Staes - PQS_peptides

Quantifiable peptide tags for targeted proteomics.

  • Organism: Homo sapiens
  • Instrument: TSQ Vantage
  • SpikeIn: Yes
Abstract
The use of protein tagging to facilitate detailed characterization of target proteins has not only revolutionized cell biology, but also enabled biochemical analysis through efficient recovery of the protein complexes wherein the tagged proteins reside. The endogenous use of these tags for detailed protein characterization is widespread in lower organisms that allow for efficient homologous recombination. With the recent advances in genome engineering, tagging of endogenous proteins is now within reach for most experimental systems, including mammalian cell lines cultures. In this work, we describe the selection of peptides with ideal mass spectrometry characteristics for use in quantification of tagged proteins using targeted proteomics. We mined the proteome of the hyperthermophile Pyrococcus furiosus to obtain two peptides that are unique in the proteomes of all known model organisms (proteotypic) and allow sensitive quantification of target proteins in a complex background. By combining these ’Proteotypic peptides for Quantification by SRM’ (PQS peptides) with epitope tags, we demonstrate their use in co-immunoprecipitation experiments upon transfection of protein pairs, or after introduction of these tags in the endogenous proteins through genome engineering. Endogenous protein tagging for absolute quantification provides a powerful extra dimension to protein analysis, allowing the detailed characterization of endogenous proteins.
Created on 5/25/16 10:44 AM

Dear visitor,

 

The attached file contains an excel document that consists of 2 worksheets.
Sheet 1 clarifies which Skyline library was used for each figure.
Sheet 2 is a legend for each Skyline file showing which replicate corresponds to a certain sample (e.g. which constructs were transfected, what cell line was used, used compound, which affinity sequence was used for pullout experiments, ...)  

 

Kind regards,

Giel Vandemoortele

  Attached Files  
   
 Skyline libraries legend PQS paper.xlsx

Clustergrammer Heatmap
Flag FileDownloadCreatedProteinsPeptidesPrecursorsTransitionsReplicates
pfu_2016-01-21_09-10-33.sky.zip (1 GB)2016-05-255821,5812,76722,08660
3_p53_mdm2_overexpression.zip (732 KB)2016-05-251242618
oPT_srmsamples130110_onlyDigests_50ul.zip (203 KB)2016-05-25115151121
endo_enriched_HA_pullouts_only.zip (243 KB)2016-05-25124196
direct_endogenous_not_enriched.zip (245 KB)2016-05-25124206
endo_enriched_flag_pullouts_only.zip (484 KB)2016-05-251241912
FLAGLCP2_GRAP2.zip (489 KB)2016-05-252242812
Mal_MyD88_Mock.zip (736 KB)2016-05-252243018
oPT_srm_3BestPep_RESULTS_Giel_LOD.zip (1 MB)2016-05-251353348

Dear visitor,

 

The skyline file "pfu_2016-01-21_09-10-33.sky.zip" present in the section "Targeted MS Runs" contains all results of the pyrococcus furiosus discovery runs.

The dat files resulting from the mascot searches are embedded as a spectral library, whereas the raw files are imported as result files. In this way all peptides can be visualized in the raw files together with the library match.

 

Kind regards,

An Staes