Robust, Quantitative, Label-free Phosphoproteomics Platform: Automated Enrichment and 2D-LC/MS/MS

摘要

Traditionally, quantitative phosphoproteomics strategies were limited to the use of stable-isotope labeled cell cultures, which is still the most commonly used sample type. Recent developments have been made using label-free techniques which make the analysis applicable to biological fluids and tissues, matrices which are not amenable to SILAC-based approaches. Here we describe an automated capillary-based enrichment strategy that achieves the robust quantitative reproducibility essential for the success of label-free workflows. For the phosphopeptide enrichment column, a fused-silica capillary was packed with titania particles to the appropriate dimension for the quantity of digested protein to be enriched. The column was placed in-line with a Waters CapLC pump, autosampler, and UV detector. Enrichment of phosphopeptides was performed through a series of injections of sample, wash buffer, and elution buffer. Quantitative reproducibility of the procedure was determined using area-under-the-curve measurements after accurate mass/retention time alignment and robust mean normalization of identified phosphopeptides from four replicate enrichments analyzed by LC/MS/MS on a Waters Synapt G1. The average CV of the 516 identified phosphopeptides was 8.7%, with an enrichment specificity of ～80%. In addition to the enrichment, the described platform also includes improvements to the LC/MS/MS analysis of enriched samples. This strategy employs the high/low pH separation of phosphopeptides that has been recently used to increase the coverage of un-enriched samples in a reasonable time frame. By adjusting the fractionation to compensate for the more acidic nature of phosphopeptides, a 3-fraction 2DLC/MS/MS method using a Waters nanoAcquity coupled to a Synapt G2 was implemented to increase the coverage of an enriched rat brain lysate from 472 to 932 over a single dimension analysis while only increasing the total analysis time by 70%. Additionally, 97% of phosphopeptides were identified in a single 2D fraction. Articles from Journal of Biomolecular Techniques : JBT are provided here courtesy of The Association of Biomolecular Resource Facilities.