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1.
Nat Biotechnol ; 39(7): 846-854, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33767396

RESUMO

Accurate quantification of the proteome remains challenging for large sample series and longitudinal experiments. We report a data-independent acquisition method, Scanning SWATH, that accelerates mass spectrometric (MS) duty cycles, yielding quantitative proteomes in combination with short gradients and high-flow (800 µl min-1) chromatography. Exploiting a continuous movement of the precursor isolation window to assign precursor masses to tandem mass spectrometry (MS/MS) fragment traces, Scanning SWATH increases precursor identifications by ~70% compared to conventional data-independent acquisition (DIA) methods on 0.5-5-min chromatographic gradients. We demonstrate the application of ultra-fast proteomics in drug mode-of-action screening and plasma proteomics. Scanning SWATH proteomes capture the mode of action of fungistatic azoles and statins. Moreover, we confirm 43 and identify 11 new plasma proteome biomarkers of COVID-19 severity, advancing patient classification and biomarker discovery. Thus, our results demonstrate a substantial acceleration and increased depth in fast proteomic experiments that facilitate proteomic drug screens and clinical studies.


Assuntos
Proteômica/métodos , Espectrometria de Massas em Tandem , Arabidopsis/metabolismo , Biomarcadores/metabolismo , COVID-19/sangue , COVID-19/diagnóstico , Linhagem Celular , Humanos , Peptídeos/análise , Proteoma/análise , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Índice de Gravidade de Doença
2.
J Am Soc Mass Spectrom ; 28(10): 2143-2150, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28717932

RESUMO

Orthogonal injection time-of-flight (orthoTOF) mass spectrometry (MS) is the most prevalent form of TOFMS, owing to its greater control over incoming ion energy, the ability to correct for aberrations in incoming ion velocity and position, and its ability to provide an entire mass spectrum within a single scan. However, the duty cycle of orthoTOFMS is low compared with scanning analyzers, which can have 100% duty cycle when measuring a single type of ion. Typical duty cycles for orthoTOFMS range from 1% to 30%, depending on instrument geometry. Generally, as instrument resolution increases, duty cycle decreases. Additionally, the greatest duty cycle is achieved for the highest m/z ion recorded in the spectrum, and decreases for all other ions as a function of m/z. In a prior publication [Loboda, A.V.; Chernushevich, I.V. J. Am. Soc. Mass Spectrom. 20, 1342-1348 (20)], a novel trapping/release method for restoring the duty cycle of a V-geometry orthoTOFMS to near 100% (referred to as "Zeno pulsing") was presented. Here, we apply that method to a W-TOF geometry analyzer with analog detection. Across a m/z range of 100-2000, sensitivity gains of ~5-20 are observed, for total ion currents approaching ~107 ions·s-1. Zeno pulsing, or similar strategies for restoring duty cycle, will continue to be important as instrument resolution in orthoTOFMS is increased through the use of ion mirrors. Graphical Abstract ᅟ.

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