Dataset Information

Evaluation of a Dual Isolation Width Acquisition (DIWA) method for isobaric labelling ratio decompression

ABSTRACT: Stable isotope labelling of peptides using isobaric reagents such as iTRAQ and TMT enables the multiplexed analysis of proteomes with deep quantitative coverage. Isobaric tagging demonstrates high precision but imperfect accuracy due to ratio underestimation caused by co-fragmentation of ions with mass-to-charge ratios within the isolation window of the targeted precursors. Prompted by empirical observations of isobaric-labelled peptide MS2 spectra, we argue that although a very narrow isolation window will result in severe loss of backbone fragment ions, rendering the spectra unsuitable for peptide identification, the reporter ion signals will remain intense enough to generate quantitative information for a significant portion of the spectra. Based on this assumption we have designed a Dual Isolation Width Acquisition (DIWA) method, in which each precursor is first fragmented with HCD using a standard isolation width for peptide identification and preliminary quantification followed by a concomitant MS2 HCD fragmentation using a much narrower isolation width for the acquisition of quantification-only spectra with reduced interference. We leverage the quantitative values obtained by the “narrow” scans to build linear regression models and apply these to decompress the fold-changes measured at the “standard” scans. Here, we evaluate the DIWA method using a two species TMT-6plex model and discuss the potential of this approach.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human) Escherichia Coli

TISSUE(S): Cell Culture

SUBMITTER: James Wright

LAB HEAD: Jyoti Choudhary

PROVIDER: PXD010571 | Pride | 2019-01-07

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	20180703_TR_Choudhary_lab_VLS_DIWA_IW01_01.raw	Raw
	20180703_TR_Choudhary_lab_VLS_DIWA_IW01_02.raw	Raw
	20180703_TR_Choudhary_lab_VLS_DIWA_IW01_03.raw	Raw
	20180703_TR_Choudhary_lab_VLS_DIWA_IW01_04.raw	Raw
	20180703_TR_Choudhary_lab_VLS_DIWA_IW01_05.raw	Raw

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Evaluation of a Dual Isolation Width Acquisition Method for Isobaric Labeling Ratio Decompression.

Roumeliotis Theodoros I TI Weisser Hendrik H Choudhary Jyoti S JS

Journal of proteome research 20190103 3

Isobaric labeling is a highly precise approach for protein quantification. However, due to the isolation interference problem, isobaric tagging suffers from ratio underestimation at the MS2 level. The use of narrow isolation widths is a rational approach to alleviate the interference problem; however, this approach compromises proteome coverage. We reasoned that although a very narrow isolation window will result in loss of peptide fragment ions, the reporter ion signals will be retained for a s ...[more]

PMID: 30576155

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Project description:A comprehensive proteome map is essential to elucidate molecular pathways and protein functions. Although great improvements in sample preparation, instrumentation and data analysis already yield-ed impressive results, current studies suffer from a limited proteomic depth and dynamic range there-fore lacking low abundant or highly hydrophobic proteins. Here, we combine and benchmark advanced micro pillar array columns (µPAC) operated at nanoflow with wide window acquisition (WWA) and the AI-based CHIMERYS search engine for data analysis to maximize chromatographic separation power, sensitivity and proteome coverage. The wide window acquisition method merges the strengths of DDA and DIA. WWA uses a broad isolation window (≥4 Th) for data-dependent precursor selection. Similar to DIA, precursor ions close to those selected are co-fragmented, producing chimeric spectra that boost IDs and improve coverage of low-abundance peptides, which would otherwise be missed. WWA is particularly powerful when combined with the AI-driven CHIMERYS search algorithm, which allows confident identification of up to eleven peptides from a single chimeric spectrum. Developed by MSAID, the CHIMERYS algorithm makes use of accurate fragment spectrum predictions trained on millions of spectra, which allows to utilize additional spectral properties such as relative signal intensities for drastically improved identification rates. Entrapment experiments were conducted searching mouse immunoprecipitation samples with MS Amanda 2.0 and CHIMERYS by applying an additional custom-made decoy database, which demonstrated excellent protein, peptide and PSM FDR control from 1% upwards for both search engines. Combining wide precursor isolation widths of 4-12 Th with the CHIMERYS search engine identified 51-74% more proteins and 59-150% more peptides for single cell, immunoprecipitation and multi-species samples in comparison to a standard proteomics workflow employing MS Amanda 2.0 and an isolation width of 1 Th. Application of different isolation widths revealed that the optimal isolation window size varies according to sample amount and complexity with 12 Th resulting in the highest number of identifications for single cell-equivalent injection amounts, while 4 Th provided best results for classical bulk inputs of 400ng. Evaluation of coefficients of variation also yielded improved results for WWA over classical DDA when assessing single cell, 40 cell and 250pg bulk HeLa lysates. Overall, WWA and CHIMERYS resulted in substantially improved proteomic depth and quantification for all sample types tested and will provide a highly valuable toolset for the proteomics community.