A universal, high recovery assay for protein quantitation through temperature programmed liquid chromatography (TPLC)

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 214 nm permits accurate estimation of total protein concentration from samples containing as little as 0.75 μg. 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 10 μg protein extracts of Escherichia coli, is shown to exceed 93%. Extinction coefficients at 214 nm were calculated across the human proteome, providing a relative standard deviation of 21% (versus 42% at 280 nm), suggesting absorbance values at 214 nm 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.

► Temperature programming increases protein recovery above 90% in reversed phase LC.
► 214 nm provides consistent response (by mass) across the proteome.
► LC–UV assay more accurately measures protein concentration versus colorimetric assays.
► LC–UV is applicable to SDS-containing samples, following detergent depletion.