title: LC-MS analysis of polyclonal human anti-Neu5Gc xeno-autoantibodies immunoglobulin G Subclass and partial sequence using multistep intravenous immunoglobulin affinity purification and multienzymatic digestion.
abstract: Human polyclonal IgG antibodies directly against the nonhuman sialic acid N-glycolylneuraminic acid (Neu5Gc) are potential biomarkers and mechanistic contributors to cancer and other diseases associated with chronic inflammation. Using a sialoglycan microarray, we screened the binding pattern of such antibodies (anti-Neu5Gc IgG) in several samples of clinically approved human IVIG (IgG). These results were used to select an appropriate sample for a multistep affinity purification of the xeno-autoantibody fraction. The sample was then analyzed via our multienzyme digestion procedure followed by nano liquid chromatography (nanoLC) coupled to linear ion trap-Fourier transform mass spectrometry (LTQ-FTMS). We used characteristic and unique peptide sequences to determine the IgG subclass distribution and thus provided direct evidence that all four IgG subclasses can be generated during a xeno-autoantibody immune response to carbohydrate Neu5Gc-antigens. Furthermore, we obtained a significant amount of sequence coverage of both the constant and variable regions. The approach described here, therefore, provides a way to characterize these clinically significant antibodies, helping to understand their origins and significance.
authors.affiliation: Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115, United States.
authors.forename: Qiaozhen
authors.initials: Q
authors.lastname: Lu
authors.forename: Vered
authors.initials: V
authors.lastname: Padler-Karavani
authors.forename: Hai
authors.initials: H
authors.lastname: Yu
authors.forename: Xi
authors.initials: X
authors.lastname: Chen
authors.forename: Shiaw-Lin
authors.initials: SL
authors.lastname: Wu
authors.forename: Ajit
authors.initials: A
authors.lastname: Varki
authors.forename: William S
authors.initials: WS
authors.lastname: Hancock
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001323:Autoantibodies / Q000737:chemistry* / Q000276:immunology*; D014408:Biomarkers, Tumor / Q000737:chemistry / Q000276:immunology; D002846:Chromatography, Affinity / Q000379:methods; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000276:immunology; D007074:Immunoglobulin G / Q000737:chemistry* / Q000276:immunology*; D016756:Immunoglobulins, Intravenous / Q000737:chemistry / Q000276:immunology; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000276:immunology; D009438:Neuraminic Acids / Q000276:immunology*; D002679:Pan troglodytes; D040081:Protein Array Analysis / Q000379:methods*
chemical_list: D001323:Autoantibodies; D014408:Biomarkers, Tumor; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D016756:Immunoglobulins, Intravenous; D009438:Neuraminic Acids; C032592:N-glycolylneuraminic acid