Quantification of serum IgG subclasses by use of subclass-specific tryptic peptides and liquid chromatography-tandem mass spectrometry.
IgG subclass measurements are routinely done by immunonephelometry (2-4). However, nephelometry is subject to antigen excess (hook effect) interference 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 recognized (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 concentration. 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 excess, has emerged as a useful tool for quantifying proteins (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 multiple proteins, as demonstrated recently by groups monitoring many proteins in a single run (14, 15). As a class of proteins, immunoglobulins lend themselves to multiplexing since they contain numerous proteotypic peptides including clone-specific variable regions, isotype-specific constant regions, and subclass-specific regions. Recently, Hong et al. (16) demonstrated that IgG glycoforms can also be monitored along with subclass-specific proteotypic peptides by use of LCMS/MS. Here we present an assay based on subclass-specific tryptic peptides and LC-MS/MS that performs on par with the current nephelometry assay and can report the concentration of all 4 IgG subclasses and total IgG in a single analysis. We also show through a smaller subset of samples that horse IgG can be used as a surrogate digestion standard as part of the quantification of human IgG subclasses and total IgG.
Materials and Methods
Ammonium bicarbonate, 2,2,2-trifluoroethanol, DL-dithiothreitol, and iodoacetamide were purchased from Sigma-Aldrich. Formic acid, water (HPLC grade), and acetonitrile (HPLC grade) were purchased from Thermo Fisher Scientific.
STANDARDS AND CONTROLS
We determined the proteotypic peptides specific for each IgG subclass, total IgG, and horse IgG using the FASTA IgG data published in the UniProt database (www.uniprot.org). Candidate peptides were then subjected to basic local alignment search tool (BLAST) sequence analysis to assure uniqueness in the human proteome. We optimized selected reaction monitoring (SRM) transitions using MRMPilot[TM] software (Applied Biosystems).
IgG Sub Standard (LC001.TB) and IgG Sub Low Control (NL001.N) were components of Kit LK001.TB (The Binding Site). Fisherbrand[TM] research-grade fetal bovine serum was purchased from Thermo Fisher Scientific. We prepared calibrators and controls by serially diluting IgG Subclass Standard or IgG Sub Low Control in bovine serum. Seven calibrators and 5 controls were prepared as outlined in Supplemental Table 1, which accompanies the online version of this article at http://www.clinchem.org/content/vol60/issue8. Binding Site reagent was chosen because it was used for our IgG subclass nephelometric assay, thus allowing for direct quantitative comparison of the methods. The reagent also provides a known amount of each subclass in 1 bottle, negating the use of 4 different calibrator curves.
We chose bovine serum as the matrix for standards and controls because our chosen peptides were specific for human IgGs and not bovine IgGs, and human serum would have to be depleted of IgG before use. Large changes in the protein content would compromise the human serum as a model for the trypsin digestion.
We prepared stable isotope-labeled internal standard (IS) peptides in-house; they were synthesized by use of [sup.15]N and [sup.13]C stable isotope-labeled amino acids shown in bold in online Supplemental Table 2. IS peptide purity was verified by HPLC and elemental analysis, and all peptides' purities were >92%. We isolated purified horse IgG from horse serum using the General Electric Healthcare Life Sciences Protein G Gravitrap and AB Buffer Kit following the manufacturer's instructions. The concentration of total IgG was measured by nephelometry and protein electrophoresis and was adjusted to 10 g/L. A proteotypic peptide specific for the horse IgG subclass 1 constant region was chosen to be monitored by SRM and served as a surrogate digestion control. Pooled human serum did not have significant concentrations of the horse IgG subclass peptide.
We added all of the labeled peptides and purified horse IgG to blanks, standards, controls, and samples before digestion as an IS mix in 50 mmol/L ammonium bicarbonate. The 1-mg/L labeled peptides were combined as follows: 15 [micro]L IgG1,13 [micro]L IgG2, 25 [micro]L IgG3, 23 [micro]L IgG4, and 15 [micro]L of the common labeled peptide. They were then diluted to 1 mL in 1% formic acid in water to give a labeled peptide mix. The final IS mix consisted of 100 [micro]L of the labeled peptide mix and 300 [micro]L of the 10-mg/L horse IgG diluted to 10 mL in 50 mmol/L ammonium bicarbonate.
Patient serum, calibrators, and controls were brought to room temperature before dispensing for digestion. We pipetted 20 [micro]L sample into a 12 X 75-mm plastic test tube along with 100 [micro]L of the 50 mmol/L ammonium bicarbonate IS mix containing labeled peptides and horse IgG protein, 100 [micro]L 2,2,2-trifluoroethanol, and 25 [micro]L of 100 mmol/L DL-dithiothreitol. Samples were mixed and placed in a rotating incubator at 55 [degrees]C for 30 min to reduce. After reduction, samples were allowed to cool and then were alkylated with 30 [micro]L of 200 mmol/L iodoacetamide while rotating in the dark at room temperature for 1 h. A volume of 50 [micro]L of reduced and alkylated sample was transferred to a 96-well plate and mixed with 200 [micro]L water, 50 [micro]L of 50 mmol/L ammonium bicarbonate, and 30 [micro]L of 1 g/L trypsin. The tray was sonicated for 1 min and placed in a rotating incubator at 37[degrees]C for 3 h. After the 3 h, we added 20 [micro]L formic acid to stop the digestion.
All samples were analyzed on an ABSciex API 5000 triple quadrupole mass spectrometer coupled to a Thermo TLX2 LC system with a LEAP HTC Pal autosampler. A volume of 20 /[micro]L digest was separated on a Waters X-Bridge C8 column with dimensions 3.0 x 30 mm, 3.5 [micro]m particle size, at 400 [micro]L/min. Mobile phase A consisted of 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile. The gradient started at 2% B and was held for 2 min before increasing to 40% over 5.5 min. At 7.5 min, B was increased over 30 s from 40% to 98% and then was held at 98% for 2 min. At 10 min, B was ramped down from 98% to 2% and then reequilibrated for 3 min for a total run time of 13.5 min.
Source conditions were as follows: Curtain Gas, 40; gas 1, 35; gas 2, 30; temperature, 600; collisionally activated dissociation gas, 12; ion spray voltage, 5500; and entrance potential, 10. Two SRM transitions were monitored for each analyte and internal standard; the transitions used for quantification are listed in online Supplemental Table 2. Scheduled SRM was used to ensure that >15 points were acquired across an LC peak. We assessed ion suppression by postcolumn infusion of the standards (17). The standards were infused through a syringe pump connected to a T to the column effluent, or t-infusion method. No ion suppression was found.
The results presented in this paper are a combination of 2 different validation studies performed a year apart. The first study is a full method validation including limits of capability, imprecision, method comparison, linearity (to include analytical measuring range and reportable range), and reference interval. We used Analyst 1.6.1 for analysis of data. We generated calibration curves for each subclass and for the total IgG. For IgG1, for example, the area counts for the IgG1-specific peptide were divided by the area counts for its labeled peptide, giving an area ratio specific for each sample. A curve was generated by use of linear 1/x regression with the known concentration of each standard (x) to its measured area ratio (y), giving a 7-point calibration curve with linear 1/x regression for quantification for IgG subclasses 1, 2, and 3 and a 6-point calibration curve with linear 1/x regression for quantification of IgG subclass 4 and total IgG. Subclass results were summed to verify agreement with total IgG. Horse IgG was used as a digest control; the peak area of peptide was monitored but not used for any calculations.
The second half of the study used a smaller set of samples for imprecision, method comparison, linearity, and reference interval. Horse IgG, as a digest standard, was used to generate calibration curves for each subclass and total IgG with linear 1/x regression. We used Analyst 1.6.2 for analysis of data. For IgG1, for example, the area counts for the IgG1-specific peptide were divided by the area counts for the peptide for horse IgG. A curve was generated by use of linear 1/x regression with the known concentration of each standard (x) to its measured area ratio (y). Subclass results were summed to verify agreement with total IgG.
We performed the clinical immunology laboratory IgG subclass assay on a Siemens BN II nephelometer (Siemens Dade Behring BN II Nephelometer) with Binding Site antisera for each individual subclass and Siemens antiserum for IgG total. The protocol prepared initial serum dilutions specific for each subclass assessment and made subsequent dilutions dependent on initial results. The 4 subclass results were summed, and if the sum was within 80%-120% of the total IgG concentration, the results were considered valid.
Each validation run consisted of a double blank (bovine serum with no IS or digest standard added), blank (bovine serum with IS and digest standard), 7 calibrators, 5 controls, and unknowns (neat and diluted 1:10 in bovine serum). We used the double blank to show that bovine serum had no interfering peaks. The background peptide-specific area counts of the blank sample were used to calculate the limit of detection, but not for quantification of the standard curves. Fig. 1 shows an example of the ion chromatograms observed for IgG1-4 subclass-specific peptides and the IgG total common peptide. We determined limits of capability [limit of detection (LOD) and limit of quantitation (LOQ)] from a mean of 14 runs (see online Supplemental Table 3). The mean of the peptide area counts from 14 runs of the blank was calculated. Three times the SD of the blank area counts was added to the mean [mean(blank) + 3SD(blank)]. This result was compared to the mean areas (14 runs) for low-concentration samples of each subclass to determine where the mean area for a specific concentration was found to be greater than the mean(blank) + 3SD(blank) and labeled as the LOD for that IgG subclass or total. LOQ was chosen as greater or equal to the LOD, taking into account imprecision criteria (<20%) and clinical needs. We assessed linearity from the mean of 14 different calibration curves. The linear range (analytical measuring range) was determined from the LOQ to where the regression line deviated from CVs by <10%, slope equal to 1.0 ([+ or -]0.1), 95% of the intercept CI spanning 0, or r > 0.98. We performed dilution studies to show that unknowns could be diluted into the linear range of the curve to allow for an expansion of the reportable range to 50 times higher than the analytical range of measurements. We performed replicate extractions for both intraassay (n = 20) and interassay (n = 14) imprecision at 3 different concentrations for each analyte. Pools were made by diluting IgG Sub Low Control in bovine serum to produce concentrations across the linear range for each subclass. Imprecision statistics (see online Supplemental Table 4) demonstrated intra- and interassay imprecision data for subclasses 1, 2, 3 and total IgG of <10%. However, IgG 4 was <10% for intraassay measurements but >10% for interassay imprecision, with values of 15% at 3.48 mg/dL, 12% at 7.47 mg/dL, and 13% at 14.4mg/dL. We compared the LC-MS/MS assay to the nephelometric assay by running 116 unique patient samples neat and diluted 1:10 in bovine serum.
Online Supplemental Fig. 1 shows the results for the linear regression analysis of subclasses 1-4 and total IgG, with the concentration found by LC-MS/MS on the y axis and the concentration found by nephelometry on the x axis. Online Supplemental Fig. 2 shows linear regression analyses comparing the calculated concentration for the total IgG found by adding all 4 subclass concentrations together to the concentration determined by use of the total IgG standard for both the nephelometric assay and the LC-MS/MS assay. A reference interval comparison was also performed by use of 20 samples (see online Supplemental Fig. 3). This graph shows the concentrations found by each method for each of the 4 subclasses and the total IgG bracketed by the reference interval that was established during the validation of the nephelometric assay.
The first validation was performed by use of labeled peptides as the internal control. The second validation study was designed to test if quantification on the basis of a surrogate protein digestion standard could produce results comparable to those of the first validation study by use of individual stable isotope-labeled IS peptides. Horse immunoglobulin was used because of its similar tertiary structure, species-specific peptides, and relatively low cost compared with synthetically derived proteins. Tables 1 and 2 show the intraassay imprecision data (n = 20) of two different serum samples that were analyzed by both LC-MS/MS and nephelometry along with interassay imprecision (n = 5) of controls run over 5 days for the LC-MS/MS assay. Both the nephelometric assay and the LCMS/MS assay showed intraassay imprecision of <10%, which agreed with previous validations. The LCMS/MS assay interassay imprecision for the small number of runs was <12%. A comparison of LCMS/MS to nephelometry was performed on 112 unique patient and 20 reference range samples. Fig. 2 shows the result from the linear regression, and Fig. 3 shows the Bland-Altman plots from the patient sample comparison. The reference range sample comparison (Fig. 4) showed that the quantification by LC-MS/MS was consistent with the nephelometry reference ranges. Comparisons of the total IgG to the IgG calculated from the sum of the 4 subclasses for both the nephelometric assay and the LC-MS/MS assay is shown in Fig. 5. Among the patient samples is a serum that contains a monoclonal IgG1 with a protein electrophoresis M-spike of 2740 mg/ dL. The nephelometric result for IgG1 was 1515 mg/dL, and the sum of the 4 subclasses was 41% of the total IgG. The LC-MS/MS IgG1 value was 2650 mg/dL. This particular monoclonal immunoglobulin was poorly recognized by the IgG1 antiserum, but the IgG1-specific peptide was accurately quantified.
Human IgG subclasses have upwards of 80% identity between the 4 different constant-region amino acid sequences. The high degree of similarity between the different classes and the similar 3-dimensional structure of the molecules can make subclass-specific antisera difficult to produce. This can translate into different results for the same sample depending on the source of the reagents used in the immune-based assay. For example, the College of American Pathologists proficiency testing survey for IgG subclasses has 2 statistically separated peer groups on the basis of the manufacturer of the reagents used by the respective laboratory. In addition to reagent differences, limited subclass-specific epitopes may result in monoclonal IgGs having variable recognition by subclass-specific antisera. As a quality tool for measurement of the IgG subclasses, 5 separate measurements are performed; IgGs 1, 2, 3, 4 and total IgG. The antiserum to total IgG recognizes a broader spectrum of epitopes and is not as sensitive to the potential absence of sites on a monoclonal IgG. If the sum of the 4 IgG subclasses differs by >20% from the total IgG concentration, the assays are repeated at higher dilutions to rule out antigen excess. During our validation studies, serum from an 82-year-old male was submitted for subclass quantification. Nephelometry resulted in a total IgG of 3720 mg/dL, but the sum of the subclass measurements accounted for only 41% of the total IgG. The serum was retested with further dilutions to rule out antigen excess, and no antigen excess was detected. LC-MS/MS analysis gave a similar total IgG of 3150 mg/dL, with the summed IgG subclass measurements of 2748 mg/dL or 87% of the total IgG. The IgG1 measurement by nephelometry was 1515 mg/dL, whereas LC-MS/MS gave an IgG1 result of 2650 mg/dL. Serum protein electrophoresis was performed on the sample, revealing a 2740 mg/dL M-spike in the y region. The monoclonal IgG1 protein had a reduced interaction with the IgG1 antisera.
The assay presented here that uses subclass-specific tryptic peptides coupled with LC-MS/MS eliminates inconsistencies observed with immunoassays. This is accomplished by monitoring a specific set of physical parameters, precursor ion and fragment ion masses, along with LC retention time, which in combination with appropriate stable isotope-labeled ISs and digestion controls provides superior specificity to immunoassays. Our data indicate that the LC-MS/MS quantification of IgG subclasses can be performed in a single analysis with sufficient precision, analytical sensitivity, and method comparability for use in the clinical laboratory. The limits of quantification are comparable with those of the nephelometer assay. The intra- and interassay imprecision data are also comparable to the imprecision of the nephelometric assay. To confidently measure the IgG subclasses, 5 separate measurements need to be performed; IgGs 1,2,3,4 and total IgG. If the sum of IgG1-4 differs by >20% from the total IgG concentration, the assays are repeated at higher dilutions to rule out antigen excess. The slope of the comparisons of summed IgG subclasses to total IgG for the nephelometric assays was 0.964, whereas the slope for the LC-MS/MS assay was 0.987, both of which were within the 20% tolerance window (Fig. 5). The quantification of all 4 subclasses and total IgG showed good correlation between nephelometry and LCMS/MS (Figs. 3 and 4), and the comparisons of the results for the 112 patient sera met our acceptance criteria for new lots of antisera regent (slope within 0.8-1.2 and [r.sup.2] > 0.9). Comparison of the 20 healthy donors (Fig. 4) also showed good correlation, indicating that the reference intervals for the LC-MS/MS assay are similar to those of the nephelometric assay.
Recent studies point to the need for accurate and precise IgG4 measurements, since IgG4 has recently been associated with IgG4-RD, a chronic fibroinflammatory condition that derives its name from its frequent association with increases in serum IgG4 concentrations and tissue infiltration with abundant [IgG4.sup.+] plasma cells (18, 19). The spectrum of IgG4-RD is still being investigated but can involve many organ systems such as autoimmune pancreatitis, IgG4-related sclerosing cholangitis, IgG4-related retroperitoneal fibrosis, IgG4-related sialadenitis, or IgG4-related orbital inflammatory pseudotumor. A recent study by Ryu et al. (20) highlighted the spectrum of disorders associated with increased serum IgG4 concentrations and concluded that a majority of patients with serum IgG4 increases do not have IgG4-RD: increase of serum IgG4 has been demonstrated in approximately 7% of patients with pancreatic cancer, 9% of patients with primary sclerosing cholangitis, and 12%-14% of patients with cholangiocarcinoma (21, 22). Khosroshahi et al. (23) recently determined that 26% of IgG4 measurements in patients with IgG4-RD were falsely low due to the prozone or hook effect.
The results presented here that use subclass-specific tryptic peptides coupled with LC-MS/MS describe an analytically sensitive and specific assay for the quantification of IgG subclasses. Automation of the preanalytic portion of the digestion step will help simplify the LC-MS/MS method and make it cost-effective as we introduce this methodology into the clinical laboratory. We conclude that the quantification of IgG subclasses by LC-MS/MS using isotope-labeled ISs specific for each subclass or a surrogate digestion standard (horse IgG) has similar performance to current nephelometric-based assays and is not subject to antigen excess artifacts. The analytic performance and reduced reagent cost of LC-MS/MS make it an attractive methodology for multiplexing IgG subclass assays.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors' Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest.
Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.
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Paula M. Ladwig,  David R. Barnidge,  Melissa R. Snyder,  Jerry A. Katzmann,  and David L. Murray  *
 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 email@example.com.
Received January 20, 2014; accepted April 17, 2014.
Previously published online at DOI: 10.1373/clinchem.2014.222208
 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.
Table 1. Intraassay (n = 20) imprecision data for for LC-MS/MS using the surrogate IS (horse IgG) for calculation of results for respective IgG subclasses and IgG total. (a) LC-MS/MS Imprecision IgG1 IgG2 IgG3 IgG4 Total Neat serum Mean, mg/dL 490 350 80 32 890 SD, mg/dL 32 29 7 2.3 52 CV, % 6.6 8.4 9.3 7.0 5.8 Dilution Mean, mg/dL 57.9 34.7 8.5 3.0 101 SD, mg/dL 2.8 1.2 0.4 0.2 3.8 CV, % 4.8 3.5 5.1 7.8 3.7 Nephelometry Imprecision IgG1 IgG2 IgG3 IgG4 Total Neat serum Mean, mg/dL 410 300 88 20 920 SD, mg/dL 11 17 5.2 0.7 32 CV, % 2.6 5.7 5.9 3.4 3.4 Dilution Mean, mg/dL 47.8 31.2 9.1 2.3 1166 SD, mg/dL 1.5 1.6 0.7 0.1 38.7 CV, % 3.2 5.1 7.3 3.7 3.3 (a) Patient serum and a1:10 dilution were digested (n = 20) and run by LC-MS/MS. Both the neat serum and dilution were also analyzed (n = 20) by nephelometry. Table 2. Interassay (n = 5) imprecision data for for LC-MS/MS using the surrogate IS (horse IgG) for calculation of results for respective IgG subclasses and IgG total. (a) Imprecision IgG1 IgG2 IgG3 IgG4 Total IgG subclass 1 Expected, mg/dL 26 52 100 200 410 Mean, mg/dL 29 58 110 210 380 SD, mg/dL 1.6 5.3 10 22 41 CV, % 5.7 9.2 9.2 10.4 10.8 IgG subclass 2 Expected, mg/dL 15 30 60 120 240 Mean, mg/dL 16 31 60 130 250 SD, mg/dL 0.9 2.8 1.3 9 18 CV, % 5.8 8.9 2.2 7.3 7.0 IgG subclass 3 Expected, mg/dL 2 5 9 18 37 Mean, mg/dL 2 5 9 19 36 SD, mg/dL 0.3 0.4 0.3 1.3 2.1 CV, % 1.11 9.7 3.3 7.0 5.8 IgG subclass 4 Expected, mg/dL 1.5 3.1 6.1 12 24 Mean, mg/dL 1.5 3.2 6.6 13 24 SD, mg/dL 0.1 0.4 0.6 1.6 1.5 CV, % 8.5 11.5 9.9 12.1 6.2 IgG total Expected, mg/dL 45 90 180 360 720 Mean, mg/dL 46 97 190 390 710 SD, mg/dL 1.7 8.7 5 30 41 CV, % 3.7 8.9 2.8 7.6 5.7 (a) Binding Site IgG Sub Low Control was run neat and serially diluted to 1:16 and analyzed with each LC-MS/MS run.
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|Title Annotation:||Proteomics and Protein Markers|
|Author:||Ladwig, Paula M.; Barnidge, David R.; Snyder, Melissa R.; Katzmann, Jerry A.; Murray, David L.|
|Date:||Aug 1, 2014|
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