title: A universal, high recovery assay for protein quantitation through temperature programmed liquid chromatography (TPLC).
abstract: As an alternative to direct UV absorbance measurements, estimation of total protein concentration is typically conducted through colorimetric reagent assays. However, for protein-limited applications, the proportion of the sample sacrificed to the assay becomes increasingly significant. This work demonstrates a method for quantitation of protein samples with high recovery. Temperature programmed liquid chromatography (TPLC) with absorbance detection at 214nm permits accurate estimation of total protein concentration from samples containing as little as 0.75μg. The method incorporates a temperature gradient from 25 to 80°C to facilitate elution of total protein into a single fraction. Analyte recovery, as measured from 1 and 10μg protein extracts of Escherichia coli, is shown to exceed 93%. Extinction coefficients at 214nm were calculated across the human proteome, providing a relative standard deviation of 21% (versus 42% at 280nm), suggesting absorbance values at 214nm provide a more consistent measure of protein concentration. These results translate to a universal protein detection strategy exhibiting a coefficient of variation below 10%. Together with the sensitivity and tolerance to contaminants, TPLC with UV detection is a favorable alternative to colorimetric assay for total protein quantitation, particularly in sample-limited applications.
authors: Department of Pathology, Dalhousie University, Halifax, NS, Canada.
authors: Dennis J
authors: DJ
authors: Orton
authors: Alan A
authors: AA
authors: Doucette
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid / Q000295:instrumentation*; D056148:Chromatography, Reverse-Phase / Q000295:instrumentation*; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000032:analysis / Q000302:isolation & purification; D006801:Humans; D008892:Milk / Q000737:chemistry; D008894:Milk Proteins / Q000032:analysis / Q000302:isolation & purification; D009113:Muramidase / Q000032:analysis / Q000302:isolation & purification; D011506:Proteins / Q000032:analysis* / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000302:isolation & purification; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
chemical_list: D029968:Escherichia coli Proteins; D008894:Milk Proteins; D011506:Proteins; D012710:Serum Albumin, Bovine; D009113:Muramidase