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Sodium-Doped 3-Amino-4-hydroxybenzoic Acid: Rediscovered Matrix for Direct MALDI Glycotyping of O-Linked Glycopeptides and Intact Mucins

by Shogo Urakami and Hiroshi Hinou

Int. J. Mol. Sci. 2023, 24(23), 16836; https://doi.org/10.3390/ijms242316836 - 28 Nov 2023

Cited by 4 | Viewed by 2471

Abstract

3-Amino-4-hydroxybenzoic acid (AHB) was the first matrix identified by glycoprotein glycan analysis using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). However, compared to commonly used matrices, such as 2,5-dihydroxybenzoic acid (DHB), AHB is less efficient at glycan ionization and lacks the ability to ionize other molecular species, such as peptides, and thus is no longer used. In this study, we focused on the glycan-selective ionization ability of AHB and its low-noise properties in the low-molecular-weight region, as we expected that these properties could be enhanced by adding sodium to AHB. Sodium-doped AHB (AHB/Na) selectively imparts sodium adduct ions onto O-glycan fragments generated by the in-source decay (ISD) of glycopeptides and glycoproteins containing O-glycans that occurs during intense laser irradiation, enabling direct O-glycan analysis. Furthermore, we demonstrated that it is possible to investigate the internal structure of each O-glycan fragment with pseudo-MS/MS/MS using the sodium adduct ion of the O-glycan-derived ISD fragments from an intact mucin mixture. Full article

(This article belongs to the Special Issue New Advances in Glycobiotechnology)

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14 pages, 8338 KB

Open AccessArticle

Transient Conformations Leading to Peptide Fragment Ion [c + 2H]⁺ via Intramolecular Hydrogen Bonding Using MALDI In-source Decay Mass Spectrometry of Serine-, Threonine-, and/or Cysteine-Containing Peptides

by Mitsuo Takayama

Molecules 2023, 28(23), 7700; https://doi.org/10.3390/molecules28237700 - 22 Nov 2023

Cited by 1 | Viewed by 1544

Abstract

The formation of a peptide fragment ion [c + 2H]⁺ was examined using ultraviolet matrix-assisted laser desorption/ionization in-source decay mass spectrometry (UV/MALDI-ISD MS). Unusually, an ISD experiment with a hydrogen-abstracting oxidative matrix 4-nitro-1-naphthol (4,1-NNL) resulted in a [c + 2H]⁺ ion when the analyte peptides contained serine (Ser), threonine (Thr), and/or cysteine (Cys) residues, although the ISD with 4,1-NNL merely resulted in [a]⁺ and [d]⁺ ions. The [c + 2H]⁺ ion observed could be rationalized through intramolecular hydrogen atom transfer (HAT), like a Type-II reaction via a seven-membered conformation involving intramolecular hydrogen bonding (HB) between the active hydrogens (–OH and –SH) of the Ser/Thr/Cys residues and the backbone carbonyl oxygen at the adjacent amino (N)-terminal side residue. The ISD of the Cys-containing peptide resulted in the [c + 2H]⁺ ions, which originated from cleavage at the backbone N-Cα bonds far from the Cys residue, suggesting that the peptide molecule formed 16- and 22-membered transient conformations in the gas phase. The time-dependent density functional theory (TDDFT) calculations of the model structures of the Ser and Cys residues indicated that the Cys residue did not show a constructive bond interaction between the donor thiol (-SH) and carbonyl oxygen (=CO), while the Ser residue formed a distinct intramolecular HB. Full article

(This article belongs to the Special Issue Emerging Technologies in Biochemical Mass Spectrometry)

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15 pages, 1225 KB

Open AccessArticle

Access of Hydrogen-Radicals to the Peptide-Backbone as a Measure for Estimating the Flexibility of Proteins Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

by Mitsuo Takayama, Keishiro Nagoshi, Ryunosuke Iimuro and Kazuma Inatomi

Int. J. Mol. Sci. 2014, 15(5), 8428-8442; https://doi.org/10.3390/ijms15058428 - 13 May 2014

Cited by 9 | Viewed by 7733

Abstract

A factor for estimating the flexibility of proteins is described that uses a cleavage method of “in-source decay (ISD)” coupled with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). The MALDI-ISD spectra of bovine serum albumin (BSA), myoglobin and thioredoxin show discontinuous intense ion peaks originating from one-side preferential cleavage at the N-Cα bond of Xxx-Asp, Xxx-Asn, Xxx-Cys and Gly-Xxx residues. Consistent with these observations, Asp, Asn and Gly residues are also identified by other flexibility measures such as B-factor, turn preference, protection and fluorescence decay factors, while Asp, Asn, Cys and Gly residues are identified by turn preference factor based on X-ray crystallography. The results suggest that protein molecules embedded in/on MALDI matrix crystals partly maintain α-helix and that the reason some of the residues are more susceptible to ISD (Asp, Asn, Cys and Gly) and others less so (Ile and Val) is because of accessibility of the peptide backbone to hydrogen-radicals from matrix molecules. The hydrogen-radical accessibility in MALDI-ISD could therefore be adopted as a factor for measuring protein flexibility. Full article

(This article belongs to the Special Issue Mass Spectrometry Application in Biology)

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