Enhanced digestion efficiency, peptide ionization efficiency, and sequence resolution for protein hydrogen/deuterium exchange monitored by fourier transform ion cyclotron resonance mass spectrometry

Hui Min Zhang, Saša Kazazic, Tanner M. Schaub, Jeremiah D. Tipton, Mark Emmett, Alan G. Marshall

Research output: Contribution to journalArticle

68 Citations (Scopus)

Abstract

Solution-phase hydrogen/deuterium exchange (HDX) monitored by high-resolution Fourier transform ion cyclotron resonance (FTICR) mass spectrometry offers a rapid method to study protein conformations and protein-protein interactions. Pepsin is usually used to digest proteins in HDX and is known for lack of cleavage specificity. To improve digestion efficiency and specificity, we have optimized digestion conditions and cleavage preferences for pepsin and protease type XIII from Aspergillus saitoi. A dilution series of the proteases was used to determine the digestion efficiency for several test proteins. Protease type XIII prefers to cleave on the C-terminal end of basic amino acids and produced the highest number of fragments and the best sequence coverage compared to pepsin or protease type XVIII from Rhizhopus. Furthermore, protease type XIII exhibited much less self-digestion than pepsin and thus is superior for HDX experiments. Many highly overlapped segments from protease type XIII and pepsin digestion, combined with high-resolution FTICR mass spectrometry, provide high sequence resolution (to as few as one or two amino acids) for the assignment of amide hydrogen exchange rate. Our H/D exchange results correlate well with the secondary and tertiary structure of myoglobin. Such assignments of highly overlapped fragments promise to greatiy enhance the accuracy and sequence resolution for determining conformational differences resulting from ligand binding or protein-protein interactions.

Original languageEnglish (US)
Pages (from-to)9034-9041
Number of pages8
JournalAnalytical Chemistry
Volume80
Issue number23
DOIs
StatePublished - Dec 1 2008
Externally publishedYes

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Cyclotron resonance
Deuterium
Ionization
Mass spectrometry
Hydrogen
Fourier transforms
Pepsin A
Ions
Peptide Hydrolases
Peptides
Proteins
Basic Amino Acids
Myoglobin
Aspergillus
Amides
Dilution
Conformations
Ion exchange
Ligands
Amino Acids

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Enhanced digestion efficiency, peptide ionization efficiency, and sequence resolution for protein hydrogen/deuterium exchange monitored by fourier transform ion cyclotron resonance mass spectrometry. / Zhang, Hui Min; Kazazic, Saša; Schaub, Tanner M.; Tipton, Jeremiah D.; Emmett, Mark; Marshall, Alan G.

In: Analytical Chemistry, Vol. 80, No. 23, 01.12.2008, p. 9034-9041.

Research output: Contribution to journalArticle

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