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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Produce Safety and Microbiology Research » Research » Publications at this Location » Publication #386949

Research Project: Elucidating the Factors that Determine the Ecology of Human Pathogens in Foods

Location: Produce Safety and Microbiology Research

Title: Sequestration of the ionizing proton in singly charged metastable protein ions generated by MALDI

Author
item Fagerquist, Clifton - Keith
item Dodd, Claire

Submitted to: International Journal of Mass Spectrometry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/22/2021
Publication Date: 10/25/2021
Citation: Fagerquist, C.K., Dodd, C.E. 2021. Sequestration of the ionizing proton in singly charged metastable protein ions generated by MALDI. International Journal of Mass Spectrometry. Available at:https://www.sciencedirect.com/science/article/pii/S1387380621002177?via%3Dihub. https://doi.org/10.1016/j.ijms.2021.116736.
DOI: https://doi.org/10.1016/j.ijms.2021.116736

Interpretive Summary: Protein identification is one of the fundamental goals of proteomic science. For top-down proteomic analysis, it is critical that proteins are successfully transferred from the condensed phase (solids or liquids) to the gas phase and ionized such that the intact protein (and any of its protein fragments) are detected and have a corresponding mass-to-charge (m/z) ratio. Protein ionization is largely determined by the ionization technique and the acidic and/or basic functional groups of specific amino acid residues present in the protein. We have detected a strong preference for ionization/protonation at arginine residues in proteins ionized by matrix-assisted laser desorption/ionization (MALDI). Arginine effectively acts as a proton sink where the proton remains immobilized. If a protein has only one arginine residue, that arginine effectively limits the number of fragment ions that are likely to be detected as each fragment must possess the arginine for the protein fragment to be ionized. By contrast when a protein has no arginine residues, the ionizing proton is distributed at many different sites in the polypeptide chain, often at lysine residues, resulting in a greater number of fragment ions detected which ultimately increases the confidence level of protein identification.

Technical Abstract: A hypothesis is proposed for the preferential ionization/protonation of arginine residues in metastable protein ions. This phenomenon has significant consequences for detection of protein fragments resulting from polypeptide backbone cleavage. Preferential ionization at arginine (when present) is based upon MS/MS analysis of singly charged/protonated metastable protein ions: YahO, CspC, CspE, B-subunit of Shiga toxin 2 and thioredoxin, that have either zero or one arginine residue in their sequences. For protein ions having no arginine, we observe multiple b/y complementary fragment ion pairs (CFIP) by MALDI-MS/MS post-source decay demonstrating that the ionizing proton is located at multiple locations amongst the packet of protein precursor ions being analyzed. By contrast, for protein ions having one arginine, we observe little or no CFIP, and almost every fragment ion has the arginine residue in its sequence. This preferential proton sequestration is presumably due to the higher gas phase basicity of arginine compared to lysine, although lysine residues are often more numerous than arginine residues. As this phenomenon has been noted previously in the ionization of tryptic peptides by MALDI where the basic residue (K or R) is located exclusively at the C-terminus (if there are no missed proteolytic cleavages), we conclude that rapid proton transfer in the gas phase occurs via protein/matrix collisions in the MALDI plume. Upon proton transfer to the side-chain of an arginine residue, the proton is effectively immobilized by its guanidino functional group. An alternative explanation is proton exchange from the ionized side-chain of a lysine to the side-chain of the arginine in the metastable ion.