title: 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.
authors.affiliation: The Dow Chemical Company, Analytical Sciences, Midland, MI 48667, USA. SJulka@dow.com
authors.forename: Samir
authors.initials: S
authors.lastname: Julka
authors.forename: Jason
authors.initials: J
authors.lastname: Folkenroth
authors.forename: Scott A
authors.initials: SA
authors.lastname: Young
mesh_terms: D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid / Q000295:instrumentation* / Q000379:methods*; D045424:Complex Mixtures / Q000737:chemistry*; D004867:Equipment Design; D006736:Horses; D016014:Linear Models; D013058:Mass Spectrometry / Q000295:instrumentation* / Q000379:methods*; D011506:Proteins / Q000032:analysis* / Q000302:isolation & purification; D015203:Reproducibility of Results
chemical_list: D045424:Complex Mixtures; D011506:Proteins