Covalent attachment and dissociative loss of sinapinic acid to/from cysteine-containing proteins from bacterial cell lysates analyzed by MALDI-TOF-TOF mass spectrometry

Abstract

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 using ␣-cyano-4-hydroxycinnamic acid (HCCA). The additional peaks appear at a mass-to-charge (m/z) ϳ208 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 (TOF-TOF) 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 facilitated by a pentacyclic transition-state followed by proton abstraction of the ␤-hydrogen of the bound SA by a sulfur lone pair followed by 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/disulfidecontaining proteins may be exploited for identification of unknown cysteine-containing proteins. (J Am Soc Mass Spectrom 2010, 21, 819 -832)

Possible Evidence of Amide Bond Formation Between Sinapinic Acid and Lysine-Containing Bacterial Proteins by Matrix-Assisted Laser Desorption/Ionization (MALDI) at 355 nm

“…SA bound by electrostatic forces is the ammonium carboxylate salt transferred into the gas phase. The continued lack of reactivity of CHCA in these experiments (as well as earlier experiments [42]) suggests that the electron-withdrawing effect of the α-cyano group of CHCA may inhibit ammonium carboxylate salt formation and/or amide bond formation. The previous hypothesis of covalent attachment of SA at the side-chain of cysteine residues does not appear to be correct based on these more definitive experiments.…”

“…The presence of adducts is often dependent on the chemical composition of the matrix and the analyte. A number of groups have reported adduction of MALDI matrices to disulfide containing peptides and proteins [39][40][41][42]. In an extensive study of peptides and digested proteins having disulfide bonds, Liu and coworkers were able to demonstrate covalent attachment of α-cyano-4-hydroxycinnamic acid (CHCA) (and other matrices) under disulfide reducing conditions at high pH.…”

“…In a previous communication [42], we reported the apparent covalent attachment and dissociative loss of sinapinic acid (SA) to/from cysteine residues in disulfide bond-containing proteins (HdeA, HdeB, and YbgS) extracted from bacterial cell lysates of E. coli at low pH and under non-disulfide reducing conditions. Covalent attachment was assumed to occur at cysteine residues as only proteins having disulfide bonds appeared to show evidence of SA adduction.…”

“…We have conducted further MALDI-TOF-MS and MALDI-MS/MS (PSD) experiments to further examine this phenomenon using an E. coli strain in which hdeA or hdeB was transcribed exogenously on a plasmid under the control of an inducible promoter in order to boost expression and thus protein ion intensity by MALDI. The higher intensity of HdeA and HdeB ions (compared with ionization from lysates of wild-type E. coli strains [42]) reveal two types of SA adduction: covalently-bound SA as well as SA bound by electrostatic forces. MS/MS (PSD) reveals dissociative loss of both types of adduction.…”

Mass spectrometry: a revolution in clinical microbiology?

“…Finally 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid or SA) is a more recent matrix. It allows the analysis of proteins of higher molecular weight than the HCCA and DHB [19] . (Table 1 ) The MALDI-TOF MS is the most promising technology for the present and the future in the microbiology laboratories.…”