Estimation of Flexible and Rigid Residues of Disulfide-Bridged and Phosphorylated Proteins Using Matrix-Assisted Laser Desorption/Ionization in-Source Decay Mass Spectrometry.

Flexible and rigid residues in disulfide-bridged and phosphorylated proteins have been estimated by using MALDI in-source decay mass spectrometry (ISD MS). The MALDI-ISD spectra of bovine α-lactoalbumin, β-lactoglobulin A, and β-casein predict that the backbone amide of Xxx-Asp/Asn/Cys/Ser/pSer and Gly-Xxx residues has higher hydrogen accessibility than other residues, while Xxx-Ile/Val residues have less accessibility. The higher hydrogen-accessible and lower accessible residues as measured by MALDI-ISD are consistent with the flexible and rigid residues determined by X-ray, nuclear magnetic resonance, and fluorescence decay methods. The disulfide bridges and phosphate groups do not prevent the estimation of flexible or rigid residues, whereas some other disulfide bridges inhibit the identification because of decreased sensitivity of ISD fragment ions. The estimation of flexible and rigid residues by means of the matrix-hydrogen accessibility can be explained by exposure or lack thereof to the hydrogen-accessible sites of intact proteins. It is proposed that MALDI-ISD is a powerful tool for identifying flexible and rigid residues of posttranslational modified proteins without the conformation information of the protein data bank.