Building Mass Spectrometry Spectral Libraries of Human Cancer Cell Lines

Faktor, Jakub; Bouchal, Pavel

doi:10.14735/amko20164s54

Cited by 6 publications

(5 citation statements)

References 9 publications

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“…In addition, several spectral library tools were also developed for identification with peptide modifications, such as QuickMod 10 and pMatch 11 for post-translational modification, Hybrid for unanticipated modifications 12 , cross-linked peptide identification 13 , and GPQuest 14 for N-linked glycopeptides. Owing to improved sensitivity and turnaround time, several sample-specific libraries have been built, such as the neuropeptide database 15 , and a database for cancer cell lines 16 .…”

Section: Introductionmentioning

confidence: 99%

“…Owing to improved sensitivity and turnaround time, several sample-specific libraries have been built, such as the neuropeptide database 15 and a database for cancer cell lines. 16 In recent years, isobaric labeling-based relative quantification approaches, such as tandem mass tag (TMT), have emerged as the mainstream method for multiplexed protein quantification. 17 In the Orbitrap instruments, high energy collisional dissociation (HCD) is implemented, in which higher energy and shorter activation time are used to generate product ions, including low-mass regions, such as TMT reporter ions, y 1 , and b 1 ions.…”

Section: ■ Introductionmentioning

confidence: 99%

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

et al. 2018

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Tandem mass tag (TMT)-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a proven approach for large-scale multiplexed protein quantification. However, the identification of TMT-labeled peptides is compromised by the labeling during traditional sequence database searches. In this study, we aim to use a spectral library search to increase the sensitivity and specificity of peptide identification for TMT-based MS data. Compared to MS/MS spectra of unlabeled peptides, the spectra of TMT-labeled counterparts usually display intensified b ions, suggesting that TMT labeling can alter product ion patterns during MS/MS fragementation. We compiled 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). A mouse TMT spectral library of similar size was also constructed. The libraries were subsequently appended with decoy spectra to evaluate the false discovery rate, which was validated by a simulated null TMT data set. The performance of the library search was further optimized by removing TMT reporter ions and selecting an appropriate library construction method. Finally, we searched a human TMT data set against the spectral library to demonstrate that the spectral library outperformed the sequence database. Both human and mouse TMT libraries were made publicly available to the research community.

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Section: Introductionmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

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

et al. 2018

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“…The spectral library sample was prepared by equimolar pooling of all samples. The spectral library was measured as described in ( Faktor and Bouchal, 2016 ). Briefly, pooled spectral library samples were measured in information-dependent mode (IDA).…”

Section: Methodsmentioning

confidence: 99%

CHIP-dependent regulation of the actin cytoskeleton is linked to neuronal cell membrane integrity

et al. 2021

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Summary CHIP is an E3-ubiquitin ligase that contributes to healthy aging and has been characterized as neuroprotective. To elucidate dominant CHIP-dependent changes in protein steady-state levels in a patient-derived human neuronal model, CHIP function was ablated using gene-editing and an unbiased proteomic analysis conducted to compare knock-out and wild-type isogenic induced pluripotent stem cell (iPSC)-derived cortical neurons. Rather than a broad effect on protein homeostasis, loss of CHIP function impacted on a focused cohort of proteins from actin cytoskeleton signaling and membrane integrity networks. In support of the proteomics, CHIP knockout cells had enhanced sensitivity to induced membrane damage. We conclude that the major readout of CHIP function in cortical neurons derived from iPSC of a patient with elevate α-synuclein, Parkinson's disease and dementia, is the modulation of substrates involved in maintaining cellular “health”. Thus, regulation of the actin cytoskeletal and membrane integrity likely contributes to the neuroprotective function(s) of CHIP.

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“…The separated peptides were ionised in the nanoelectrospray. The sample for a spectral library was measured in IDA mode as described in Faktor and Bouchal, 2016 [57]. Brie y, the full scan was set to cover a m/z range from 400 Th to 1200 Th and following, in the MS/MS mode, the top 20 most intensive precursors were selected and fragmented during each cycle.…”

Section: Spectral Library Measurement and Swath Ms Data Acquisitionmentioning

confidence: 99%

Characterization of the AGR2-NPM3 axis uncovers the AGR2 involvement in PD-L1 regulation in colorectal cancer

Martisova,

Faktor,

Sosolikova

et al. 2024

Preprint

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Despite extensive research, the molecular role of AGR2 in the progression and metastasis of colorectal cancer (CRC) has not been fully characterized. We used quantitative mass spectrometry (SWATH MS) to identify differentially expressed proteins in paired CRC cell models of the SW480 and SW620 cell lines in response to AGR2 protein level manipulation. Relying on the results from SWATH MS and subsequent immunochemical validation, we selected NMP3 as the top candidate protein associated with AGR2 in CRC tumour cells in our screen. RT‒qPCR and immunochemical analysis confirmed the involvement of AGR2-mediated regulation of NPM3 at the transcriptional and posttranscriptional levels. Since PD-L1 is a constituent of the NPM3 regulatory axis, we aimed to correlate the changes in PD-L1 to the differential expression of AGR2 in our cell models. We found that AGR2 positively regulates PD-L1 levels in both SW480 and SW620 cell lines; additionally, several different CRC patient transcriptome cohorts confirmed the association of AGR2 with PD-L1. Our work reveals a new AGR2-NPM3 regulatory axis and the involvement of AGR2 in the regulation of PD-L1, which paves the way for the association of AGR2 with immune evasion in CRC cells.

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Building Mass Spectrometry Spectral Libraries of Human Cancer Cell Lines

Cited by 6 publications

References 9 publications

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

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

CHIP-dependent regulation of the actin cytoskeleton is linked to neuronal cell membrane integrity

Characterization of the AGR2-NPM3 axis uncovers the AGR2 involvement in PD-L1 regulation in colorectal cancer

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