GeLC‐FAIMS‐MS workflow for in‐depth middle‐down proteomics

Middle‐down proteomics (MDP) is an analytical approach in which protein samples are digested with proteases such as Glu‐C to generate large peptides (>3 kDa) that are analyzed by mass spectrometry (MS). This method is useful for characterizing high‐molecular‐weight proteins that are difficult to...

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Bibliographic Details
Published in:Proteomics (Weinheim) 2024-02, Vol.24 (3-4), p.e2200431-n/a
Main Authors: Takemori, Ayako, Kaulich, Philipp T., Konno, Ryo, Kawashima, Yusuke, Hamazaki, Yuto, Hoshino, Ayuko, Tholey, Andreas, Takemori, Nobuaki
Format: Article
Language:English
Subjects:
AnExSP
Digestion
Electrophoresis
Electrophoretic mobility
Exosomes
Fractionation
Glu‐C digestion
Integrins
Ionic mobility
LC‐FAIMS
Liquid chromatography
Mass spectrometry
Mass spectroscopy
Molecular weight
PEPPI‐MS
Peptides
Polyacrylamide
Proteins
proteoform
Proteomics
Separation
Workflow
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Description
Summary:Middle‐down proteomics (MDP) is an analytical approach in which protein samples are digested with proteases such as Glu‐C to generate large peptides (>3 kDa) that are analyzed by mass spectrometry (MS). This method is useful for characterizing high‐molecular‐weight proteins that are difficult to detect by top‐down proteomics (TDP), in which intact proteins are analyzed by MS. In this study, we applied GeLC‐FAIMS‐MS, a multidimensional separation workflow that combines gel‐based prefractionation with LC‐FAIMS MS, for deep MDP. Middle‐down peptides generated by optimized limited Glu‐C digestion conditions were first size‐fractionated by polyacrylamide gel electrophoresis, followed by C4 reversed‐phase liquid chromatography separation and additional ion mobility fractionation, resulting in a significant increase in peptide length detectable by MS. In addition to global analysis, the GeLC‐FAIMS‐MS concept can also be applied to targeted MDP, where only proteins in the desired molecular weight range are gel‐fractionated and their Glu‐C digestion products are analyzed, as demonstrated by targeted analysis of integrins in exosomes. In‐depth MDP achieved by global and targeted GeLC‐FAIMS‐MS supports the exploration of proteoform information not covered by conventional TDP by increasing the number of detectable protein groups or post‐translational modifications (PTMs) and improving the sequence coverage.
ISSN:1615-9853
1615-9861
DOI:10.1002/pmic.202200431