Dataset Information

Mining Missing Membrane Proteins by High-pH Reverse Phase StageTip Fractionation and Validation by Multiple Reaction Monitoring Mass Spectrometry

ABSTRACT: Several significant efforts have been made in the past decade to map all human proteome. However, more than 3000 proteins are grouped as missing having no evidence of translation. Membrane protein annotation of the current missing proteins list showed 34%to be missing membrane proteins, which are challenging even in standard shotgun proteomics. So as to find missing membrane proteins, we employed a highly sensitive and efficient peptide pre-fractionation of membrane enriched sample from 11 non-small cell lung cancer (NSCLC) cell lines of different EGFR mutation status by High-pH Reverse Phase StageTip. Besides, we analyzed 20 tumor and adjacent normal lung cancer tissue membrane samples to enhance identification coverage. With the use of multiple search engines and FDR analysis: X!Tandem and Comet followed by MAYU through TPP as well as Mascot followed PeptideShaker for FDR estimation, we were able to confidently identify 7701 proteins from which 5120 (66%) were annotated to be membrane proteins with below 1% FDR at protein, peptide and PSMs level having at least one unique peptide of 7 or more amino acid residues. Of this 181 proteins belong to the missing proteins in neXtProt (09-2014 release) and 75 are annotated to be missing membrane proteins. Furthermore we were able to validate 8 selected missing proteins (7 membrane proteins with TMH domain) using 10 synthetic peptides assisted multiple reaction monitoring (MRM) mass spectrometry. This study indicates membrane sub-proteome focus with efficient pre-fractionation and bioinformatics filtering followed by targeted validation by MRM is useful approach to assist mapping of all human proteome.

REANALYSED by: PAe005379PAe005374PAe005364PAe005358PAe005449PAe005443PAe005416PAe005414PAe005413PAe005336

INSTRUMENT(S): TripleTOF 5600, Synapt MS

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Lung

SUBMITTER: Baby Rorielyn Dimayacyac-Esleta

LAB HEAD: Baby Rorielyn T. Dimayacyac-Esleta

PROVIDER: PXD002224 | Pride | 2015-07-27

REPOSITORIES: Pride

ACCESS DATA

Publications

Mining Missing Membrane Proteins by High-pH Reverse-Phase StageTip Fractionation and Multiple Reaction Monitoring Mass Spectrometry.

Kitata Reta Birhanu RB Dimayacyac-Esleta Baby Rorielyn T BR Choong Wai-Kok WK Tsai Chia-Feng CF Lin Tai-Du TD Tsou Chih-Chiang CC Weng Shao-Hsing SH Chen Yi-Ju YJ Yang Pan-Chyr PC Arco Susan D SD Nesvizhskii Alexey I AI Sung Ting-Yi TY Chen Yu-Ju YJ

Journal of proteome research 20150806 9

Despite significant efforts in the past decade toward complete mapping of the human proteome, 3564 proteins (neXtProt, 09-2014) are still "missing proteins". Over one-third of these missing proteins are annotated as membrane proteins, owing to their relatively challenging accessibility with standard shotgun proteomics. Using nonsmall cell lung cancer (NSCLC) as a model study, we aim to mine missing proteins from disease-associated membrane proteome, which may be still largely under-represented. ...[more]

PMID: 26202522

Similar Datasets

Project description:The current state of proteomics requires a choice between targeted and discovery methods. The former provides exceptional quantitation and data completeness, but only with few analytes. Discovery proteomics can identify hundreds and thousands of proteins in a sample, but with poor quantitation and data completeness. We have established and optimized a method that combines targeted and data-independent acquisition for absolute quantitation of all plasma proteins in a single sequential window acquisition of all theoretical fragment ions (SWATH) acquisition run using a panel of spike-in standards (SIS). We compared the absolute quantitation (AQ) of SWATH and high-resolution multiple-reaction monitoring (MRM-HR) acquisition methods using the 100 protein PlasmaDive SIS panel spiked into human plasma. SWATH provided equivalent quantitation and differentially abundant protein profiles as MRM-HR. Absolute quantities of the SIS peptides from the SWATH data were used to estimate the absolute quantities (eAQ) for all the proteins in the run. The eAQ values provided similar quantitation and differentially abundant protein profiles as AQ and protein group (PG) values, and the eAQ method was the only scheme where all selected proteins were verified by MRM-HR. We applied the eAQ method to a cohort of 16 non-small cell lung cancer (NSCLC) patients receiving immunotherapeutics and found that fibronectin (FN1) and proteoglycan 4 (PRG4) were useful markers for predicting patients who would stay on these agents for at least 6 months (area under the curve of 0.8438 and 0.6735, respectively). Thirteen additional plasma samples confirmed that FN1 and PRG4 are putative biomarkers (positive predictive value is 100% and 85.7%, respectively) for a prolonged (>6 months) response to immunotherapeutic agents. In conclusion, we describe an optimized method for absolute quantification of all proteins in a SWATH run, and this approach is amenable for the identification of plasma biomarkers.

Dataset Information

Mining Missing Membrane Proteins by High-pH Reverse Phase StageTip Fractionation and Validation by Multiple Reaction Monitoring Mass Spectrometry

Publications

Mining Missing Membrane Proteins by High-pH Reverse-Phase StageTip Fractionation and Multiple Reaction Monitoring Mass Spectrometry.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure