LC-MS analysis of polyclonal human anti-Neu5Gc Xeno-autoantibodies IgG subclass and partial sequence using multi-step IVIG affinity purification and multi-enzymatic digestion

摘要

Human polyclonal IgG antibodies directly against the non-human 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 multi-step affinity purification of the xeno-autoantibody fraction. The sample was then analyzed via our multi-enzyme digestion procedure followed by nanoLC coupled to 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.