Parallel reaction monitoring on a Q Exactive mass spectrometer increases reproducibility of phosphopeptide detection in bacterial phosphoproteomics measurements.
J Proteomics
; 189: 60-66, 2018 10 30.
Article
em En
| MEDLINE
| ID: mdl-29605292
ABSTRACT
Increasing number of studies report the relevance of protein Ser/Thr/Tyr phosphorylation in bacterial physiology, yet the analysis of this type of modification in bacteria still presents a considerable challenge. Unlike in eukaryotes, where tens of thousands of phosphorylation events likely occupy more than two thirds of the proteome, the abundance of protein phosphorylation is much lower in bacteria. Even the state-of-the-art phosphopeptide enrichment protocols fail to remove the high background of abundant unmodified peptides, leading to low signal intensity and undersampling of phosphopeptide precursor ions in consecutive data-dependent MS runs. Consequently, large-scale bacterial phosphoproteomic datasets often suffer from poor reproducibility and a high number of missing values. Here we explore the application of parallel reaction monitoring (PRM) on a Q Exactive mass spectrometer in bacterial phosphoproteome analysis, focusing especially on run-to-run sampling reproducibility. In multiple measurements of identical phosphopeptide-enriched samples, we show that PRM outperforms data-dependent acquisition (DDA) in terms of detection frequency, reaching almost complete sampling efficiency, compared to 20% in DDA. We observe a similar trend over multiple heterogeneous phosphopeptide-enriched samples and conclude that PRM shows a great promise in bacterial phosphoproteomics analyses where reproducible detection and quantification of a relatively small set of phosphopeptides is desired. SIGNIFICANCE:
Bacterial phosphorylated peptides occur in low abundance compared to their unmodified counterparts, and are therefore rarely reproducibly detected in shotgun (DDA) proteomics measurements. Here we show that parallel reaction monitoring complements DDA analyses and makes detection of known, targeted phosphopeptides more reproducible. This will be of significance in replicated MS measurements that have a goal to reproducibly detect and quantify phosphopeptides of interest.Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Fosfopeptídeos
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Proteínas de Bactérias
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Proteoma
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Proteômica
Tipo de estudo:
Diagnostic_studies
/
Evaluation_studies
Idioma:
En
Revista:
J Proteomics
Assunto da revista:
BIOQUIMICA
Ano de publicação:
2018
Tipo de documento:
Article
País de afiliação:
Alemanha