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Rapid High-pH Reverse Phase StageTip for Sensitive Small-Scale Membrane Proteomic Profiling

ABSTRACT: To increase the coverage of membrane protein identification,we developed high-pH reverse phase (Hp-RP) combined with stop-and-go extraction tip (StageTip) technique, as a fast (∼15 min.). Almost 5000 proteins (∼60% membrane proteins) can be identified in only 10 μg of membrane protein digests, showing the superior sensitivity of the Hp-RP StageTip approach.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Baby Rorielyn T. Dimayacyac-Esleta and Yu-Ju Chen

PROVIDER: PXD004904 | JPOST Repository | Thu Sep 08 00:00:00 BST 2016

REPOSITORIES: jPOST

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			Action	DRS
	RP1_F1_150820_inj02.raw	Raw
	RP1_F2_150820_inj02.raw	Raw
	RP1_F3_150820_inj02.raw	Raw
	RP1_F4_150820_inj02.raw	Raw
	RP1_F5_150820_inj02.raw	Raw

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Rapid High-pH Reverse Phase StageTip for Sensitive Small-Scale Membrane Proteomic Profiling.

Dimayacyac-Esleta Baby Rorielyn T BR Tsai Chia-Feng CF Kitata Reta Birhanu RB Lin Pei-Yi PY Choong Wai-Kok WK Lin Tai-Du TD Wang Yi-Ting YT Weng Shao-Hsing SH Yang Pan-Chyr PC Arco Susan D SD Sung Ting-Yi TY Chen Yu-Ju YJ

Analytical chemistry 20151120 24

Membrane proteins are crucial targets for cancer biomarker discovery and drug development. However, in addition to the inherent challenges of hydrophobicity and low abundance, complete membrane proteome coverage of clinical specimen is usually hindered by the requirement of large amount of starting materials. Toward comprehensive membrane proteomic profiling for small amounts of samples (10 μg), we developed high-pH reverse phase (Hp-RP) combined with stop-and-go extraction tip (StageTip) techni ...[more]

PMID: 26554430

Similar Datasets

Rapid High-pH Reverse Phase StageTip for Sensitive Small-Scale Membrane Proteomic Profiling

Project description:Membrane proteins are crucial targets for cancer biomarker discovery and drug development. However, in addition to the inherent challenges of hydrophobicity and low abundance, complete membrane proteome coverage of clinical specimen is usually hindered by the requirement of large amount of starting materials. Towards comprehensive membrane proteomic profiling for small amounts of samples (< 10 µg), we developed high-pH reverse phase (Hp-RP) combined with stop-and-go extraction tip (StageTip) technique, as a fast (~ 15 mins.), sensitive, reproducible, high-resolution and multiplexed fractionation method suitable for accurate quantification of the membrane proteome. This approach provided almost 2-fold enhanced detection of peptides within transmembrane helix domains compared with strong anion exchange (SAX) and strong cation exchange (SCX) StageTip techniques. More than 5000 proteins (63% membrane proteins) can be identified in only 10 µg of membrane protein digests, showing the superior sensitivity of the Hp-RP StageTip approach. The Hp-RP StageTip method enabled in-depth membrane proteome profiling of 11 lung cancer cell lines harboring different EGFR mutation status, which resulted in the identification of 3983 annotated membrane proteins. This provides the largest collection of reference peptide spectral data for lung cancer membrane sub-proteome. Finally, relative quantification of membrane proteins between Gefitinib-resistant and -sensitive lung cancer cell lines revealed several upregulated membrane proteins with key roles in lung cancer progression.

2015-11-16 | PXD001548 | Pride

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

Project description: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.

2015-07-27 | PXD002224 | Pride

Highly sensitive phosphoproteomics by tailoring solid-phase extraction to electrostatic repulsion-hydrophilic interaction chromatography

Project description:The dataset contains the data of key experiments for developing a novel and highly sensitive bottom-up phosphoproteomics strategy termed ERLIC-SCX/RP-LC-MS. We compared two SPE types (RP, SCX) for an ERLIC run with 7 fractions. The results were used to design a workflow for highly sensitive bottom-up phosphoproteomics (third experiment) combing ERLIC in 21 fractions with the fractions 1-9 cleaned by SCX-SPE and the fractions 10-21 by RP-SPE. Up to 50% of the fractions were subjected to LC-MS/MS analysis with 45 h of total MS time per replicate.

2014-12-02 | PXD001381 | Pride

Genome-wide Ifh1p occupancy at the OX growth phase and RC quiescent phase of the yeast metabolic cycle

Project description:We found ribosomal transcription factor Ifh1p is dynamically acetylated and phosphorylated in response to nutrient cues. ChIP-seq data revealed dynamic binding to ribosomal genes (RP) during the OX growth phase of the yeast metabolic cycle (YMC) when RP genes are highly induced, and weaker binding in the RC quiescent-like phase. Besides RP genes, our ChIP-seq data also reveals binding of Ifh1p to non-RP genes such as translation factors and metabolic genes.

2013-07-05 | GSE39147 | GEO

Genome-wide Ifh1p occupancy at the OX growth phase and RC quiescent phase of the yeast metabolic cycle

2013-07-05 | E-GEOD-39147 | biostudies-arrayexpress

The plasma proteome is favorably modified by a high protein diet but not by additional resistance training in older adults - a 17-week randomized controlled trial

Project description:Background: The age-related loss of muscle mass significantly contributes to the development of chronic diseases, loss of mobility and dependency on others, yet could be improved by an optimized lifestyle. Objective: The goal of this randomized controlled trial was to compare the influence of a habitual diet (CON) with either a diet containing the recommended protein intake (RP) or a high protein intake (HP), both with and without strength training, on the plasma proteome in older adults. Methods: 136 women and men (65-85 years) were randomly assigned to three intervention groups. CON continued their habitual diet; participants of the HP and RP group consumed either high protein or standard foods. After 6 weeks of dietary intervention, HP and RP groups additionally started a strength training intervention twice per week for 8 weeks. 24h dietary recalls were performed every 7 to 10 days. Body composition was assessed and blood taken. Plasma proteomics were assessed with LC-MS. Results: Participants of the HP group doubled their baseline protein intake from 0.80±0.31 g/kg BW/d to 1.63±0.36 g/kg BW/d; RP increased the protein intake from 0.89±0.28 g/kg BW/d to 1.06±0.26 g/kg BW/d. The CON group kept the protein intake stable throughout the study. Combined exercise and HP initiated notable changes, resulting in a reduction in bodyfat and increased muscle mass. Proteomics analyses revealed 14 significantly affected proteins by HP diet, regulating innate immune system, lipid transport and blood coagulation, yet the additional strength training did not elicit further changes. Conclusions: Combined HP and resistance exercise in healthy older adults seem to induce favorable changes in the body composition. Changes in the plasma proteome due to the high protein diet point to a beneficial impact for the innate immune system, lipid transport and blood coagulation system, all of which are involved in chronic disease development.

2022-10-15 | PXD033493 | Pride

TMT-based proteomics analysis of arachidonic acid (ARA) treated and control oocytes

Project description:Arachidonic acid (ARA) treated and control oocytes in 3 replicates were subjected to the 6-plex TMT labeling, HP-RP fractionation, and LC-MS/MS analysis.

2022-02-15 | PXD018753 | Pride

Alteration in gene expression in the lung following neonatal RSV infection

Project description:mRNA expression data from BALB/c mice which were infected intranasally with Respiratory Syncytial Virus (or Hep-2 cell lysate control) at 1 week old and challenged with PBS or house dust mite (HDM) extract as adults. Experimental groups: RH – neonatal RSV, adult HDM, RP – neonatal RSV, adult PBS, HH – neonatal Hep-2, adult HDM and HP – neonatal Hep-2, adult PBS.

2019-03-19 | E-MTAB-3654 | biostudies-arrayexpress

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

Project description: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. To increase identification coverage, we employed Hp-RP StageTip prefractionation of membrane-enriched samples from 11 NSCLC cell lines. Analysis of membrane samples from 20 pairs of tumor and adjacent normal lung tissue was incorporated to include physiologically expressed membrane proteins. Using multiple search engines (X!Tandem, Comet, and Mascot) and stringent evaluation of FDR (MAYU and PeptideShaker), we identified 7702 proteins (66% membrane proteins) and 178 missing proteins (74 membrane proteins) with PSM-, peptide-, and protein-level FDR of 1%. Through multiple reaction monitoring using synthetic peptides, we provided additional evidence of eight missing proteins including seven with transmembrane helix domains. This study demonstrates that mining missing proteins focused on cancer membrane subproteome can greatly contribute to map the whole human proteome. All data were deposited into ProteomeXchange with the identifier PXD002224.

| S-EPMC4761227 | biostudies-literature

TMT-based proteomics analysis of GV, GVBD and MII oocytes

Project description:Two replicates of GV, GVBD, and MII oocytes were subjected to the 6-plex TMT labeling, HP-RP fractionation, and LC-MS/MS analysis. Two labeling experiments were performed for the total four replicates of each stage of oocytes.

2022-02-15 | PXD018777 | Pride

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