Difference in mass analysis using labeled lysines (DIMAL-K): a new, efficient proteomic quantification method applied to the analysis of astrocytic secretomes.
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.
Delcourt
Nicolas
N
Département de Neurobiologie, Institut de Génomique Fonctionnelle, 141 rue de la cardonille, 34094 Montpellier Cedex 5, France.
Jouin
Patrick
P
Poncet
Joël
J
Demey
Emmanuelle
E
Mauger
Eric
E
Bockaert
Joël
J
Marin
Philippe
P
Galéotti
Nathalie
N
D000818:Animals; D001253:Astrocytes / Q000737:chemistry*; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional; D007553:Isotope Labeling; D008239:Lysine / Q000737:chemistry*; D051379:Mice; D010446:Peptide Fragments / Q000032:analysis*; D020543:Proteome / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D010446:Peptide Fragments; D020543:Proteome; D008239:Lysine