Systematic Errors in Peptide and Protein Identification and Quantification by Modified Peptides

Bogdanow, Boris; Zauber, Henrik; Selbach, Matthias

doi:10.1074/mcp.m115.055103

Cited by 66 publications

(61 citation statements)

References 41 publications

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“…Additionally, when a modified peptide matches a precursor mass, all possible localization combinations have to be tested and scored. It should also be noted that a substantial fraction of the spectra that end up as unidentified may arise from modified peptides …”

Section: Management Of the Search Spacementioning

confidence: 99%

“…should also be noted that a substantial fraction of the spectra that end up as unidentified may arise from modified peptides. 68,69 To illustrate this effect, we used the example dataset of the Figure 4B shows the number of peptides to evaluate in every search space enlargement condition listed above. As expected, the total number of peptides evaluated follows the increase in median number of peptides per precursor.…”

Section: Protein Sequence Databasesmentioning

confidence: 99%

See 1 more Smart Citation

Anatomy and evolution of database search engines—a central component of mass spectrometry based proteomic workflows

Verheggen

Ræder

Berven

et al. 2017

Mass Spectrometry Reviews

106

104

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Sequence database search engines are bioinformatics algorithms that identify peptides from tandem mass spectra using a reference protein sequence database. Two decades of development, notably driven by advances in mass spectrometry, have provided scientists with more than 30 published search engines, each with its own properties. In this review, we present the common paradigm behind the different implementations, and its limitations for modern mass spectrometry datasets. We also detail how the search engines attempt to alleviate these limitations, and provide an overview of the different software frameworks available to the researcher. Finally, we highlight alternative approaches for the identification of proteomic mass spectrometry datasets, either as a replacement for, or as a complement to, sequence database search engines.

show abstract

Section: Management Of the Search Spacementioning

confidence: 99%

Section: Protein Sequence Databasesmentioning

confidence: 99%

Anatomy and evolution of database search engines—a central component of mass spectrometry based proteomic workflows

Verheggen

Ræder

Berven

et al. 2017

Mass Spectrometry Reviews

106

104

View full text Add to dashboard Cite

show abstract

“…As shown in a number of studies, restricted proteome search engines cannot identify peptides with unexpected modifications, which is a major reason underlying the low identification rate [8][9][10][11] . Therefore, a feasible solution involves enlarging the search space to retrieve more peptide candidates with any type of modification.…”

Section: Introductionmentioning

confidence: 99%

Open-pFind enables precise, comprehensive and rapid peptide identification in shotgun proteomics

Yang

et al. 2018

Preprint

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Shotgun proteomics has grown rapidly in recent decades, but a large fraction of tandem mass spectrometry (MS/MS) data in shotgun proteomics are not successfully identified. We have developed a novel database search algorithm, Open-pFind, to efficiently identify peptides even in an ultra-large search space which takes into account unexpected modifications, amino acid mutations, semi-or non-specific digestion and co-eluting peptides. Tested on two metabolically labeled MS/MS datasets, Open-pFind reported 50.5-117.0% more peptide-spectrum matches (PSMs) than the seven other advanced algorithms. More importantly, the Open-pFind results were more credible judged by the verification experiments using stable isotopic labeling. Tested on four additional large-scale datasets, 70-85% of the spectra were confidently identified, and high-quality spectra were nearly completely interpreted by Open-pFind. Further, Open-pFind was over 40 times faster than the other three open search algorithms and 2-3 times faster than three restricted search algorithms. Re-analysis of an entire human proteome dataset consisting of ~25 million spectra using Open-pFind identified a total of 14,064 proteins encoded by 12,723 genes by requiring at least two uniquely identified peptides. In this search results, Open-pFind also excelled in an independent test for false positives based on the presence or absence of olfactory receptors. Thus, a practical use of the open search strategy has been realized by Open-pFind for the truly global-scale proteomics experiments of today and in the future.

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“…3B and C). Due to the fact that the presence of multiple modifications dramatically increases the database search space, the MS-based identification of peptides harboring several modifications is still challenging (41,42). Thus, we assume that only a subgroup of pUL69 phosphorylation sites could be detected by MS-based analyses.…”

Section: Discussionmentioning

confidence: 99%

Phosphosite Analysis of the Cytomegaloviral mRNA Export Factor pUL69 Reveals Serines with Critical Importance for Recruitment of Cellular Proteins Pin1 and UAP56/URH49

Thomas

Müller

Horn

et al. 2020

J Virol

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Human cytomegalovirus (HCMV) encodes the viral mRNA export factor pUL69, which facilitates the cytoplasmic accumulation of mRNA via interaction with the cellular RNA helicase UAP56 or URH49. We reported previously that pUL69 is phosphorylated by cellular CDKs and the viral CDK-like kinase pUL97. Here, we set out to identify phosphorylation sites within pUL69 and to characterize their importance. Mass spectrometry-based phosphosite mapping of pUL69 identified 10 serine/threonine residues as phosphoacceptors. Surprisingly, only a few of these sites localized to the N terminus of pUL69, which could be due to the presence of additional posttranslational modifications, like arginine methylation. As an alternative approach, pUL69 mutants with substitutions of putative phosphosites were analyzed by Phos-tag SDS-PAGE. This demonstrated that serines S46 and S49 serve as targets for phosphorylation by pUL97. Furthermore, we provide evidence that phosphorylation of these serines mediates cis/trans isomerization by the prolyl isomerase Pin1, thus forming a functional Pin1 binding motif. Surprisingly, while abrogation of the Pin1 motif did not affect the replication of recombinant cytomegaloviruses, mutation of serines next to the interaction site for UAP56/URH49 strongly decreased viral replication. This was correlated with a loss of UAP56/URH49 recruitment. Intriguingly, the critical serines S13 and S15 were located within a sequence resembling the UAP56 binding motif (UBM) of cellular mRNA adaptor proteins like REF and UIF. We propose that betaherpesviral mRNA export factors have evolved an extended UAP56/URH49 recognition sequence harboring phosphorylation sites to increase their binding affinities. This may serve as a strategy to successfully compete with cellular mRNA adaptor proteins for binding to UAP56/URH49. IMPORTANCE The multifunctional regulatory protein pUL69 of human cytomegalovirus acts as a viral RNA export factor with a critical role in efficient replication. Here, we identify serine/threonine phosphorylation sites for cellular and viral kinases within pUL69. We demonstrate that the pUL97/CDK phosphosites within alpha-helix 2 of pUL69 are crucial for its cis/trans isomerization by the cellular protein Pin1. Thus, we identified pUL69 as the first HCMV-encoded protein that is phosphorylated by cellular and viral serine/threonine kinases in order to serve as a substrate for Pin1. Furthermore, our study revealed that betaherpesviral mRNA export proteins contain extended binding motifs for the cellular mRNA adaptor proteins UAP56/URH49 harboring phosphorylated serines that are critical for efficient viral replication. Knowledge of the phosphorylation sites of pUL69 and the processes regulated by these posttranslational modifications is important in order to develop antiviral strategies based on a specific interference with pUL69 phosphorylation.

show abstract

Systematic Errors in Peptide and Protein Identification and Quantification by Modified Peptides

Cited by 66 publications

References 41 publications

Anatomy and evolution of database search engines—a central component of mass spectrometry based proteomic workflows

Anatomy and evolution of database search engines—a central component of mass spectrometry based proteomic workflows

Open-pFind enables precise, comprehensive and rapid peptide identification in shotgun proteomics

Phosphosite Analysis of the Cytomegaloviral mRNA Export Factor pUL69 Reveals Serines with Critical Importance for Recruitment of Cellular Proteins Pin1 and UAP56/URH49

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