A Stable Isotopic Pre-digestion Labeling Method for Protein Quantitative Analysis Using Matrix-assisted Laser Desorption/ionization Mass Spectrometry

As an extension of our previous work, here a strategy was demonstrated for protein identification and quantification analyses utilizing a combination of stable isotope chemical labeling with subsequent denaturation, enzymatic digestion and matrix assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). Using d₀]‐ and d₆]‐4,6‐dimethoxy‐2‐(methylsulfonyl)pyrimidine (d₀]‐/d₆]‐DMMSP), stable isotopic labels were incorporated before digestion. The comparative samples were combined before labeling after digestion, thus biases resulting from differences in sample digestion were avoided and the higher accuracy of quantification could be attained. The labeling was spatial‐selective to particular residues of cysteine, lysine, and tyrosine before denaturation, which could lead to a better universality of the strategy for cysteine‐free proteins. In addition, some lysine residues were blocked after labeling, the partly destroyed recognition sites could simplify the trypsin hydrolysates and hence facilitate the MS complexity. Together, our one‐step labeling strategy combined several desirable properties such as spatial‐selective labeling, reliability of quantitative results, simplification of analysis of complex systems and direct analysis with minimum sample handling. Our results demonstrate the usefulness of the method for analyzing lysozyme in egg white. The method was expected to provide a new powerful tool for comparative proteome research.