Flexible Xxx^|^#8211;Asp/Asn and Gly^|^#8211;Xxx Residues of Equine Cytochrome c in Matrix-Assisted Laser Desorption/Ionization In-Source Decay Mass Spectrometry

Abstract

e backbone exibility of a protein has been studied from the standpoint of the susceptibility of amino acid residues to in-source decay (ISD) in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Residues more susceptible to MALDI-ISD, namely Xxx-Asp/Asn and Gly-Xxx, were identi ed from the discontinuous intense peak of c′-ions originating from speci c cleavage at N-C α bonds of the backbone of equine cytochrome c. e identity of the residues susceptible to ISD was consistent with the known exible backbone amides as estimated by hydrogen/deuterium exchange (HDX) experiments. e identity of these exible amino acid residues (Asp, Asn, and Gly) is consistent with the fact that these residues are preferred in exible secondary structure free from intramolecular hydrogen-bonded structures such as α-helix and β-sheet.e MALDI-ISD spectrum of equine cytochrome c gave not only intense N-terminal side c′-ions originating from N-C α bond cleavage at Xxx-Asp/Asn and Gly-Xxx residues, but also C-terminal side complement z′-ions originating from the same cleavage sites. e present study implies that MALDI-ISD can give information about backbone exibility of proteins, comparable with the protection factors estimated by HDX.

Analysis of Flexibility of Proteins by means of Positive and Negative Ion MALDI In-Source Decay Mass Spectrometry

“…1), discontinuous intense fragment peaks (cleavage site) were observed at c 24 (Gly-Lys), c 30 (Pro-Asn), c 34 (Gly-Lys), c 37 (Gly-Arg), c 42 (Gln-Ala), c 45 (Gly-Phe), c 49 ( r-Asp), c 51 (Ala-Asn), and c 53 (Lys-Asn) in both positive and negative ions. e C-terminal side z′ 35 (Glu69-Asn70), and z 35 -matrix ions were also observed in both ion modes, as was previously reported in positive mode, 9) although here we did not assign the peaks of z′-ions and z-matrix ions. In the ISD spectra of myoglobin (Fig.…”

“…e susceptibility for each amino acid residue in those proteins was estimated from the intensity values of c-ions, Int(c), observed in the ISD spectra. 9) We show that in both positive and negative ion MALDI-ISD of proteins Asp, Asn, Gly, and Cys residues are more susceptible than the rest. e susceptible Asp, Asn, Gly, and Cys residues are compared to the exible residues identi ed by means of the B-factor, 19) the turn preferential factor 20) and the protection factors.…”

“…8) In a previous work, we reported that amino acid residues susceptible to ISD (Asp, Asn, and Gly) in equine cytochrome c were partly consistent with them being in exible regions as identi ed by NMR-HDX. 9) MALDI-ISD can measure the exibility of proteins by means of the interaction between carbonyl oxygens on the peptide backbone and hydrogen radicals from matrix molecules, while the exibility in NMR-HDX can be measured by the rate of HDX reaction between backbone amide hydrogens and environmental water molecules. e exible amino acid residues identi ed from the NMR-HDX of ubiquitin and cytochrome c have been reported to be Asp, Asn, Gly, Lys, r, Ile, and Met.…”

MALDI In-Source Decay of Protein

“…Furthermore, the recent report of a new matrix, 5-amino-1-naphthol (5,1-ANL), 59,60) suitable for both positiveand negative-ion MALDI-ISD of intact proteins including phosphorylated-peptides and -proteins has enabled a top-down analysis of intact proteins to be performed by using MALDI-ISD/CID experiments. 61,62) e 5,1-ANL and related-derivatives 1,5-diaminonaphthalene (1,5-DAN) 30,63) and 1,5-dihydroxynaphthalene (1,5-DHN) 60) are useful for MALDI-ISD of proteins (Scheme 11 right).…”

Photochemical Reactions of Aminonaphthols Caused by Laser Desorption/Ionization

“…20,21) 5-Amino-1-naphthol (5,1-ANL) as a MALDI matrix has the capability of hydrogen releasing reagent to generate the ISD fragments. [22][23][24] e 5,1-ANL has less volatility (more durability under vacuum condition) than another reductive matrix 1,5-diaminonaphtarene (1,5-DAN) 25) and without any sweet spots in the MALDI experiments. On the other hand, the chemical 5,1-ANL has been noticed from the standpoint of the polymer-modi ed electrodes.…”