+ Site Statistics
References:
54,258,434
Abstracts:
29,560,870
PMIDs:
28,072,757
+ Search Articles
+ PDF Full Text Service
How our service works
Request PDF Full Text
+ Follow Us
Follow on Facebook
Follow on Twitter
Follow on LinkedIn
+ Subscribe to Site Feeds
Most Shared
PDF Full Text
+ Translate
+ Recently Requested

Improving protein and proteome coverage through data-independent multiplexed peptide fragmentation



Improving protein and proteome coverage through data-independent multiplexed peptide fragmentation



Journal of Proteome Research 9(7): 3621-3637



Performance differences in protein and proteome characterization achieved by data-independent acquisition (DIA) LC/MS(E) and data-dependent acquisition (DDA) LC/MS/MS approaches were investigated. LC/MS(E) is a novel mode of generating product ion data for all coeluting precursors in parallel as opposed to LC/MS/MS where coeluting precursors must be serially fragmented one at a time. During LC/MS(E) analysis, alternating MS scans of "normal" and "elevated" collision energy are collected at regular intervals, providing nearly a 100% duty cycle for precursor detection and fragmentation because all precursors are fragmented across their full chromatographic elution profile. This is in contrast to DDA-based MS/MS where serial selection of precursor ions is biased toward interrogation and detection of the highest abundance sample components by virtue of the intensity-driven interrogation scheme employed. Both modes of acquisition were applied to a simple four-protein standard mixture with a 16-fold dynamic range in concentration, an in-gel digest of the Arabidopsis thaliana protein FLS2 purified by immunoprecipitation, and a solution-digested tomato leaf proteome sample. Dramatic improvement for individual protein sequence coverage was obtained for all three samples analyzed by the DIA approach, particularly for the lowest abundance sample components. In many instances, precursors readily detected and identified during DIA were either interrogated by MS/MS during DDA at inopportune points in their chromatographic elution profiles resulting in poor quality product ion spectra or not interrogated at all. Detailed evaluation of both DDA and DIA raw data and timing of the MS-to-MS/MS switching events clearly revealed the fundamental limitations of serial MS/MS interrogation and the advantages of parallel fragmentation by DIA for more comprehensive protein identification and characterization which holds promise for enhanced isoform and post-translational modification analysis.

Please choose payment method:






(PDF emailed within 0-6 h: $19.90)

Accession: 053754897

Download citation: RISBibTeXText

PMID: 20450226

DOI: 10.1021/pr100144z


Related references

Multiplexed and data-independent tandem mass spectrometry for global proteome profiling. Mass Spectrometry Reviews 33(6): 452-470, 2015

Improving deep proteome and PTMome coverage using tandem HILIC-HPRP peptide fractionation strategy. Analytical and Bioanalytical Chemistry 411(2): 459-469, 2018

Use of an integrated MS--multiplexed MS/MS data acquisition strategy for high-coverage peptide mapping studies. Rapid Communications in Mass Spectrometry 21(5): 730-744, 2007

Novel parallelized quadrupole/linear ion trap/Orbitrap tribrid mass spectrometer improving proteome coverage and peptide identification rates. Analytical Chemistry 85(24): 11710-4, 2014

In-Depth Proteome Coverage by Improving Efficiency for Membrane Proteome Analysis. Analytical Chemistry 89(10): 5179-5185, 2017

Comprehensive absolute quantification of the cytosolic proteome of Bacillus subtilis by data independent, parallel fragmentation in liquid chromatography/mass spectrometry (LC/MS(E)). Molecular & Cellular Proteomics 13(4): 1008-1019, 2014

Improved proteome coverage by using high efficiency cysteinyl peptide enrichment: the human mammary epithelial cell proteome. Proteomics 5(5): 1263-1273, 2005

Database searching and accounting of multiplexed precursor and product ion spectra from the data independent analysis of simple and complex peptide mixtures. Proteomics 9(6): 1696-1719, 2009

Comparison of data analysis parameters and MS/MS fragmentation techniques for quantitative proteome analysis using isobaric peptide termini labeling (IPTL). Analytical and Bioanalytical Chemistry 404(4): 1103-1114, 2013

Exploring the Arabidopsis proteome: influence of protein solubilization buffers on proteome coverage. International Journal of Molecular Sciences 16(1): 857-870, 2015

Improving proteome coverage on a LTQ-Orbitrap using design of experiments. Journal of the American Society for Mass Spectrometry 22(4): 773-783, 2011

Improving Proteome Coverage by Reducing Sample Complexity via Chromatography. Advances in Experimental Medicine and Biology 919: 83-143, 2017

Top-down protein identification of proteasome proteins with nanoLC-FT-ICR-MS employing data-independent fragmentation methods. Proteomics 14(10): 1271-1282, 2014

PepSeeker: a database of proteome peptide identifications for investigating fragmentation patterns. Nucleic Acids Research 34(Database Issue): D649-D654, 2005

A probabilistic framework for peptide and protein quantification from data-dependent and data-independent LC-MS proteomics experiments. Omics 16(9): 468-482, 2013