Difference in mass analysis using labeled lysines (DIMAL-K): a new, efficient proteomic quantification method applied to the analysis of astrocytic secretomes |
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Authors: | Delcourt Nicolas Jouin Patrick Poncet Joël Demey Emmanuelle Mauger Eric Bockaert Joël Marin Philippe Galéotti Nathalie |
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Institution: | Département de Neurobiologie, Institut de Génomique Fonctionnelle, 141 rue de la cardonille, 34094 Montpellier Cedex 5, France. |
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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 microg). We showed that DIMAL-K was more sensitive and accurate than densitometric image analysis and allowed the detection and quantification of novel proteins. |
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