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Wang et al. Microbiome Res Rep 2024;3:39 Microbiome Research
DOI: 10.20517/mrr.2024.21
Reports

Original Article Open Access

Assessing fecal metaproteomics workflow and small
protein recovery using DDA and DIA PASEF mass

spectrometry

1
1,#
2,3
2,3
1,2
Angela Wang 1,2,# , Emily E F Fekete , Marybeth Creskey , Kai Cheng , Zhibin Ning , Annabelle Pfeifle ,
1,2
Xuguang Li , Daniel Figeys 2,3 , Xu Zhang 1,3
1
Regulatory Research Division, Biologic and Radiopharmaceutical Drugs Directorate, Health Products and Food Branch, Health
Canada, Ottawa K1A 0K9, Ontario, Canada.
2
Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa K1H 8M5,
Ontario, Canada.
3
School of Pharmaceutical Sciences, Faculty of Medicine, University of Ottawa, Ottawa K1H 8M5, Ontario, Canada.
#
Authors contributed equally.
Correspondence to: Dr. Xu Zhang, Regulatory Research Division, Biologic and Radiopharmaceutical Drugs Directorate, Health
Products and Food Branch, Health Canada, 251 Sir Frederick Banting Driveway, Ottawa K1A 0K9, Ontario, Canada. E-mail:
xu.zhang@hc-sc.gc.ca
How to cite this article: Wang A, Fekete EEF, Creskey M, Cheng K, Ning Z, Pfeifle A, Li X, Figeys D, Zhang X. Assessing fecal
metaproteomics workflow and small protein recovery using DDA and DIA PASEF mass spectrometry. Microbiome Res Rep
2024;3:39. https://dx.doi.org/10.20517/mrr.2024.21
Received: 15 Mar 2024 First Decision: 25 Apr 2024 Revised: 17 Jun 2024 Accepted: 25 Jun 2024 Published: 3 Jul 2024
Academic Editor: Marco Ventura Copy Editor: Pei-Yun Wang Production Editor: Pei-Yun Wang

Abstract
Aim: This study aims to evaluate the impact of experimental workflow on fecal metaproteomic observations,
including the recovery of small and antimicrobial proteins often overlooked in metaproteomic studies. The
overarching goal is to provide guidance for optimized metaproteomic experimental design, considering the
emerging significance of the gut microbiome in human health, disease, and therapeutic interventions.
Methods: Mouse feces were utilized as the experimental model. Fecal sample pre-processing methods (differential
centrifugation and non-differential centrifugation), protein digestion techniques (in-solution and filter-aided), data
acquisition modes (data-dependent and data-independent, or DDA and DIA) when combined with parallel
accumulation-serial fragmentation (PASEF), and different bioinformatic workflows were assessed.

Results: We showed that, in DIA-PASEF metaproteomics, the library-free search using protein sequence database
generated from DDA-PASEF data achieved better identifications than using the generated spectral library.

© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0
International License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, sharing,
adaptation, distribution and reproduction in any medium or format, for any purpose, even commercially, as
long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and
indicate if changes were made.

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