The Importance of Denaturing Conditions for Protein and Peptide Quantification by Tryptophan Fluorescence

Tryptophan fluorescence is utilized in the proteomics field to determine protein and peptide content, but without appropriate guidelines for its routine use. Using bovine serum albumin (BSA) and TYK-nu cell samples, we discovered that only sufficiently denatured samples resulted in accurate protein measurements. For BSA, this was achieved with 2% sodium dodecyl sulfate, or by combining other solubilization reagents (8 M urea or 1% sodium deoxycholate) with reducing agents. In contrast, accurate protein readings from TYK-nu cell samples occurred only when sodium dodecyl sulfate was utilized for protein denaturation, irrespective of reducing agent usage. To demonstrate its versatility, tryptophan fluorescence was used to quantify protein and peptide levels during an in-solution digestion of BSA, whereupon the sample dilution step reported a 50% decrease in measurable protein. However, the subsequent enzymatic digestion largely mitigated this outcome. Finally, tryptophan fluorescence and colorimetric protein assays were conducted to calculate conversion factors for BSA and a soy flour sample, which facilitated accurate protein determinations from tryptophan fluorescence readings alone. These studies demonstrate the wide applicability of the tryptophan fluorescence assay for proteomic studies, provided samples are adequately denatured to avoid protein content overestimation.