Spectral Library Search Improves Assignment of TMT Labeled MS/MS Spectra

Shen, Jianqiao; Pagala, Vishwajeeth; Breuer, Alex Michael; Peng, Junmin; Ma, Bin; Wang, Xusheng

doi:10.1021/acs.jproteome.8b00594

Cited by 22 publications

(24 citation statements)

References 30 publications

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“…A human TMT spectral library of 401,168 unique peptides of high quality from millions of peptide-spectrum matches in tens of profiling projects, matching to 14,048 nonredundant proteins (13,953 genes) [25] was used with MSPepSearch. Fixed search parameters included trypsin, one missed cleavage allowed, TMT-six plex modification and carbamidomethylation at cysteine, while methionine oxidation, N-terminal carbamylation and N-terminal acetylation were set as dynamic.…”

Section: Discussionmentioning

confidence: 99%

Basement membrane proteins in various arterial beds from individuals with and without type 2 diabetes mellitus: a proteome study

Steffensen

Iversen

Hansen

et al. 2021

Cardiovasc Diabetol

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Background Basement membrane (BM) accumulation is a hallmark of micro-vessel disease in diabetes mellitus (DM). We previously reported marked upregulation of BM components in internal thoracic arteries (ITAs) from type 2 DM (T2DM) patients by mass spectrometry. Here, we first sought to determine if BM accumulation is a common feature of different arteries in T2DM, and second, to identify other effects of T2DM on the arterial proteome. Methods Human arterial samples collected during heart and vascular surgery from well-characterized patients and stored in the Odense Artery Biobank were analysed by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). We included ascending thoracic aortas (ATA) (n = 10 (type 2 DM, T2DM) and n = 10 (non-DM)); laser capture micro-dissected plaque- and media compartments from carotid plaques (n = 10 (T2DM) and n = 9 (non-DM)); and media- and adventitia compartments from ITAs (n = 9 (T2DM) and n = 7 (non-DM)). Results We first extended our previous finding of BM accumulation in arteries from T2DM patients, as 7 of 12 pre-defined BM proteins were significantly upregulated in bulk ATAs consisting of > 90% media. Although less pronounced, BM components tended to be upregulated in the media of ITAs from T2DM patients, but not in the neighbouring adventitia. Overall, we did not detect effects on BM proteins in carotid plaques or in the plaque-associated media. Instead, complement factors, an RNA-binding protein and fibrinogens appeared to be regulated in these tissues from T2DM patients. Conclusion Our results suggest that accumulation of BM proteins is a general phenomenon in the medial layer of non-atherosclerotic arteries in patients with T2DM. Moreover, we identify additional T2DM-associated effects on the arterial proteome, which requires validation in future studies.

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Section: Discussionmentioning

confidence: 99%

Basement membrane proteins in various arterial beds from individuals with and without type 2 diabetes mellitus: a proteome study

Steffensen

Iversen

Hansen

et al. 2021

Cardiovasc Diabetol

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“…There are two types of tagging strategies, which use either in vivo markers such as SILAC, or in vitro markers, such as iTRAQ [24]. Tandem mass tag (TMT)-labeling high-throughput screening technology [25], which uses isotopic tagging of the N-terminal amino group of the polypeptide and the amino group of lysine side chains. This technique can identify and compare the protein expression profiles of up to ten different samples simultaneously and be coupled with LC-MS for tandem analysis [26].…”

Section: Discussionmentioning

confidence: 99%

Proteomics and Bioinformatics Analysis of Cartilage in Post-Traumatic Osteoarthritis in a Mini-Pig Model of Anterior Cruciate Ligament Repair

Che

Gao

et al. 2020

Med Sci Monit

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Background: Although osteoarthritis (OA) is a degenerative disease that is increasingly common with age, the pathogenesis of post-traumatic OA (PTOA) is poorly understood. This study aimed to undertake proteomics and bioinformatics analysis of cartilage in PTOA in a mini-pig model of anterior cruciate ligament repair (ACLR). Material/Methods: The mini-pig model of PTOA involved autologous orthotopic ACLR. Screening and identification of differentially expressed proteins in the knee joint cartilage in the OA cartilage group were compared with the control group using tandem mass tag (TMT)-labeling liquid chromatography with tandem mass spectrometry (LC-MS-MS). A protein expression level >1.2 fold-change represented protein upregulation and <0.83 fold-change represented protein down-regulation Bioinformatics analysis included Gene Ontology (GO) functional annotation and the Kyoto Encyclopedia of Genes and Genome (KEGG) pathway enrichment analysis to determine the biological functions and pathways of proteins showing altered expression profiles associated with OA. Results: There were 2,950 proteins screened from the knee cartilage tissues of the OA model group using quantitative TMT-labeling LC-MS-MS. There were 491 proteins identified with altered expression profiles, 198 proteins were upregulated and 293 proteins were down-regulated in the OA cartilage group. GO function and KEGG pathway enrichment analysis of the 491 proteins identified their functions in cellular processes, metabolic processes, and biological regulation. Conclusions: Proteomics and bioinformatics analysis of cartilage in PTOA in a mini-pig model of ACLR identified OA-related proteins.

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“…We applied the simple premise that if plasma is a repository of molecules reflecting the biological and physiological status of the human body (95), then DIA MS (here SWATH) should be able to detect all spectra (96) including low abundance protein biomarkers. Although there are a number of ways to investigate this, including multiple library free approaches (97), in silico spectral libraries (98), labelling (99), spiking (100) or synthetic peptide approaches (101), we chose to use a library approach using a recombinant protein library of 36 low-medium abundance since it was hoped that a more comprehensive tryptic peptide representation for each protein could be achieved.…”

Section: Discussionmentioning

confidence: 99%

A Recombinant Protein Biomarker DDA Library Increases DIA Coverage of Low Abundance Plasma Proteins

Ahn

Kamath

Mohamedali

et al. 2020

Preprint

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Credible detection and quantification of low abundance proteins from human blood plasma is a major challenge in precision medicine biomarker discovery when using mass spectrometry (MS). Here, we employed a mixture of recombinant proteins in DDA libraries to subsequently detect cancer-associated low abundance plasma proteins using SWATH/DIA. The exemplar DDA recombinant protein spectral library (rPSL) was derived from tryptic digestion of 36 human recombinant proteins that had been previously implicated as possible cancer biomarkers in both our own and other studies. The rPSL was then used to identify proteins from non-depleted colorectal cancer (CRC) plasmas by SWATH-MS. Most (32/36) of the proteins in the rPSL were reliably identified in plasma samples, including 8 proteins (BTC, CXCL10, IL1B, IL6, ITGB6, TGFα, TNF, TP53) not previously detected using high-stringency MS in human plasmas according to PeptideAtlas. The rPSL SWATH-MS protocol was compared to DDA-MS using MARS-depleted and post-digestion peptide fractionated plasmas (here referred to as a human plasma DDA library). Of the 32 proteins identified using rPSL SWATH, only 12 were identified using DDA-MS. The 20 additional proteins exclusively identified by using the rPSL approach with SWATH were mostly lower abundance (i.e., <10ng/ml) plasma proteins. To mitigate FDR concerns, and replicating a more typical approach, the DDA rPSL was also merged into a human plasma DDA library. When SWATH identification was repeated using this merged library, the majority (33/36) of low abundance plasma proteins from the rPSL could still be identified using high-stringency HPP Guidelines v3.0 protein inference criteria.

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Spectral Library Search Improves Assignment of TMT Labeled MS/MS Spectra

Cited by 22 publications

References 30 publications

Basement membrane proteins in various arterial beds from individuals with and without type 2 diabetes mellitus: a proteome study

Basement membrane proteins in various arterial beds from individuals with and without type 2 diabetes mellitus: a proteome study

Proteomics and Bioinformatics Analysis of Cartilage in Post-Traumatic Osteoarthritis in a Mini-Pig Model of Anterior Cruciate Ligament Repair

A Recombinant Protein Biomarker DDA Library Increases DIA Coverage of Low Abundance Plasma Proteins

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