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

Qiaozhen Lu, Vered Padler-Karavani, Hai Yu, Xi Chen, Shiaw Lin Wu, Ajit Varki, William S. Hancock

Research output: Contribution to journalArticle

22 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)2761-2768
Number of pages8
JournalAnalytical Chemistry
Volume84
Issue number6
DOIs
StatePublished - Mar 20 2012

ASJC Scopus subject areas

  • Analytical Chemistry

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