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Open AccessArticle

Optimization of Data-Independent Acquisition Mass Spectrometry for Deep and Highly Sensitive Proteomic Analysis

1
Department of Applied Genomics, Kazusa DNA Research Institute, Chiba 292-0818, Japan
2
Laboratory for Gut Homeostasis, RIKEN Center for Integrative Medical Sciences, Kanagawa 230-0045, Japan
3
Department of Microbiology and Immunology, Keio University School of Medicine, Tokyo 160-8582, Japan
4
Department of Pediatric Surgery, Faculty of Medicine, The University of Tokyo, Tokyo 113-8655, Japan
5
Laboratory for Microbiome Sciences, RIKEN Center for Integrative Medical Sciences, Kanagawa 230-0045, Japan
6
Graduate School of Advanced Science and Engineering, Waseda University, Tokyo 169-8555, Japan
7
Laboratory for Integrative Genomics, RIKEN Center for Integrative Medical Sciences, Kanagawa 230-0045, Japan
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2019, 20(23), 5932; https://doi.org/10.3390/ijms20235932
Received: 29 October 2019 / Revised: 20 November 2019 / Accepted: 23 November 2019 / Published: 26 November 2019
(This article belongs to the Collection Advances in Proteomic Research)
Data-independent acquisition (DIA)-mass spectrometry (MS)-based proteomic analysis overtop the existing data-dependent acquisition (DDA)-MS-based proteomic analysis to enable deep proteome coverage and precise relative quantitative analysis in single-shot liquid chromatography (LC)-MS/MS. However, DIA-MS-based proteomic analysis has not yet been optimized in terms of system robustness and throughput, particularly for its practical applications. We established a single-shot LC-MS/MS system with an MS measurement time of 90 min for a highly sensitive and deep proteomic analysis by optimizing the conditions of DIA and nanoLC. We identified 7020 and 4068 proteins from 200 ng and 10 ng, respectively, of tryptic floating human embryonic kidney cells 293 (HEK293F) cell digest by performing the constructed LC-MS method with a protein sequence database search. The numbers of identified proteins from 200 ng and 10 ng of tryptic HEK293F increased to 8509 and 5706, respectively, by searching the chromatogram library created by gas-phase fractionated DIA. Moreover, DIA protein quantification was highly reproducible, with median coefficients of variation of 4.3% in eight replicate analyses. We could demonstrate the power of this system by applying the proteomic analysis to detect subtle changes in protein profiles between cerebrums in germ-free and specific pathogen-free mice, which successfully showed that >40 proteins were differentially produced between the cerebrums in the presence or absence of bacteria. View Full-Text
Keywords: DIA; SWATH; deep proteomics; label-free quantification; single shot; overlaping window DIA DIA; SWATH; deep proteomics; label-free quantification; single shot; overlaping window DIA
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Kawashima, Y.; Watanabe, E.; Umeyama, T.; Nakajima, D.; Hattori, M.; Honda, K.; Ohara, O. Optimization of Data-Independent Acquisition Mass Spectrometry for Deep and Highly Sensitive Proteomic Analysis. Int. J. Mol. Sci. 2019, 20, 5932.

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