JBEI - Workflow

A rapid methods development workflow for high-throughput quantitative proteomic applications

  • Organism: Saccharomyces cerevisiae, Pseudomonas putida KT2440, Escherichia coli
  • Instrument: 6460 Triple Quadrupole LC/MS
  • SpikeIn: No
  • Keywords: discovery proteomics, targeted proteomics, iRT calculator, methods development
  • Lab head: Chris Petzold
Abstract
Recent improvements in the speed and sensitivity of liquid chromatography-mass spectrometry systems have driven significant progress toward system-wide characterization of the proteome of many species. These efforts create large proteomic datasets that provide insight into biological processes and identify diagnostic proteins whose abundance changes significantly under different experimental conditions. Yet, these system-wide experiments are typically the starting point for hypothesis-driven, follow-up experiments to elucidate the extent of the phenomenon or the utility of the diagnostic marker, wherein many samples must be analyzed. Transitioning from a few discovery experiments to quantitative analyses on hundreds of samples requires significant resources both to develop sensitive and specific methods as well as analyze them in a high-throughput manner. To aid these efforts, we developed a workflow using data acquired from discovery proteomic experiments, retention time prediction, and standard-flow chromatography to rapidly develop targeted proteomic assays. With this workflow, one can target peptides in scheduled/dynamic acquisition methods from a shotgun proteomic dataset downloaded from online repositories, validate with appropriate control samples or standard peptides, and begin analyzing hundreds samples in only a few minutes.
Experiment Description
In this work we describe a workflow using data acquired from shotgun proteomic experiments and retention time prediction methods to rapidly develop high-throughput targeted proteomic assays. This workflow simplifies validation of peptides identified from shotgun proteomic experiments and significantly reduces development time of high-throughput quantitative SRM assays. It is enabled by highly reproducible peptide retention times from standard-flow chromatography systems, comprehensive spectral libraries produced from DDA experiments, and tools developed for Skyline, such as the iRT calculator. The workflow is instrument agnostic and performs well by using online resources from other labs to inform target peptide selection
Created on 10/11/18 11:33 PM
Clustergrammer Heatmap
Flag FileDownloadCreatedProteinsPeptidesPrecursorsTransitionsReplicates
Figure 2 - S.cerevisiae_IRT_Evaluation_500P_140 minutes_2018-09-10_14-34-48.sky.zip (3 MB)2018-10-112205005002,0002
Figure 2 - S.cerevisiae_IRT_Evaluation_500P_5 minutes_2018-09-10_14-30-24.sky.zip (1 MB)2018-10-112205005002,00012
Table 1 - P.putida F1_lib_2018-07-20_13-43-33.sky.zip (439 MB)2018-10-111,4835,2815,28115,8432
Figure 2 - S.cerevisiae_IRT_Evaluation_500P_30 minutes_2018-09-10_14-32-25.sky.zip (1 MB)2018-10-112205005002,0004
Figure 2 - EC_IRT_Evaluation_500P_30 minutes_2018-09-10_12-23-54.sky.zip (1 MB)2018-10-112145005001,9694
Figure 2 - EC_IRT_Evaluation_500P_5minutes_2018-09-10_11-31-08.sky.zip (905 KB)2018-10-112145005001,96910
Figure 2 - EC_IRT_Evaluation_500P_140 minutes_2018-09-10_12-23-16.sky.zip (4 MB)2018-10-112145005001,9692
Figure 3 - C. glutamine targets_amino acid biosynthesis_2018-09-07_15-21-38.sky.zip (182 KB)2018-10-111236412021
Figure 3 - P.putida targets_aromatic compound degradation_2018-09-07_15-16-37.sky.zip (155 KB)2018-10-111433331741
Figure 3 - R.toruloides targets_fatty acid biosynthesis_2018-09-07_15-09-20.sky.zip (247 KB)2018-10-112351512671
Figure 4 - Quantitation of pathway proteins of P. putida cultures grown in different carbon sources_2018-09-07_14-36-49.sky.zip (7 MB)2018-10-11791711719809
Table 1 - A.tumefaciens_lib_2018-07-20_13-31-12.sky.zip (60 MB)2018-10-114831,3651,3654,0952
Table 1 - E.coli DH5a_2018-07-20_11-19-56.sky.zip (830 MB)2018-10-111,0176,9949,33127,9936
Table 1 - S.cerevisiae_fusion_2018-07-20_15-39-51.sky.zip (3 GB)2018-10-114,18432,47635,322105,9615
Table 1 - R. torulodias_lib_2018-07-20_13-00-21.sky.zip (172 MB)2018-10-116821,9031,9035,7094
Table 1 - P. putida KT2440_2018-07-20_13-39-56.sky.zip (12 MB)2018-10-115491,4981,4984,4941
Table 1 - C. glutamicum_lib_2018-07-20_13-25-34.sky.zip (47 MB)2018-10-113581,1231,1233,3692