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

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.

Figure 1.

Positive ion matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) spectrum of bovine serum albumin (Mr 66430.3) obtained with 5-amino-1-naphthol matrix. The inset represents tertiary structure obtained by X-ray crystallography (PDB: 4F5S). The upper line represents primary and secondary structure (wave: α-helix, convex: turn, straight: bend or empty).

Figure 2.

Positive ion MALDI-ISD spectrum of equine apo-myoglobin (Mr 16951.4) obtained with 5-amino-1-naphthol matrix. The inset represents tertiary structure obtained by X-ray crystallography (PDB: 2FRF). The upper line represents primary and secondary structure (wave: α-helix, convex: turn, straight: bend or empty). Adapted from [18] with permissions from Springer, copyright 2014.

Figure 3.

Positive ion MALDI-ISD spectrum of human thioredoxin with HisTag (Mr 13,769.6) obtained with 5-amino-1-naphthol matrix. The inset represents tertiary structure obtained by X-ray crystallography (PDB (protein data bank): 1AUC). The upper line represents primary and secondary structure (wave: α-helix, arrow: β-strand, convex: turn, straight: bend or empty).

Figure 4.

The sites of carbonyl groups at (a) Xxx-Asp20, Xxx-Asp44 and Gly35-Xxx residues obtained from the X-ray crystallography structure of myoglobin (PDB: 2FRF); (b) Xxx-Cys32 and Xxx-Cys73 residues of thioredoxin (PDB: 1AUC).

Scheme 1.

(a) Interaction between matrix active-hydrogens and carbonyl oxygens of the peptide backbone, hydrogen transfer from matrix to the backbone and the formation of hypervalent radical species of protein. (b) Pathways of N-Cα Bond Cleavage of the Backbone to generate Fragment c/z• and c•/z Pairs and the Nomenclature of ISD (in-source decay) Fragments and (c) Formation of ISD Fragments w, z′ and z-ANL.

Scheme 2.

Thermochemical Values ΔG (kJ/mol) and ΔH (kJ/mol) for the Formation of ISD Fragments c/z• and c•/z pairs obtained from ab initio Calculations.

Scheme 3.

Thermochemical Values ΔH (kJ/mol) and ΔG (kJ/mol) for the Formation of c/z•. Pairs Originating from Cleavages at the N-Cα bond of Both Sides of n-th Residues Xxx-X and X-Xxx.

Scheme 4.

Steric hindrance by Ile and Val residues to form preliminary hydrogen-Bonding between matrix active-hydrogens and carbonyl oxygens of the backbone. The arrow represents hydrogen-bonding.