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

Abstract

We previously reported the apparent formation of matrix adducts of 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid or SA) via covalent attachment to disulfide bond-containing proteins (HdeA, Hde, and YbgS) from bacterial cell lysates ionized by matrix-assisted laser desorption/ionization (MALDI) time-of-flight-time-of-flight tandem mass spectrometry (TOF-TOF-MS/MS) and post-source decay (PSD). We also reported the absence of adduct formation when using α-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 formation of a solid phase 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 electronwithdrawing effect of the α-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 MS/MS (PSD). 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).

What Do We See in Spectra?: Assignment of High-Intensity Peaks of Cutibacterium and Staphylococcus Spectra of MALDI-TOF Mass Spectrometry by Interspecies Comparative Proteogenomics

“…Teramoto et al have identified two of a dozen prominent peaks in the spectra of Cutibacterium acnes that are used in subspecies discrimination-a CsbD-like protein and a 7 kD antitoxin [4]-and this is the first report of this kind in bacterial MALDI-TOF spectra. The postsource decay (PSD) effect in the commonly used setting for MALDI-TOF species identification, which includes linear mode with α-cyano-4-hydroxycinnamic acid (HCCA/CHCA) as the matrix, is faint [5,6] and can be ignored as an origin of prominent peaks. Therefore, the ions consisting of such peaks should be proton-added protein molecules without a PSD effect.…”

Bacteriophage cell lysis of Shiga toxin‐producing Escherichia coli for top‐down proteomic identification of Shiga toxins 1 & 2 using matrix‐assisted laser desorption/ionization tandem time‐of‐flight mass spectrometry

“…The peak at m/z 9955 is a matrix adduct peak of HdeA. [47] The B-subunit of Stx1a is observed at m/z 7695 which is close to its [M+H] + theoretical value of m/z 7691.7. The identity of the peak at m/z 7695 was further confirmed by MS/MS-PSD as shown in Fig.…”

“…The peak at m / z 7275 is the bacterial cold shock‐like protein: CspC. The peak at m / z 9955 is a matrix adduct peak of HdeA . The B‐subunit of Stx1a is observed at m / z 7695 which is close to its [M+H] + theoretical value of m / z 7691.7.…”

Mass spectrometry of nitroxide-terminated poly(4-vinylpyridine): A case of unwanted reactive MALDI

“…The MS/MS spectrum obtained for [I 12 +H] + at m/z 1349.7 (Fig. 2a) exhibited the same four product ion series as described for P2VP (Fig.…”

“…For example, this approach allowed the number of NH 2 end-groups in synthetic polymers to be determined from mass increments observed in MALDI mass spectra obtained for the so-formed Schiff bases compared to the underivatized original species [9]. In mass spectrometry of peptides, specific reactivity of the MALDI matrix was utilized to decipher the number of targeted amino acid residues from the mass of peptide-matrix covalent adducts, as reported for cysteine with ␣,␤-unsaturated matrices (such as ␣-cyanohydroxycinnamic acid, CHCA) [10,11] or for lysine with sinapinic acid (SA) [12].…”