Glycoproteins account for the majority of the proteins located at the cell surface and in the
extracellular environment. During carcinogenesis the normal function of these glycoproteins as
binding ligands for cell-cell adhesion, extracellular matrix molecules, signaling receptors, and
immune cells becomes altered. Many of the steps in metastatic progression involve re-organization
and degradation of the glycoproteins in the extracellular matrix. Hence, glycan and glycoprotein
antigens still comprise the majority of FDA approved diagnostic cancer biomarkers. Our group
has recently developed multiple N-linked glycan imaging mass spectrometry workflows to
characterize the glycomes in clinically relevant tumor tissues, biofluids and cells, as well as
individually targeted glycoproteins by immunocapture. In this proposal, application of a new click
chemistry reagent specific for targeting of a2,3 sialic acid antigens, like sialyl Lewis A/CA19-9,
sialyl Lewis X and sTRA, will be used to improve isolation and sequencing of tumor-associated
glycopeptides from FFPE tissues, biofluids and cells. Standard proteases and new ECM-targeted
proteases will be used in the analyses. To accomplish this, three Specific Aims are proposed: 1.
Isolate, identify and co-localize sialic acid glycoproteins from clinical FFPE cancer tissues; 2.
Isolate and identify sialic acid glycoproteins in plasma cancer samples; 3. Isolate and identify
sialic acid glycoproteins in cancer cell lines with high levels of a2,3 sialic antigens. The
carbohydrate-targeted click capture reagents developed can be applied directly to any FFPE tissue,
cell or biofluid of interest using simple chemical reactions. The combination of tools developed
herein will facilitate the identification of specific glycopeptide and glycoprotein species in relevant
cancer samples which can be applied across the spectrum of cancer prevention, progression,
prognosis, and biomarker applications.