insights into the complexity of proteome dynamics in tendon, likely 13 required to maintain tissue homeostasis. 14

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Heterogeneity of proteome dynamics between connective tissue phases of adult tendon

Simpson

Choi²,

Wang³

et al. 2020

Preprint

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“…This could reflect differences in extractability of previously synthesised heterotrimeric and homotrimeric collagen (I) molecules due to altered crosslinking (Pfeiffer et al, 2005). In this study, we used an extraction method for the insoluble pellet previously shown to increase the identification of collagenous proteins (Ashraf Kharaz et al, 2017) However, it may be that pepsinisation of the insoluble pellet (Peffers et al, 2014) to break mature crosslinks between telopeptides would have released more homotrimeric collagen (I), and hence alpha-1(I) chain (COL1A1) peptides from Dupuytren's tissue.…”

Section: Discussionmentioning

confidence: 99%

“…Combined flow-throughs were acidified with Trifluoroacetic acid to 0.2% (v/v). Incubation media was bound to and digested off Strataclean beads as previously described (Angi et al, 2016;Ashraf Kharaz et al, 2017). A yeast enolase peptide standard was added to each sample, though this was not utilised for later quantification.…”

Section: Quantitative Real-time Pcr (Qpcr)mentioning

confidence: 99%

Active synthesis of type I collagen homotrimer in Dupuytren’s fibrosis is unaffected by anti-TNF-α treatment

Williamson

Cooper

Lee

et al. 2020

Preprint

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Dupuytren's disease is a common fibroproliferative disease of the palmar fascia of the hand with severe cases treated surgically. The rate of disease recurrence following treatment is high and a continual production of matrisomal proteins could lead to disease recurrence. There is no animal model for Dupuytren's disease, but analysis of the surgically excised tissue provides an accessible means to study the mechanisms of human tissue fibrosis. Here we sought to determine how new synthesis and the composition of matrisomal proteins in Dupuytren's differs from normal palmar fascia samples, using metabolic labelling approaches and proteomics. Model non-fibrotic, but fibrous connective tissues, equine flexor tendon and canine cranial cruciate ligament, were used to analyse active collagen-1 protein synthesis in development, ageing and degenerative disease, where it was restricted to early development and ruptured tissue. Dupuytren's tissue was shown to actively synthesise type I collagen, a proportion of which comprised abnormal collagen (I) homotrimer (mean 14.3% ± 14.4), as well as fibronectin, matrix metalloproteinases (MMP2, MMP3) and their inhibitors; Tissue Inhibitor of Metalloproteinases 2 (TIMP2). Insulin-Like Growth Factor Binding Protein 7 (IGFBP7) was actively synthesised by Dupuytren's as well as control tissue. Label-free analysis implicated the TGFβ pathway in the matrisomal profile of Dupuytren's tissue whilst myocilin, a Wnt-pathway regulator, was noticeably more abundant in control samples. No collagen (I) neopeptides representing the major collagenase cleavage site were identified, however periostin neopeptides were abundant in Dupuytren's tissue and gelatin neopeptides in both tissue types. Synthesis of MMP-resistant collagen-1 homotrimer, together with altered β and Wnt signalling environments, could contribute to the persistence of the fibrotic tissue and disease recurrence following treatment.

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“…Proteomics. Proteomics was performed as reported in previous studies (3,27,40) Raw spectra were converted to mgf files using Proteome Discovery (Thermo Scientific, Loughborough, UK) and resulting files were searched against the UniProt mouse sequence database using a Mascot server (Matrix Science, London, UK). Search parameters used were: peptide mass tolerance, 10ppm; fragment mass tolerance, 0.01Da; +1,+2,+3 ions; missed cleavages 1; instrument type ESI-TRAP.…”

Section: Methodsmentioning

confidence: 99%

Multiomics Analysis of the mdx/mTR Mouse Model of Duchenne Muscular Dystrophy

Pelt

Kharaz

Sarver

et al. 2019

Preprint

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Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disease characterized by extensive muscle weakness. Patients with DMD lack a functional dystrophin protein, which transmits force and organizes the cytoskeleton of skeletal muscle. Multiomic studies evaluate combined changes in the transcriptome, proteome, and metabolome, and have been proposed as a way to obtain novel insight about disease processes from preclinical models.We therefore sought to use this approach to study pathological changes in dystrophic muscles.We evaluated hindlimb muscles of male mdx/mTR mice, which lack a functional dystrophin protein and have deficits in satellite cell abundance and proliferative capacity. Wild type (WT) C57BL/6J mice served as controls. Muscle fiber contractility was measured, along with changes in the transcriptome using RNA sequencing, and in the proteome, metabolome, and lipidome using mass spectroscopy. While mdx/mTR mice displayed gross pathological changes and continued cycles of degeneration and regeneration, we found no differences in fiber contractility between strains. However, there were numerous changes in the transcriptome and proteome related to protein balance, contractile elements, extracellular matrix, and metabolism. There was only a 53% agreement in fold change data between the proteome and transcriptome, highlighting the need to study protein abundance along with gene expression measures. Numerous changes in markers of skeletal muscle metabolism were observed, with dystrophic muscles exhibiting elevated glycolytic metabolites. These findings highlight the utility of multiomics in studying muscle disease, and provide additional insight into the pathological changes in dystrophic muscles that might help to guide evidence-based exercise prescription in DMD patients.

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Comparison between chaotropic and detergent‐based sample preparation workflow in tendon for mass spectrometry analysis

Cited by 26 publications

References 39 publications

Heterogeneity of proteome dynamics between connective tissue phases of adult tendon

Heterogeneity of proteome dynamics between connective tissue phases of adult tendon

Active synthesis of type I collagen homotrimer in Dupuytren’s fibrosis is unaffected by anti-TNF-α treatment

Multiomics Analysis of the mdx/mTR Mouse Model of Duchenne Muscular Dystrophy

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