Sample preparation is a critical part of the proteomic analysis workflow. Such protocols invariably involve protein solubilisation and extraction, followed by chemical reduction and alkylation, enzymatic digestion, and peptide clean-up steps. Due to the multistage nature of proteomic sample preparation, sample losses incurred after each step accumulate to diminish protein recovery and analytical sensitivity, generating biases regarding the presence of specific protein groups.
The severity of protein loss can be difficult to measure as protein assays suffer from interference caused by reagent use. It is known that the bicinchoninic acid assay is affected by reducing agents, while the Bradford assay is sensitive to commonly used surfactants. Additionally, protein losses are generally evaluated after the clean-up process, offering no insight as to the extent of loss occurring in each individual step.
Here we present an overview of the use of a reported fluorescent tryptophan assay for the investigation of different aspects of the sample preparation process for bovine serum albumin (BSA), as a model protein, with the goal of achieving an improved understanding of protein losses across concentration ranges, and to allow for further optimisation of each individual step in the workflow. Additionally, the influence of common cell lysis conditions (e.g. SDC, SDS, RIPA) on protein loss is investigated, and their impact on later stages of sample preparation will be a particular focus of the work.