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Technology Project 4

PI: David Graham          Co-PIs: Hui Zhang, Jennifer Van Eyk

Cell Surface and Secretory Pathway Tool Development

The plasma membrane proteome is an exceedingly difficult subproteome to study in part due to lack of appropriate tools. Our group will focus on i) plasma membrane proteins that face the extracellular space (cell surface proteins), ii) the secretory pathway, representing the plasma membrane, membrane organelles and membrane micro-domains, iii) the proteins associated with membrane micro-domains and v) the variation in the glycan profile both at the protein and cell level.  O-GlcNAc mod

Overall Research Plan: We will optimize our glycoprotein technologies to cardiac muscle to: i) focus on protein identification and quantification each modified peptide and ii) determine if the glycans themselves are altered and which proteins are associated with these modifications and iii) the difference in the plasma membrane verses targeted membrane microdomains.

Goal 1: Using quantitative mass spectrometry to distinguish and characterize the cell surface (plasma membrane) verses the secretory pathway proteomes. (Graham, co-PI Van Eyk)

In the heart, the plasma membrane is the location of adrenergic receptors, ion channels (NCX, L-type Ca2+ channel etc.), adenylate cyclases, gap junctions and adherent junctions. In contrast to general methods, we have focused our efforts on specifically targeting either cell surface proteins or secretory pathway. Our highly complementary strategies addressing the plasma membrane or secretory pathway involve the use of oligosaccharides as “chemical handles” since most, if not all cell surface and secretory path proteins are glycosylated. 

The first technology is termed cell surface capturing (CSC-technology) and is described in the figure below. The second technology targets specific subclasses by introducing specific metabolic precursors into cells that contain an alkane or alkyne.  Precursors are reacted with an azide-reactive group conjugated to a biotin bead by a cycloaddition reaction, termed a “click” reaction.

CSCRationale: To date, the cardiac myocyte cell surface and secretory pathway proteome has not been determined.

Overall Research Goal: The goal is to uncover the modifications and protein content changes that drive the phenotypic alterations in these subproteomes.

Goal 2: Development of lectin arrays for characterization of i) proteins with specific glycan and ii) the glycosylation status of the intact cell (Graham, co-PI Zhang)

Rationale: Lectin arrays can be used for easy enrichment of classes of glycoproteins prior to MS and also for whole cell glycome profiling. The large protein or protein complexes are captured on these arrays are digested in situ and identified by mass fingerprinting (Mascot) using MALDI TOF TOF instruments.  Each array includes one or more spots acting as blanks, enabling subtraction of peaks due to lectin and trypsin.  Lectin arrays show some specify. The presence (and to some degree the quantity) of multiple analytes on the same spot can be accessed directly by mass spectrometry, as each component carries mass as an inherent label.