A Statistical Model for Identifying Proteins by Tandem Mass Spectrometry

Nesvizhskii, Alexey I.; Keller, Andrew; Kolker, Eugene; Aebersold, Ruedi

doi:10.1021/ac0341261

Cited by 4,385 publications

(3,941 citation statements)

References 42 publications

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“…Peptide identifications were accepted if they could be established with less than 1.0% false discovery by the Scaffold Local false discovery rate (FDR) algorithm and contained at least two identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm (Nesvizhskii, Keller, Kolker & Aebersold, 2003). …”

Section: Methodsmentioning

confidence: 99%

Oxidation resistance 1 is a novel senolytic target

et al. 2018

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SummaryThe selective depletion of senescent cells (SCs) by small molecules, termed senolytic agents, is a promising therapeutic approach for treating age‐related diseases and chemotherapy‐ and radiotherapy‐induced side effects. Piperlongumine (PL) was recently identified as a novel senolytic agent. However, its mechanism of action and molecular targets in SCs was unknown and thus was investigated. Specifically, we used a PL‐based chemical probe to pull‐down PL‐binding proteins from live cells and then mass spectrometry‐based proteomic analysis to identify potential molecular targets of PL in SCs. One prominent target was oxidation resistance 1 (OXR1), an important antioxidant protein that regulates the expression of a variety of antioxidant enzymes. We found that OXR1 was upregulated in senescent human WI38 fibroblasts. PL bound to OXR1 directly and induced its degradation through the ubiquitin‐proteasome system in an SC‐specific manner. The knockdown of OXR1 expression by RNA interference significantly increased the production of reactive oxygen species in SCs in conjunction with the downregulation of antioxidant enzymes such as heme oxygenase 1, glutathione peroxidase 2, and catalase, but these effects were much less significant when OXR1 was knocked down in non‐SCs. More importantly, knocking down OXR1 selectively induced apoptosis in SCs and sensitized the cells to oxidative stress caused by hydrogen peroxide. These findings provide new insights into the mechanism by which SCs are highly resistant to oxidative stress and suggest that OXR1 is a novel senolytic target that can be further exploited for the development of new senolytic agents.

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Section: Methodsmentioning

confidence: 99%

Oxidation resistance 1 is a novel senolytic target

et al. 2018

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“…The peptide identifications were accepted if they could be established at greater than 99.0% probability as assigned by the Peptide Prophet algorithm [49]. The protein identifications were accepted if they could be established at greater than 95% probability as assigned by the Protein Prophet algorithm [50]; were based on at least 2 identified peptides; and were detected in at least two out of three replicates (both biological and technical).…”

Section: Methodsmentioning

confidence: 99%

Comparative proteomics of twoMycoplasma hyopneumoniaestrains andMycoplasma flocculareidentified potential porcine enzootic pneumonia determinants

et al. 2018

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Mycoplasma hyopneumoniae and Mycoplasma flocculare are genetically similar bacteria, which coinhabit the porcine respiratory tract. These mycoplasmas share most of the known virulence factors, but, while M. hyopneumoniae causes porcine enzootic pneumonia (PEP), M. flocculare is a commensal species. To identify potential PEP determinants and provide novel insights on mycoplasma-host interactions, the whole cell proteomes of two M. hyopneumoniae strains, one pathogenic (7448) and other non-pathogenic (J), and M. flocculare were compared. A cell fractioning approach combined with mass spectrometry (LC-MS/MS) proteomics was used to analyze cytoplasmic and surface-enriched protein fractions. Average detection of ~ 50% of the predicted proteomes of M. hyopneumoniae 7448 and J, and M. flocculare was achieved. Many of the identified proteins were differentially represented in M. hyopneumoniae 7448 in comparison to M. hyopneumoniae J and M. flocculare, including potential PEP determinants, such as adhesins, proteases, and redox-balancing proteins, among others. The LC-MS/MS data also provided experimental validation for several genes previously regarded as hypothetical for all analyzed mycoplasmas, including some coding for proteins bearing virulence-related functional domains. The comprehensive proteome profiling of two M. hyopneumoniae strains and M. flocculare provided tens of novel candidates to PEP determinants or virulence factors, beyond those classically described.

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“…Protein probabilities were assigned by the Protein Prophet algorithm. 59 Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Normalization was performed iteratively (across samples and spectra) by subtracting the average ratios in log-space.…”

Section: Methodsmentioning

confidence: 99%

Transfer-RNA-mediated enhancement of ribosomal proteins S6 kinases signaling for cell proliferation

et al. 2018

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While transfer-RNAs (tRNAs) are known to transport amino acids to ribosome, new functions are being unveiled from tRNAs and their fragments beyond protein synthesis. Here we show that phosphorylation of 90-kDa RPS6K (ribosomal proteins S6 kinase) was enhanced by tRNALeu overexpression under amino acids starvation condition. The phosphorylation of 90-kDa RPS6K was decreased by siRNA specific to tRNALeu and was independent to mTOR (mammalian target of rapamycin) signaling. Among the 90-kDa RPS6K family, RSK1 (ribosomal S6 kinase 1) and MSK2 (mitogen-and stress-activated protein kinase 2) were the major kinases phosphorylated by tRNALeu overexpression. Through SILAC (stable isotope labeling by/with amino acids in cell culture) and combined mass spectrometry analysis, we identified EBP1 (ErbB3-binding protein 1) as the tRNALeu-binding protein. We suspected that the overexpression of free tRNALeu would reinforce ErbB2/ErbB3 signaling pathway by disturbing the interaction between ErbB3 and EBP1, resulting in RSK1/MSK2 phosphorylation, improving cell proliferation and resistance to death. Analysis of samples from patients with breast cancer also indicated an association between tRNALeu overexpression and the ErbB2-positive population. Our results suggested a possible link between tRNALeu overexpression and RSK1/MSK2 activation and ErbB2/ErbB3 signaling.

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A Statistical Model for Identifying Proteins by Tandem Mass Spectrometry

Cited by 4,385 publications

References 42 publications

Oxidation resistance 1 is a novel senolytic target

Oxidation resistance 1 is a novel senolytic target

Comparative proteomics of twoMycoplasma hyopneumoniaestrains andMycoplasma flocculareidentified potential porcine enzootic pneumonia determinants

Transfer-RNA-mediated enhancement of ribosomal proteins S6 kinases signaling for cell proliferation

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