Chemical reagents and methods for detection and quantification of proteins in complex mixtures
a technology of complex mixtures and chemical reagents, applied in the field of analytical biochemistry, can solve the problems of not being able to focus on selected proteins, not being able to use the previously described methods to determine the absolute quantity of proteins in a sample,
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example i
Chemical Structure and Synthesis of VICAT Reagents
[0110]This example describes a synthetic scheme for VICAT reagents.
[0111]The structure of exemplary VICAT reagents and their corresponding synthetic schemes are shown in FIGS. 1, 2 and 4. A representative reagent contains the biotin and iodoacetyl features that are present in the ICAT reagents (Gygi et al., Nature Biotechnol. 17:994-999 (1999) and also contains a tetramethylrhodamine to make the reagent highly colored and fluorescent. The iodoacetyl group is attached to an ethylenediamine unit, which is attached to the rest of the VICAT reagent via a photolabile 2-nitrobenzyloxycarbonyl group. Exposure of the VICAT-peptide conjugate to long wavelength UV light (using a handheld lamp) for several minutes leads to photocleavage of the bond between the benzyl carbon and the bridging ester oxygen, yielding Peptide-S—CH2CONHCH2CH2NH—COOH. The latter undergoes spontaneous decarboxylation (Olejnik et al., Proc. Natl. Acad. Sci. USA 92:7590-...
example ii
Labeling, Detection and Cleavage of a Peptide Sample
[0131]This example describes a procedure for labeling protein samples with VICAT reagents.
[0132]Cell or tissue protein are reduced, and cysteine-containing proteins are tagged with a VICAT reagent. The sample is submitted to proteolysis, for example, with trypsin, to generate the peptide mixture. The proteolytic digestion can be carried out, for example, overnight at a suitable temperature for the protease used, such as 22-37° C. for trypsin. A defined amount of one or more internal standards, peptides tagged with isotopically substituted VICAT reagent, are added to the proteolytic digest. These internal standards are readily prepared by solid-phase synthesis of the peptides of interest (matching in sequence to the peptides generated by proteolysis of the proteins of interest) and reacting the cysteine of these synthetic peptides with the isotopically heavy VICAT reagent.
[0133]The peptide mixture derived from the biological sample ...
example iii
Isoelectric Focusing and ESI-MS Analysis of Peptides
[0140]This example describes the analysis of peptides labeled with a VICAT reagent using isoelectric focusing and ESI-MS.
[0141]The peptide mixture consisting of the tagged peptides from the protein sample mixture and the differentially isotopically tagged internal standard are often very complex and need to be separated prior to ESI-MS analysis. Any of a number of peptide separation methods can be applied to the sample, for example, chromatography, electrophoresis, and the like. Among the multitude of mature peptide separation methods available, isoelectric focusing (IEF) in polyacrylamide gels with immobilized pH gradients is particularly useful for the following reasons: i) The method is robust, relatively inexpensive, highly reproducible and easily multiplexed (Righetti et al., Methods. Enzymol. 270:235-55 (1996)). IEF strips covering different pH ranges are commercially available (for example, Pharmacia-Amersham Biotech; Piscat...
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