|Fagerquist, Clifton - Keith|
|GARBUS, BRANDON - Intuitive Surgical, Inc|
|WILLIAMS, KATHERINE - University Of California|
|Harden, Leslie - Les|
Submitted to: Journal of American Society for Mass Spectrometry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/14/2010
Publication Date: 1/25/2010
Citation: Fagerquist, C.K., Garbus, B., Williams, K., Bates, A.H., Harden, L.A. 2010. Covalent attachment and dissociative loss of sinapinic acid to/from cysteine-containing proteins from bacterial cell lysates analyzed by MALDI-TOF-TOF mass spectrometry. Journal of American Society for Mass Spectrometry. 21,819-832.
Interpretive Summary: We report the binding of sinapinic acid (SA) to specific protein biomarkers from bacterial cell lysates of pathogenic and non-pathogenic strains of E. coli analyzed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). Only protein biomarkers having cysteine amino acid residues appeared to bind SA. Evidence for attachment of SA is supported by the appearance of additional peaks in the MS spectra when using SA which were not present when using other MALDI matrices. The "additional" peaks appeared at 208 mass-to-charge (m/z) units higher to that of a more abundant protein ion peak. Protein biomarkers were identified by tandem mass spectrometry (MS/MS) using a MALDI time-of-flight/time-of-flight (TOF/TOF) mass spectrometer and top-down proteomics using web-based in-house developed software. Three protein biomarkers (each containing two cysteine residues) were identified as having apparent reactivity to SA: the acid stress chaperone-like proteins HdeA and HdeB and the homeobox protein (or YbgS protein). No protein was found to react with SA that did not have cysteine residues in its primary sequence. MS and MS/MS fragment ions were consistent with the attachment of SA by a thio-ester bond. MS/MS of a protein biomarker ion (with a bound SA) revealed fragment ion peaks with and without SA suggesting dissociative loss of SA. We propose dissociative loss of SA by a molecular ion rearrangement leading to cleavage of the thio-ester bond. The difference in reactivity of SA versus other MALDI matrices toward cysteine-containing proteins may be utilized for identification of unknown cysteine-containing protein biomarkers.
Technical Abstract: Portions of this work were presented earlier as an oral presentation on June 2nd 2009 at the 57th American Society of Mass Spectrometry Conference (May 31-June 4, 2009, Philadelphia, PA). We report covalent attachment via a thiol ester linkage of 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid or SA) to cysteine-containing protein biomarkers from bacterial cell lysates of E. coli analyzed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry when using SA as the matrix. Evidence to support this conclusion is the appearance of additional peaks in the MS spectra when using SA which are absent when usine alpha-cyano-4-hydroxycinnamic acid (HCCA). The "additional" peaks appear at a mass-to-charge (m/z) approximately 208 greater to the m/z of a more abundant protein ion peak. Protein biomarkers were identified by tandem mass spectrometry (MS/MS) using a MALDI time-of-flight/time-of-flight mass spectrometer and top-down proteomics. Three protein biomarkers: HdeA, HdeB and homeobox or YbgS (each containing two cysteine residues) were identified as having reactivity to SA. Non-cysteine-containing protein biomarkers showed no evidence of reactivity to SA. MS ions and MS/MS fragment ions were consistent with covalent attachment of SA via a thiol ester linkage to the side-chain of cysteine residues. MS/MS of a protein biomarker ion with a covalently attached SA revealed fragment ion peaks suggesting dissociative loss SA. We propose dissociative loss of SA is initiated by a pentacyclic transition state followed by proton abstraction of the beta-hydrogen of the bound SA by a sulfur atom lone pair followed by cleavage of the thiol ester bond and dissociative loss of 3-(4-hydroxy-3,5-dimethoxyphenyl)prop-2-ynal. The apparent reactivity of SA to cysteine/disulfide-containing proteins may complicate identification of such proteins, however the apparent differential reactivity of SA and HCCA toward cysteine/disulfide-containing proteins may be exploited for identification of unknown cysteine-containing proteins.