AI Article Synopsis

  • * The study demonstrates that using a microflow LC-MS/MS system can significantly enhance proteome coverage, successfully identifying around 9,000 proteins from root samples and over 7,000 from mouse and human tissues in just 3 hours.
  • * The iBAQ method showed a dynamic range of protein quantification across 5 orders of magnitude, with a median coefficient of variation below 20%, indicating that this strategy is effective for large-scale proteome analysis and has potential for further advancements.

Article Abstract

A current trend in proteomics is to acquire data in a "single-shot" by LC-MS/MS because it simplifies workflows and promises better throughput and quantitative accuracy than schemes that involve extensive sample fractionation. However, single-shot approaches can suffer from limited proteome coverage when performed by data dependent acquisition (ssDDA) on nanoflow LC systems. For applications where sample quantities are not scarce, this study shows that high proteome coverage can be obtained using a microflow LC-MS/MS system operating a 1 mm i.d. × 150 mm column, at a flow-rate of 50 μL/min and coupled to an Orbitrap HF-X mass spectrometer. The results demonstrate the identification of ∼9 000 proteins from 50 μg of protein digest from roots, 7 500 from mouse thymus, and 7 300 from human breast cancer cells in 3 h of analysis time in a single run. The dynamic range of protein quantification measured by the iBAQ approach spanned 5 orders of magnitude and replicate analysis showed that the median coefficient of variation was below 20%. Together, this study shows that ssDDA by μLC-MS/MS is a robust method for comprehensive and large-scale proteome analysis and which may be further extended to more rapid chromatography and data independent acquisition approaches in the future.̀.

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Source
http://dx.doi.org/10.1021/acs.analchem.1c00738DOI Listing

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