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United States Department of Agriculture

Agricultural Research Service

Research Project: Molecular Biology of Human Pathogens Associated with Food

Location: Produce Safety and Microbiology Research

Title: Possible evidence of amide bond formation between sinapinic acid and lysine-containing bacterial proteins by matrix-assisted laser desorption/ionization (MALDI) at 355 nm

Authors
item Fagerquist, Clifton
item Sultan, Omar -
item Carter, Michelle

Submitted to: Journal of American Society for Mass Spectrometry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 3, 2012
Publication Date: October 2, 2012
Citation: Fagerquist, C.K., Sultan, O., Carter, M.Q. 2012. Possible evidence of amide bond formation between sinapinic acid and lysine-containing bacterial proteins by matrix-assisted laser desorption/ionization (MALDI) at 355 nm. Journal of American Society for Mass Spectrometry. 23(12):2102-2114.

Interpretive Summary: Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry(MS)is increasingly utilized to characterize bacteria including foodborne pathogens. Characterization often involves the ionization and identification of highly abundant proteins and protein toxins. Occasionally, chemical reactions may occur between the MALDI matrix and the proteins being ionized. These reactions can result in attachment of a matrix molecule to a protein and appear as satellite peaks next to a more abundant non-adducted protein ion peak in a MS spectrum. It is critical to identify such matrix adducted peaks when they occur so that they are not mis-assigned as a "new" protein or utilized as part of a bacterial "fingerprint" for identification. We have identified two types of matrix adduction of sinapinic acid (SA) matrix that can occur to proteins having lysine residues. The more abundant adduct peak is due to amide bond formation between the SA and the side-chain amine group of lysine residues. A dry ammonium carboxylate salt formed during MALDI sample preparation under acidic conditions is a precursor to amide bond formation. Intense laser irradiation converts the protein-matrix salt complex to a covalent amide bond with loss of water (dehydration). The other adduct peak results from transfer of the protein-matrix salt complex from the solid to the gas phase.

Technical Abstract: We previously reported the apparent formation of matrix adducts of 3,5-dimethoxy-4-hydroxy-cinnamic acid (sinapinic acid or SA) via covalent attachment to disulfide bond-containing proteins (HdeA, HdeB and YbgS) from bacterial cell lysates ionized by matrix-assisted laser desorption/ionization (MALDI) and analyzed by time-of-flight-time-of-flight(TOF-TOF) post-source decay (PSD) tandem mass spectrometry (MS/MS). We also reported the absence of adduct formation when using alpha-cyano-4-hydroxycinnamic acid (CHCA) matrix. Further mass spectrometric analysis of disulfide-intact and disulfide-reduced over-expressed HdeA and HdeB proteins from lysates of gene-inserted E. coli plasmids suggests covalent attachment of SA occurs not at cysteine residues but at lysine residues. In this revised hypothesis, the attachment of SA is preceded by the formation of a dry ammonium carboxylate salt between SA and accessible lysine residues of the protein during sample preparation under acidic conditions. Laser irradiation at 355 nm of the dried sample spot results in equilibrium retrogradation followed by nucleophilic attack by the amine group of lysine at the carbonyl group of SA and subsequent amide bond formation and loss of water. The absence of CHCA adducts suggests that the electron-withdrawing effect of the alpha-cyano group of this matrix may inhibit salt formation and/or amide bond formation. This revised hypothesis is supported by dissociative loss of SA (-224 Da) and the amide-bound SA (-206 Da) from SA-adducted HdeA and HdeB ions by PSD-MS/MS. It is proposed that cleavage of the amide-bound SA from the lysine side-chain occurs via rearrangement involving a pentacyclic transition state followed by hydrogen abstraction/migration and loss of 3-(4-hydroxy-3,5-dimethoxyphenyl)prop-2-ynal (206 Da).

Last Modified: 7/23/2014
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