Deciphering the IgG glycosylation code of Alzheimer's Disease - Antibodies are critical components of the adaptive immune system. While their simplest mechanism of action is neutralization mediated by their hypervariable Fab domains, most of their functional effects are a result of their constant Fc domains engaging host Fc receptors to recruit and stimulate the immune system. For IgG antibodies, Fc domains engage Fc γ receptors (FcγRs) and complement C1q to induce antibody-mediated effector functions that direct the immune response. A conserved N-linked glycan in the IgG Fc domain at residue Asn297 is overwhelmingly the most important molecular determinant of Fc receptor binding and the induction of antibody- mediated effector functions. IgG antibody glycosylation is: (1) heterogeneous – a range of glycan chemical structures are appended to any given antibody and differentially glycosylated IgG antibodies, or glycoforms, each bind with distinct affinities to Fc receptors to induce unique effector function signals in both substance and strength; (2) commonly asymmetric – half or more of the IgG protein homodimers in a given antibody preparation have chemically distinct glycans linked to the Asn297 residues on each of the two Fc protomers; and (3) correlative to the time course and intensity of numerous diseases, including certain aspects of Alzheimer’s Disease. Current, high-throughput methods to determine the glycosylation states of IgG antibodies rely on the liberation of the glycans from the antibody protein. Thus, by these methods, it is impossible to determine which IgG glycoforms, the immunologically-relevant molecules, and whether they are symmetrically or asymmetrically glycosylated, exist in a mixture of antibodies; only the proportion of particular glycans in a sample can be determined. IgG glycoforms, as intact heterogeneously and often asymmetrically glycosylated homodimeric glycoproteins are the biologically-relevant molecule in the immune system. Accordingly, all information to date correlating IgG glycosylation to disease and vaccine states is to glycan content in bulk. We have therefore developed an approach to measure IgG antibody glycosylation using intact glycoprotein liquid chromatography- mass spectroscopy (LC-MS) to quantify all IgG glycoforms, regardless of the content and symmetry of their Asn297-linked glycans. We call our method WIgGWAM for Whole Immunoglobulin Glycoprofiling With Asymmetric Monitoring and have demonstrated its proof-of-concept. We propose to decipher the glycosylation code of Alzheimer’s Disease by applying our WIgGWAM workflow to cohorts of Alzheimer’s Disease patient plasma and cerebrospinal fluid samples and age-, gender- and race-matched healthy control subjects. We will also further advance our technology to overcome remaining technical challenges in order to realize its full implementation and broad utility. This work is significant because it will provide the first ever technological tool for comprehensive, spatially faithful glycoprofiling of IgG antibodies; and for its application to Alzheimer’s Disease patient samples. The studies are innovative in that they will leverage intact glycoprotein mass spectrometry techniques in unprecedented ways in order to decipher the glycosylation code of Alzheimer’s Disease.