This study reports a global glycoproteomic analysis of pancreatic cancer cells that describes how flux through the sialic acid biosynthetic pathway selectively modulates a subset of N-glycosylation sites found within cellular proteins. These results provide evidence that sialoglycoprotein patterns are not determined exclusively by the transcription of biosynthetic enzymes or the availability of N-glycan sequons; instead, bulk metabolic flux through the sialic acid pathway has a remarkable ability to increase the abundance of certain sialoglycoproteins while having a minimal impact on others. Specifically, of 82 glycoproteins identified through a mass spectrometry and bioinformatics approach, ϳ31% showed no change in sialylation, ϳ29% exhibited a modest increase, whereas ϳ40% experienced an increase of greater than twofold. The surfaces of mammalian cells are covered with a dense layer of carbohydrates, collectively known as the glycocalyx, that influence many aspects of the interaction between a cell and its microenvironment. To date, the biosynthesis of cell surface displayed glycans has been thought to be controlled largely by individual glycosyltransferases based on the assumption that flux through the metabolic pathways that supply activated nucleotide sugar donors (the substrates for these enzymes) is not a limiting factor. For example, this premise has been used in mathematical models of sialylation (1), where concentrations of CMP-Neu5Ac in the lumen of the Golgi were assumed to be much higher than the K m of sialyltransferases (2). In the past several years, the idea that nucleotide sugars, exemplified by CMP-Neu5Ac (shown in Fig. 1A), are not a limiting or controlling factor in glycosylation has garnered one major and unambiguous exception, notably that changes in flux through the hexosamine biosynthetic pathway (HBP) 1 can alter UDP-GlcNAc levels with profound consequences on the branching of N-linked glycans (3). By changing the valence of these glycoconjugates, flux through the HBP can alter the galectin lattice and affect a host of downstream biological events including cancer progression (4); cell differentiation and proliferation (3); and autoimmunity, metabolic syndromes, and aging (5).In this report, we demonstrate that the HBP is not unique in its ability to control surface glycoproteins via bulk metabolic flux. In particular, counter to earlier assumptions that flux through the sialic acid pathway does not significantly alter the sialylation of individual glycans (2), analysis of two "high-demand" sialoglycans (i.e. polysialylated NCAM (6) and podocalyxin (7)) suggested that fluctuations in the intracellular concentrations of sialic acid and the corresponding supply of CMP-Neu5Ac critically affected their production. In the current report, we used a global cell level approach to investigate whether these two examples were outliers or whether metabolic flux controls the surface display of sialic acid with fine resolution across a wide range of N-linked glycoproteins. We found that the sialylat...