Quantification of Serum IgG Subclasses by Use of Subclass-Specific Tryptic Peptides and Liquid Chromatography–Tandem Mass Spectrometry Paula M. Ladwig, 1 David R. Barnidge, 1 Melissa R. Snyder, 1 Jerry A. Katzmann, 1 and David L. Murray 1* BACKGROUND: Measurement of IgG subclasses is a use- ful tool for investigation of humoral immune defi- ciency in the presence of total IgG within reference in- tervals and IgG4-related disease. Nephelometry has been the method of choice for quantification. We de- scribe 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 inter- nal standards or a surrogate digest standard for quan- tification and does not rely on antigen/antibody reactions. METHODS: We combined serum with labeled internal peptide standards and intact purified horse IgG. Sam- ples 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 cali- brator 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. © 2014 American Association for Clinical Chemistry The most abundant immunoglobulin class in human serum is IgG, which consists of 4 subclasses defined by unique amino acid sequences within the  heavy chain constant region. Of the total IgG, approximately 65% is IgG1, 25% is IgG2, 6% is IgG3, and 4% is IgG4. Mea- suring IgG subclass concentrations is a useful diagnos- tic measurement in patients with IgG concentrations within reference intervals who show symptoms of hu- moral or combined immunodeficiency (1). It is also useful in other subclass-related disorders such as IgG4- related disease (IgG4-RD). 2 IgG subclass measurements are routinely done by immunonephelometry (2–4). However, nephelome- try is subject to antigen excess (hook effect) interfer- ence at high protein concentrations. In antigen excess, the molar ratio of antigen to antibody is increased to the point where the antibodies can no longer cross-link to form large light-scattering complexes, causing a falsely low value. The hook effect has long been recog- nized (4, 5 ), and current automated protein analyzers have been optimized to detect and mediate the hook effect (6–8). The wide distribution of IgG subclass concentrations, however, as well as the potential for abnormal monoclonal immunoglobulin molecules, can confound accurate determination of IgG subclass concentrations. The 4 IgG subclass measurements are usually summed and compared to the total IgG con- centration. With this method, antigen excess that was not flagged by the instrumentation can often be detected. The use of proteotypic peptides along with liquid chromatography–tandem mass spectrometry (LC-MS/ MS), a technique that does not suffer from antigen ex- cess, has emerged as a useful tool for quantifying pro- teins (9 –12 ). Although clinical applications of this methodology have focused on the quantification of a single protein per sample, there are examples of clinical assays that monitor multiple tryptic peptides (13 ). As the methodology matures, there will be an increase in clinical assays with the methodology to monitor mul- tiple proteins, as demonstrated recently by groups 1 Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN. * Address correspondence to this author at: Mayo Clinic, 200 First St SW, Rochester, MN 55905. E-mail murray.david@mayo.edu. Received January 20, 2014; accepted April 17, 2014. Previously published online at DOI: 10.1373/clinchem.2014.222208 © 2014 American Association for Clinical Chemistry 2 Nonstandard abbreviations: IgG4-RD, IgG4-related disease; LC-MS/MS, liquid chromatography–tandem mass spectrometry; BLAST, basic local alignment search tool; SRM, selected reaction monitoring; IS, internal standard; LOD, limit of detection; LOQ, limit of quantitation. Clinical Chemistry 60:8 1080–1088 (2014) Proteomics and Protein Markers 1080