ABRF-sPRG 2020: Single Injection Targeted Data Acquisition of a Stable Isotope Labeled Phosphopeptide Standard.

The mission of the ABRF Proteomics Standards Research Group (sPRG) is to identify and implement technical standards that reflect the ABRF's commitment to accuracy, clarity, and consistency in the field of proteomics. There is broad interest in quantifying protein phosphorylation alterations in cellular signaling pathways, but their transient nature and low abundance makes their analysis challenging. Here we follow up on our sPRG heavy-labeled phosphopeptide standard study designed to address issues encountered in phosphopeptide experiments. This standard is constructed of 150 heavy-labeled phosphopeptides spanning seven different signaling pathways and will be useful to test the effectiveness of phosphopeptide enrichment workflows, as an internal instrument and chromatography calibrant, and as a pre-built biological assay for a wide variety of signaling pathways. In our initial characterization and validation of this standard (sPRG 2018-2019 study), we mixed the standard into an activated HeLa tryptic digest and distributed the mixture to participants with optimized and standardized enrichment and acquisition methods. Data independent acquisition (DIA) was performed on 8 gas phase fractionated injections. As an extension of this initial characterization and to prepare for a larger biological study, we present progress towards a universal single inject targeted data acquisition method using our standard which aims to be accurate, sensitive, scalable, and easily implemented and executed without the need for retention time scheduling. Two approaches were taken that both target the heavy isotope labeled internal standard peptides and trigger a mass offset scan of the endogenous light. The first method, QuanDirect, monitors for heavy y1 fragments and can be implemented on tribrid instrument platforms. The second method, SureQuant, monitors for select fragment ions and can be implemented on both tribrid and hybrid instruments.