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.
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.
Graduate School of Nanobioscience, Mass Spectrometry Laboratory, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan. takayama@yokohama-cu.ac.jp.
Mitsuo
M
Takayama
Graduate School of Nanobioscience, Mass Spectrometry Laboratory, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan. knagoshi@yokohama-cu.ac.jp.
Keishiro
K
Nagoshi
Graduate School of Nanobioscience, Mass Spectrometry Laboratory, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan. n135202e@yokohama-cu.ac.jp.
Ryunosuke
R
Iimuro
Graduate School of Nanobioscience, Mass Spectrometry Laboratory, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan. i100078g@yokohama-cu.ac.jp.
Kazuma
K
Inatomi
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D018360:Crystallography, X-Ray; D005609:Free Radicals / Q000737:chemistry*; D006859:Hydrogen / Q000737:chemistry; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000032:analysis / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013816:Thermodynamics; D013879:Thioredoxins / Q000737:chemistry / Q000378:metabolism
D005609:Free Radicals; D009211:Myoglobin; D010455:Peptides; D012710:Serum Albumin, Bovine; D013879:Thioredoxins; D006859:Hydrogen