Method for quantifying proteins and isoforms thereof

摘要

A method for quantifying metallothionein protein isomers is described herein. Such metallothionein isomer protein quantification is useful for detecting and monitoring disease. As illustrated herein, protein quantification is a more accurate measure of metallothionein induction than is mRNA quantification.

主权项

1. A method comprising:
isolating total protein from a biological test sample; reducing proteins in the isolated total protein; alkylating proteins in the isolated total protein with an alkylating agent to generate alkylated proteins; cleaving the alkylated proteins to generate alkylated peptides; mixing the alkylated peptides with a known amount of labeled standard metallothionein peptide to generate a peptide mixture; separating labeled standard metallothionein peptide and metallothionein isomer peptides from the peptide mixture by methods comprising weak retention on strong cation exchange (SCX) chromatography to generate a metallothionein peptide-containing fraction; reducing methionine sulfoxides in the metallothionein peptide-containing fraction to generate reduced peptides; separating the reduced peptides from impurities by reversed phase high pressure liquid chromatography (HPLC); and quantifying an amount of a metallothionein isomer peptide in the test sample by determining the quantity of metallothionein isomer peptide(s) in the sample relative to the known amount of labeled standard metallothionein peptide using mass spectroscopy, wherein the metallothionein isomer peptide is an N-terminal peptide from cleavage of a metallothionein protein isomer selected from the group consisting of metallothionein-1A, metallothionein-1B, metallothionein-1E, metallothionein-1F, metallothionein-1G1, metallothionein-1G2, metallothionein-1H, metallothionein-1L, metallothionein-1M, metallothionein-1X, metallothionein-2, metallothionein-3, and metallothionein-4.