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Increasing the throughput of sensitive proteomics by plexDIA

ABSTRACT: Mass-spectrometry methods enable high-throughput proteomics with large input samples, but the depth and throughput of protein analysis remain limited for smaller samples. We aimed to increase throughput for analyzing limited samples while achieving high proteome coverage and quantitative accuracy. Thus, we developed a general experimental and computational framework, plexDIA, for simultaneously multiplexing the analysis of both peptides and samples. Multiplexed analysis with plexDIA increases throughput multiplicatively with the number of labels without reducing protein coverage or quantitative accuracy. Specifically, 3-plex nonisobaric labeling of sub-microgram samples increases the number of quantitative protein ratios by about 3-fold, enabling the quantification of over 25,000 protein data points per hour of active gradient on a first-generation Q Exactive instrument. Furthermore, plexDIA increases the consistency of protein detection and quantification across samples and reduces missing data by over 2-fold. We applied plexDIA to quantify proteome dynamics during the cell division cycle in cells isolated based on their DNA content. The high sensitivity and accuracy of plexDIA detected many classical cell cycle proteins and discovered new ones. These results establish a general framework for increasing the throughput of highly sensitive and quantitative protein analysis.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Escherichia Coli (ncbitaxon:562) Saccharomyces Cerevisiae (ncbitaxon:4932) Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Nikolai Slavov

PROVIDER: MSV000088302 | MassIVE | Tue Nov 02 15:14:00 GMT 2021

SECONDARY ACCESSION(S): PXD029531

REPOSITORIES: MassIVE

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Increasing the throughput of sensitive proteomics by plexDIA

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