title: Quantification of serum IgG subclasses by use of subclass-specific tryptic peptides and liquid chromatography--tandem mass spectrometry.
abstract: BACKGROUND
  Measurement of IgG subclasses is a useful tool for investigation of humoral immune deficiency in the presence of total IgG within reference intervals and IgG4-related disease. Nephelometry has been the method of choice for quantification. We describe an LC-MS/MS method that can multiplex all 4 subclasses along with total IgG by use of either IgG subclass-specific peptide stable isotope-labeled internal standards or a surrogate digest standard for quantification and does not rely on antigen/antibody reactions.
  
  
  METHODS
  We combined serum with labeled internal peptide standards and intact purified horse IgG. Samples were denatured, reduced, alkylated, and digested. We analyzed the digested serum by LC-MS/MS for IgG subclasses 1-4 and total IgG.
  
  
  RESULTS
  We assayed 112 patient sera by LC-MS/MS and immunonephelometry. The mean of the slopes and R(2) values for IgG1, IgG2, IgG3, IgG4, and IgG were 1.18 and 0.93, respectively. Interassay imprecision for the LC-MS/MS method was <15% for total IgG and subclasses and was slightly improved by use of a calibrator peptide from an exogenous horse IgG. Summed total IgG correlated with the measured total IgG within 10%. Reference intervals and analytical measuring range were all similar to our previous validation data for the immunonephelometry assays.
  
  
  CONCLUSIONS
  Total IgG and IgG subclasses 1, 2, 3, and 4 can be quantified by LC-MS/MS with performance comparable to nephelometry.
authors.affiliation: Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN.
authors.forename: Paula M
authors.initials: PM
authors.lastname: Ladwig
authors.affiliation: Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN.
authors.forename: David R
authors.initials: DR
authors.lastname: Barnidge
authors.affiliation: Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN.
authors.forename: Melissa R
authors.initials: MR
authors.lastname: Snyder
authors.affiliation: Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN.
authors.forename: Jerry A
authors.initials: JA
authors.lastname: Katzmann
authors.affiliation: Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN. murray.david@mayo.edu.
authors.forename: David L
authors.initials: DL
authors.lastname: Murray
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood* / Q000737:chemistry; D010449:Peptide Mapping; D012015:Reference Standards; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014357:Trypsin / Q000737:chemistry*
chemical_list: D007074:Immunoglobulin G; D014357:Trypsin