Selective detection of 2‐nitrobenzenesulfenyl‐labeled peptides by matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry using a novel matrix

Abstract

The 2-nitrobenzenesulfenyl (NBS) method, which is useful for quantitative proteome analysis, is based on stable isotope labeling of tryptophan residues with NBS chloride ((12)C(6)-NBSCl or (13)C(6)-NBSCl). We found that 3-hydroxy-4-nitrobenzoic acid (3H4NBA) is a more suitable matrix than 2,5-dihydroxybenzoic acid (DHB) for detecting NBS-labeled peptides by MALDI-quadrupole IT (QIT)-TOF MS . Furthermore, NBS-labeled peptides were selectively ionized and detected in a mixture of NBS-labeled and unlabeled peptides. Labeled paired peaks were easily detected without enrichment, nonpaired labeled peaks were clearly distinguished from unlabeled contaminating peptides, and nitrotyrosine-containing peptides were also selectively detected on the 3H4NBA matrix, while by-product-peaks arising from nitrobenzene moieties were suppressed. The use of 3H4NBA as a comatrix with CHCA improved the sensitivity of detection while substantially retaining the selectivity of 3H4NBA. The 3H4NBA matrix offers great advantages in terms of simplicity, sensitivity, and usability when used for the NBS method and for MALDI-TOF MS analysis applied to compounds having a nitrobenzene ring.

Nitration is exclusive to defense-related PR-1, PR-3 and PR-5 proteins in tobacco leaves

“…The MS and MS/MS data were subjected to a FASTA search against the SwissProt and GenBank databases using the following parameters: one missed cleavage was allowed; oxidation of methionine (C15.99 Da) and pyroglutamination of the amino-terminal glutamine (¡17.06 Da) were set as variable modifications, and carbamidomethylation of cysteine (C57.02 Da) was set as a fixed modification. Additionally, some peptides were analyzed using a matrix-assisted laser desorption ionization-time of flight mass spectrometer (MALDI-TOF MS) according to Matsuo et al (2006) 17 as described in Supplemental materials and methods. In this study, unless noted otherwise, all MS analyses were performed using Q-TOF MS.…”

“…However, no nitrated peptides were detected in the tryptic digests of tobacco leaf proteins by the present Q-TOF analyses. We therefore analyzed the nitrated peptides using MALDI-TOF MS according to Matsuo et al (2006). 17 Tobacco leaves were exposed to 4.0 § 0.4 ppm 15 N-labeled NO 2 (51.6 atom% 15 N) for 8 h in light.…”

“…We therefore analyzed the nitrated peptides using MALDI-TOF MS according to Matsuo et al (2006). 17 Tobacco leaves were exposed to 4.0 § 0.4 ppm 15 N-labeled NO 2 (51.6 atom% 15 N) for 8 h in light. 14 Leaf proteins were extracted from exposed plants and subjected to 2D PAGE and immunoblotting using the anti-NT antibody as described above, except that iodoacetamide was omitted from the SDS buffer so that cysteine residues were not carbamidomethylated.…”

“…Nonetheless, we thought our present finding important in light of the reported fact that PR proteins play a vital role in plant defense/immune systems. 17,18 However, in order to clarify whether apoplastic / vacuolar proteins are nitratale, immuno-electron microscopic evidence 25 to show localization of nitrated proteins in the apoplast / the vacuole in tobacco leaves following exposure to NO 2 is absolutely required.…”

“…17,18 They are inducible upon pathogen infection and biotic/abiotic stress and play a role in the surveillance mechanism in the plant immune system. 18 They are currently classified into 17 families.…”