U of Tuebingen - Ecoli_PRM_Phosphoproteomics

Parallel Reaction Monitoring on a Q Exactive Mass Spectrometer Increases Reproducibility of Phosphopeptide Detection in Bacterial Phosphoproteomics Measurements

  • Organism: E. coli
  • Instrument: Q exactive HF
  • SpikeIn: No
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.
Created on 4/19/18, 8:27 AM
Clustergrammer Heatmap
Flag FileDownloadCreatedProteinsPeptidesPrecursorsTransitionsReplicates
Figure4_Sample1_Ecoli_phospho_PRM_results_2018-03-05_15-26-51.sky.zip2018-04-1920212113715
Figure4_Sample2_Ecoli_phospho_PRM_results_2018-03-05_15-23-54.sky.zip2018-04-1965798560215
Figure1_Ecoli_proteome_unmodified_peptides_PRM_2018-03-09_12-54-04.sky.zip2018-04-198080808754
Figure2_Sample1_Ecoli_phospho_PRM_results_2018-03-05_15-17-37.sky.zip2018-04-192021211375
Figure2_Sample2_Ecoli_phospho_PRM_results_2018-03-05_15-15-19.sky.zip2018-04-196579856025