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

Anal Chem. 2012 Mar 20;84(6):2761-8. doi: 10.1021/ac2030893. Epub 2012 Feb 28.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Autoantibodies / chemistry*
  • Autoantibodies / immunology*
  • Biomarkers, Tumor / chemistry
  • Biomarkers, Tumor / immunology
  • Chromatography, Affinity / methods
  • Humans
  • Immunoglobulin Fab Fragments / chemistry
  • Immunoglobulin Fab Fragments / immunology
  • Immunoglobulin G / chemistry*
  • Immunoglobulin G / immunology*
  • Immunoglobulins, Intravenous / chemistry
  • Immunoglobulins, Intravenous / immunology
  • Mass Spectrometry / methods
  • Molecular Sequence Data
  • Neoplasms / immunology
  • Neuraminic Acids / immunology*
  • Pan troglodytes
  • Protein Array Analysis / methods*


  • Autoantibodies
  • Biomarkers, Tumor
  • Immunoglobulin Fab Fragments
  • Immunoglobulin G
  • Immunoglobulins, Intravenous
  • Neuraminic Acids
  • N-glycolylneuraminic acid