Proteomic challenges: sample preparation techniques for microgram-quantity protein analysis from biological samples

Abstract

Proteins regulate many cellular functions and analyzing the presence and abundance of proteins in biological samples are central focuses in proteomics. The discovery and validation of biomarkers, pathways, and drug targets for various diseases can be accomplished using mass spectrometry-based proteomics. However, with mass-limited samples like tumor biopsies, it can be challenging to obtain sufficient amounts of proteins to generate high-quality mass spectrometric data. Techniques developed for macroscale quantities recover sufficient amounts of protein from milligram quantities of starting material, but sample losses become crippling with these techniques when only microgram amounts of material are available. To combat this challenge, proteomicists have developed micro-scale techniques that are compatible with decreased sample size (100 μg or lower) and still enable excellent proteome coverage. Extraction, contaminant removal, protein quantitation, and sample handling techniques for the microgram protein range are reviewed here, with an emphasis on liquid chromatography and bottom-up mass spectrometry-compatible techniques. Also, a range of biological specimens, including mammalian tissues and model cell culture systems, are discussed.

Figure 1

Acetone precipitation efficiency varies with salt concentration. The protein yield from an acetone precipitation in a complex mixture correlates with protein concentration, dielectric strength of the solution, and intrinsic protein charge. The overall model proposed is an ion-pairing model for organic solvents. With this protocol, efficiencies of 100% can be achieved for acetone precipitation. Figure adapted from Crowell, et al. (2013) [70].

Figure 2

Typical workflow for gel-based mass spectrometry analysis. The gel is used to separate whole protein in one or two dimensions. After destaining, the proteins are excised from the gel and subjected to enzymatic proteolysis. Peptides can then be analyzed via mass spectrometry. Figure adapted from Aebersold et al. (2003) [9].

Figure 3

Enhanced filter-aided sample preparation (FASP) workflow. Samples are prepared in 4% sodium dodecyl sulfate (SDS) and diluted in 8 M urea to dissociate SDS from the proteins. Filter units are passivated overnight with 5% Tween-20, followed by thorough washing in MS-grade water. Diluted samples are applied to the filter units for buffer exchanges, eliminating contaminants. Proteins are alkylated with urea present, followed by successive buffer exchanges. Proteins are digested with surfactants present, then liberated with centrifugation. Extraction with organic solvent leaves behind pure peptides for LC–MS analysis. This procedure reduces losses about 300-fold. Figure adapted from Erde et al. (2014) [104].