We propose to optimize and disseminate targeted mass spectrometric workflows for the quantification of glycopeptides and software for data interpretation. Recent developments enable efficient SWATH DIA of glycopeptides under adjusted fragmentation conditions. The sensitivity of these assays can be further improved in targeted LC-MS/MS-MRM and HR-PRM workflows. We will generate a knowledgebase of glycopeptides and the transitions needed for the development and application of the targeted glycopeptide quantification workflows. We will generate software tools necessary to develop glycopeptide transitions and analyze spectra from targeted quantification workflows with respect to these glycopeptide transitions. We will also make the analytical protocols for the development of targeted workflows for glycopeptide SWATH DIA, HR-PRM, and LC-MS/MS-MRM assays available to the research community. The availability of targeted quantification resources will enable, for the first time, exploration of glycoproteins in the context of disease pathophysiology by the broad research community. Furthermore, mass spectrometric assays for quantification of site specific protein glycoforms will drive research in the characterization of glycosylation pathways and systems glycoscience. Reliable quantification of site-specific glycoproteoforms will begin a new era of targeted clinical assays that have the potential to change the current diagnostic paradigm and to identify new therapeutic targets among the many glycoprotein targets that remain largely unexplored.
Reliable mass spectrometric quantification of glycopeptides in complex samples is now within reach due to improved analytical protocols. We propose a knowledgebase of glycopeptides and their transitions to enable targeted quantification of glycopeptides, software for data interpretation, and the dissemination of optimized workflows and assay optimization protocols. Together, these project outcomes will enable adoption of targeted workflows for the quantification of site-specific glycoform of proteins by researchers outside the analytical glycoscience community.