Two dimensional liquid chromatography–ultraviolet/mass spectrometric (2DLC–UV/MS) analyses for quantitation of intact proteins in complex biological matrices

Abstract

A conventional scale online two dimensional liquid chromatography-ultraviolet/mass spectrometric (2DLC–UV/MS) method was developed for simultaneous quantitation of intact proteins. A series of valve switches were utilized between the two LC dimensions and the mass spectrometer to resolve and confirm the proteins of interest from a complex biological matrix. Two model proteins, myoglobin and serum albumin were simultaneously resolved and quantitated from Escherichia coli lysate using a strong anion-exchange chromatography and reversed-phase chromatography as the first and second dimension respectively. The method validation consisted of evaluating linearity, precision, and accuracy. A linear relationship ( R 2 > 0.99) between the concentrations of the two proteins and peak areas was observed over the concentration range; 12.0–120.4 μg/mL and 8.5–85.4 μg/mL for serum albumin and myoglobin, respectively. The average RSD of peak areas for intra-day and inter-day analyses were 5.9% and 9.4% for myoglobin and 6.2% and 10.1% for serum albumin respectively. Over the linear range, the recoveries ranged from −15.4 to 9.0% for serum albumin and −2.5 to 9.4% for myoglobin. The system presented in this work is amenable to a quality control environment for evaluation and quantitation of expression levels of multiple target proteins. To our knowledge, this represents the first 2DLC–UV/MS method depicting the viability of simultaneous quantitation of more than one intact protein from complex biological mixtures in a single run.