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

Abstract

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 [d 0 ]-and [d 6 ]-4,6-dimethoxy-2-(methylsulfonyl)pyrimidine ([d 0 ]-/[d 6 ]-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.

Analysis of the differentially expressed low molecular weight peptides in human serum via an N‐terminal isotope labeling technique combining nano‐liquid chromatography/matrix‐assisted laser desorption/ionization mass spectrometry

“…The spiked serum samples regularly showed the singly charged ion pairs with a 6 Da mass difference in MALDI‐MS, which not only minimized operational errors in quantitation but also simplified interpretation of the mass spectra. The intrinsic pyrimidine ring of the labeling reagent increased the peak intensities of target peptides in MS resulting fromthe increased gas‐phase basicity of the derivatized N‐terminus . For each spiked sample, the peak intensity ratios between [ d 0 ]‐DMPITC‐labeled peptides and their corresponding [ d 6 ]‐DMPITC‐labeled internal standards represent the relative amounts of [ d 0 ]‐DMPITC‐labeled peptides.…”

Analysis of low molecular weight compounds by MALDI-FTICR-MS

“…9), was developed for comparative quantification of proteins [183,184] and peptides [185]. Then, [d 0 ]/[d 6 ]-DMMSP were used as derivatization agent for the studies of different expression levels of keratins in tongue coating samples of Hepatitis B patients by MALDI-MS and western-blot analysis.…”

Integration of High Accuracy N-Terminus Identification in Peptide Sequencing and Comparative Protein Analysis Via Isothiocyanate-Based Isotope Labeling Reagent with ESI Ion-trap TOF MS

“…]-4,6-dimethoxy-2-(methylsulfonyl)pyrimidine (DMMSP) has been applied as protein modification reagent with high reactivity and sensitivity owing to the introduction of pyrimidine ring [27][28][29][30] …”