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Issue 16, 2018
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Enzyme-containing spin membranes for rapid digestion and characterization of single proteins

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Abstract

Proteolytic digestion is an important step in characterizing protein sequences and post-translational modifications (PTMs) using mass spectrometry (MS). This study uses pepsin- or trypsin-containing spin membranes for rapid digestion of single proteins or simple protein mixtures prior to ultrahigh-resolution Orbitrap MS analysis. Centrifugation of 100 μL of pretreated protein solutions through the functionalized membranes requires less than 1 min and conveniently digests proteins into large peptides that aid in confirming specific protein sequence variations and PTMs. Peptic and tryptic peptides from spin digestion of apomyoglobin and four commercial monoclonal antibodies (mAbs) typically cover 100% of the protein sequences in direct infusion MS analysis. Increasing the spin rate leads to a higher fraction of large peptic peptides for apomyoglobin, and MS analysis of peptic and tryptic peptides reveals mAb PTMs such as N-terminal pyroglutamate formation, C-terminal lysine clipping and glycosylation. Relative to overnight in-solution digestion of mAbs, spin digestion yields higher sequence coverages. Spin-membrane digestion followed by infusion MS readily differentiates a mAb to the Ebola virus from a related antibody that differs by addition of a single amino acid.

Graphical abstract: Enzyme-containing spin membranes for rapid digestion and characterization of single proteins

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Publication details

The article was received on 25 May 2018, accepted on 13 Jul 2018 and first published on 17 Jul 2018


Article type: Paper
DOI: 10.1039/C8AN00969D
Citation: Analyst, 2018,143, 3907-3917
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    Enzyme-containing spin membranes for rapid digestion and characterization of single proteins

    W. Liu, Y. Pang, H. Tan, N. Patel, G. Jokhadze, A. Guthals and M. L. Bruening, Analyst, 2018, 143, 3907
    DOI: 10.1039/C8AN00969D

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