Difference in Mass Analysis Using Labeled Lysines (DIMAL-K)

Abstract

Here we describe an original strategy for unbiased quantification of protein expression called difference in mass analysis using labeled lysine (K) (DIMAL-K). DIMAL-K is based on the differential predigestion labeling of lysine residues in complex protein mixtures. The method is relevant for proteomic analysis by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. Protein labeling on lysine residues uses two closely related chemical reagents, S-methyl thioacetimidate and S-methyl thiopropionimidate. Using protein standards, we demonstrated that 1) the chemical labeling was quantitative, specific, and rapid; 2) the differentially labeled proteins co-migrated on two-dimensional gels; and 3) the identification by mass fingerprinting and the relative quantification of the proteins were possible from a single MALDI-TOF mass spectrum. The power of the method was tested by comparing and quantifying the secretion of proteins in normal and proinflammatory astrocytic secretomes (20 g). We showed that DIMAL-K was more sensitive and accurate than densitometric image analysis and allowed the detection and quantification of novel proteins. Molecular & Cellular Proteomics 4:1085-1094, 2005.Since the last decade, MS has become one of the most powerful techniques in proteomics. Two-dimensional (2-D) 1 PAGE in combination with MALDI-TOF analysis provides a sensitive method for protein identification. An important challenge in proteomics deals with quantification of relative expression levels of individual proteins in two different biological samples. In this context, quantification by mass spectrometry has recently become a major alternative strategy to analysis based on densitometric or fluorescence technologies (1-4). Despite extensive work accomplished in the field of quantification, only few strategies have been associated with the widely used 2-D PAGE/MALDI.Quantification by mass spectrometry requires the production of differentially labeled peptides from two sets of proteins. The difference in expression levels is obtained by measuring the relative intensities of MS signals of the mixed labeled peptides. Different strategies have already been described (for reviews, see Refs. 2 and 5-8). These strategies differ in 1) the pre-or postdigestion labeling, 2) the choice of the labeled function (thiol, primary amine, or carboxylic acid), 3) the use of stable isotopes or alternative chemicals, and 4) the type of MS analysis (LC-MS/MS or MALDI-TOF MS). Predigestion labeling allows for greater quantification accuracy than postdigestion labeling. Indeed introducing the chemical modification early in the experimental procedure reduces differential loss of proteins during further biochemical steps and guarantees accuracy of the quantification. Predigestion labeling often involves stable isotope incorporation through either metabolic labeling or chemical labeling on a specific residue of proteins. Metabolic labeling, such as the stable isotope labeling by amino acids in cell culture (SILAC) method, is introduced in vivo ear...

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