Sample-Enrichment Approaches: Immunodepletion
Human serum and plasma are an attractive biological matrix for clinical biomarker discovery studies due to the relative ease and low cost of sample collection, however it has an incredibly large dynamic range (at least 1010) and relatively few (~14) proteins make up 95% of the protein mass. The most common approach for increasing the number of proteins that can be quantified in serum/plasma using mass spectrometry is to utilize commercially available immunoaffinity columns packed with immobilized antibodies to deplete the most abundant proteins.
While the effectiveness of this approach to increase the number of unique protein identifications has been well described in the literature, the reproducibility of the method when deployed for a biomarker study is unfortunately most often completely ignored as a contributing factor to quantitative variability in the results.
Moreover, methods to assess reproducibility and efficiency of the immunodepletion step are not readily available. Through our experience across several clinical biomarker discoveries studies, we have developed three independent QC metrics aimed at assessing the immunodepletion process:
1) Protein assay QC. Perform Bradford assays on both the predepleted sample and flow-through (nondepleted) fraction. This allows measurement of initial column loading and final protein yield, and importantly, the overall fraction of the sample that was removed during depletion.
2) For liquid-chromatography column based immunodepletions, the peak areas (A280) for unbound and bound fractions are recorded, and a graphical display of the bound versus unbound AUC allows easy visualization of sample outliers.
3) Utilize SDS-PAGE gel separations to analyze aliquots of the immunodepleted sample, with protein bands visualized with coomassie blue staining and quantitated using image densitometry. This allows for a high-level visualization of the most abundant proteins, and gross detection of outliers. For instance, hemolysis during sample isolation will yield an intense hemoglobin band. This approach is also useful for detecting proteins differentially depleted over the course of a study due to nonspecific binding to the immunodepletion column.
Sample-Enrichment Approaches: Global Phosphopeptide Enrichment
Sample-enrichment strategies are necessary in order to access low-abundance protein or peptide species. Many approaches are available in the literature for protein and peptide enrichment, using antibodies or resin-based capture approaches, but few of the methods have associated metrics for reproducibility of the technique.
As an example, phosphorylation is a post-translational modification (PTM) of particular interest due to its role in cell signaling. The low stoichiometry of phosphorylated peptides relative to non-modified peptides necessitates enrichment strategies for these specific peptides (most often TiO2 or IMAC). The enrichment is known to be sensitive to a number of experimental conditions therefore establishing robust methodologies for enrichment as well as internal QC metrics are critical to maintaining a reproducible enrichment.
Critical practices to maintaining reproducible phosphopeptide enrichment include the following:
1) Keep constant the amount of input material versus the binding capacity of the resin, and the final concentration of enrichment-modifying compounds (e.g., glycolic acid or dihydroxybenzoic acid).
2) When possible, utilize the same enrichment column to enrich multiple samples, because constant binding capacity is so important.
3) Perhaps most important is to assess the reproducibility of the enrichment by spiking a known quantity of an exogenous phosphorylated protein into the lysate prior to sample processing.
Our practice is to spike bovine alpha-casein at 25 fmol per ug total lysate prior to sample digestion. Quantitative analysis of this protein within biological background across the sample cohort provides an internal standard to measure the digestion and enrichment reproducibility.
With proper consideration and control of analytical processes, label-free quantitative proteomics measurements can be performed with remarkable consistency and accuracy across experiments and laboratories. We believe that by keeping sample-preparation processes as simple as possible, standardizing protocols, and employing appropriate quality control metrics, proteomic approaches will mature such that mass spectrometry based proteomics will realize its potential as a powerful translational research tool.