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.