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

Mitsuo Takayama,Ryunosuke Iimuro,Keishiro Nagoshi,Kazuma Inatomi

doi:10.3390/ijms15058428

Mitsuo Takayama, Ryunosuke Iimuro + Show 2 more

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https://doi.org/10.3390/ijms15058428

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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.

Highlights

Mass spectrometry (MS) is an indispensable tool for analyzing biological molecules such as protein, nucleic acid, saccharides, hormones, lipid and neurotransmitters in living organisms
The B-factor and turn preference factor are based on X-ray crystallography in the crystal phase, while the protection factor is based on the hydrogen/deuterium exchange (HDX) reaction in nuclear magnetic resonance (NMR) and the fluorescence decay factor can be obtained from protein molecules in solution
It had been believed that mass spectrometry (MS) is in a disadvantageous position for obtaining information about flexibility or secondary and tertiary structures of protein molecules, here we present a factor for estimating the flexible amino acid residues in intact proteins by using a method of in-source decay (ISD) coupled with matrix-assisted laser desorption/ionization (MALDI) MS

Summary

Introduction

Mass spectrometry (MS) is an indispensable tool for analyzing biological molecules such as protein, nucleic acid, saccharides, hormones, lipid and neurotransmitters in living organisms. Matrix-assisted laser desorption/ionization (MALDI) [1,2] and electrospray ionization (ESI) [3,4] have become widely recognized as powerful soft ionization methods for identifying the expressed proteome. Both MALDI MS and ESI MS have outstanding abilities that allow identification of proteins due to their rapidity and high sensitivity, it is more difficult to glean the kind of information about secondary and tertiary structures that can be determined by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. The B-factor which relates to side chain mobility suggests that Asp, Asn, Gly, Pro, Lys, Glu, Gln and Ser residues have a more flexible nature than other residues [15]

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