Cost-effective generation of precise label-free quantitative proteomes in high-throughput by microLC and data-independent acquisition.
Sci Rep
; 8(1): 4346, 2018 03 12.
Article
em En
| MEDLINE
| ID: mdl-29531254
ABSTRACT
Quantitative proteomics is key for basic research, but needs improvements to satisfy an increasing demand for large sample series in diagnostics, academia and industry. A switch from nanoflowrate to microflowrate chromatography can improve throughput and reduce costs. However, concerns about undersampling and coverage have so far hampered its broad application. We used a QTOF mass spectrometer of the penultimate generation (TripleTOF5600), converted a nanoLC system into a microflow platform, and adapted a SWATH regime for large sample series by implementing retention time- and batch correction strategies. From 3 µg to 5 µg of unfractionated tryptic digests that are obtained from proteomics-typical amounts of starting material, microLC-SWATH-MS quantifies up to 4000 human or 1750 yeast proteins in an hour or less. In the acquisition of 750 yeast proteomes, retention times varied between 2% and 5%, and quantified the typical peptide with 5-8% signal variation in replicates, and below 20% in samples acquired over a five-months period. Providing precise quantities without being dependent on the latest hardware, our study demonstrates that the combination of microflow chromatography and data-independent acquisition strategies has the potential to overcome current bottlenecks in academia and industry, enabling the cost-effective generation of precise quantitative proteomes in large scale.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Peptídeos
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Saccharomyces cerevisiae
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Cromatografia Líquida
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Proteoma
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Proteínas de Saccharomyces cerevisiae
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Proteômica
Tipo de estudo:
Health_economic_evaluation
Limite:
Humans
Idioma:
En
Ano de publicação:
2018
Tipo de documento:
Article