Mass Spectrometry–Based Method Targeting Ig Variable Regions for Assessment of Minimal Residual Disease in Multiple Myeloma
2020; Elsevier BV; Volume: 22; Issue: 7 Linguagem: Inglês
10.1016/j.jmoldx.2020.04.002
ISSN1943-7811
AutoresCarlo Martins, Sarah Huet, San San Yi, Maria Stella Ritorto, Ola Landgren, Ahmet Doğan, Jessica R. Chapman,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoMultiple myeloma is a systemic malignancy of monoclonal plasma cells that accounts for 10% of hematologic cancers. With development of highly effective therapies for multiple myeloma, minimal residual disease (MRD) assessment has emerged as an important end point for management decisions. Currently, serologic assays lack the sensitivity for MRD assessment, and invasive bone marrow sampling with flow cytometry or molecular methods has emerged as the gold standard. We report a sensitive and robust targeted mass spectrometry proteomics method to detect MRD in serum, without the need of invasive, sequential bone marrow aspirates. The method detects Ig-derived clonotypic tryptic peptides predicted by sequencing the clonal plasma cell Ig genes. A heavy isotope-labeled Ig internal standard is added to patient serum at a known concentration, the Ig is enriched in a light chain type specific manner, and proteins are digested and analyzed by targeted mass spectrometry. Peptides from the constant regions of the λ or κ light chains, Ig heavy chains, and clonotypic peptides unique to the patient monoclonal Igs are targeted. This technique is highly sensitive and specific for the patient-specific monoclonal Igs, even in samples negative by multiparametric flow cytometry. Our method can accurately and precisely detect monoclonal protein in serum of patients treated for myeloma and has broad implications for management of hematologic patients. Multiple myeloma is a systemic malignancy of monoclonal plasma cells that accounts for 10% of hematologic cancers. With development of highly effective therapies for multiple myeloma, minimal residual disease (MRD) assessment has emerged as an important end point for management decisions. Currently, serologic assays lack the sensitivity for MRD assessment, and invasive bone marrow sampling with flow cytometry or molecular methods has emerged as the gold standard. We report a sensitive and robust targeted mass spectrometry proteomics method to detect MRD in serum, without the need of invasive, sequential bone marrow aspirates. The method detects Ig-derived clonotypic tryptic peptides predicted by sequencing the clonal plasma cell Ig genes. A heavy isotope-labeled Ig internal standard is added to patient serum at a known concentration, the Ig is enriched in a light chain type specific manner, and proteins are digested and analyzed by targeted mass spectrometry. Peptides from the constant regions of the λ or κ light chains, Ig heavy chains, and clonotypic peptides unique to the patient monoclonal Igs are targeted. This technique is highly sensitive and specific for the patient-specific monoclonal Igs, even in samples negative by multiparametric flow cytometry. Our method can accurately and precisely detect monoclonal protein in serum of patients treated for myeloma and has broad implications for management of hematologic patients. Multiple myeloma (MM) is a systemic plasma cell malignancy characterized by neoplastic monoclonal plasma cell infiltrates in the bone marrow. The disease accounts for 1.7% of all cases of cancer and 10% of all hematologic malignancies and is the second most prevalent hematologic malignancy in the United States.1Siegel R.L. Miller K.D. Jemal A. Cancer statistics, 2016.CA Cancer J Clin. 2016; 66: 7-30Crossref PubMed Scopus (22038) Google Scholar The neoplastic plasma cells produce a monoclonal Ig, or M-protein, which has long been used as a biomarker in serum or urine.2Landgren O. MRD testing in multiple myeloma: from a surrogate marker of clinical outcomes to an every-day clinical tool.Semin Hematol. 2018; 55: 1-3Crossref PubMed Scopus (21) Google Scholar,3Landgren O. Lu S.X. Hultcrantz M. MRD testing in multiple myeloma: the main future driver for modern tailored treatment.Semin Hematol. 2018; 55: 44-50Crossref PubMed Scopus (26) Google Scholar This biomarker is extensively used for diagnosis and monitoring of MM by a variety of techniques, including protein electrophoresis (serum protein electrophoresis or urine protein electrophoresis), immunofixation, or free light chain assays.4Kyle R.A. Gertz M.A. Witzig T.E. Lust J.A. Lacy M.Q. Dispenzieri A. Fonseca R. Rajkumar S.V. Offord J.R. Larson D.R. Plevak M.E. Therneau T.M. Greipp P.R. Review of 1027 patients with newly diagnosed multiple myeloma.Mayo Clin Proc. 2003; 78: 21-33Abstract Full Text Full Text PDF PubMed Scopus (1659) Google Scholar However, with the development of new therapeutic strategies that achieve deeper and sustained responses, their utility and effectiveness in disease monitoring has diminished.5Anderson K.C. Auclair D. Kelloff G.J. Sigman C.C. Avet-Loiseau H. Farrell A.T. Gormley N.J. Kumar S.K. Landgren O. Munshi N.C. Cavo M. Davies F.E. Di Bacco A. Dickey J.S. Gutman S.I. Higley H.R. Hussein M.A. Jessup J.M. Kirsch I.R. Little R.F. Loberg R.D. Lohr J.G. Mukundan L. Omel J.L. Pugh T.J. Reaman G.H. Robbins M.D. Sasser A.K. Valente N. Zamagni E. The role of minimal residual disease testing in myeloma treatment selection and drug development: current value and future applications.Clin Cancer Res. 2017; 23: 3980-3993Crossref PubMed Scopus (65) Google Scholar Consequently, monitoring of minimal residual disease (MRD) has emerged as an important clinical end point to assess response to therapy and to predict long-term clinical outcome.6Kumar S. Paiva B. Anderson K.C. Durie B. Landgren O. Moreau P. et al.International Myeloma Working Group consensus criteria for response and minimal residual disease assessment in multiple myeloma.Lancet Oncol. 2016; 17: e328-e346Abstract Full Text Full Text PDF PubMed Scopus (1381) Google Scholar Current methods for detection and diagnosis of MRD include immunohistochemistry, flow cytometry, quantitative PCR, and next-generation sequencing. These methods have high sensitivity for detection of MRD, ranging from one MM cell per 103 to 106 bone marrow cells.7Mailankody S. Korde N. Lesokhin A.M. Lendvai N. Hassoun H. Stetler-Stevenson M. Landgren O. Minimal residual disease in multiple myeloma: bringing the bench to the bedside.Nat Rev Clin Oncol. 2015; 12: 286-295Crossref PubMed Scopus (86) Google Scholar However, these methods depend on invasive bone marrow aspirates. Highly sensitive, specific, and noninvasive methods for detection of low-level M-protein after treatment are not currently available in the clinical setting. Efforts have been made to use mass spectrometry (MS)–based proteomics for monitoring the circulating M-protein in the serum as a surrogate marker for the presence of neoplastic plasma cells. This has been done on intact, reduced, and digested M-protein. For example, κ and λ light chain–specific enrichment was used upstream of matrix-assisted laser desorption/ionization MS for measurement of the intact Ig heavy and light chains, enabling the calculation of heavy/light chain ratio in both serum and urine with a limit of detection of 20 mg/dL.8Mills J.R. Kohlhagen M.C. Dasari S. Vanderboom P.M. Kyle R.A. Katzmann J.A. Willrich M.A. Barnidge D.R. Dispenzieri A. Murray D.L. Comprehensive assessment of M-proteins using nanobody enrichment coupled to MALDI-TOF mass spectrometry.Clin Chem. 2016; 62: 1334-1344Crossref PubMed Scopus (94) Google Scholar Another method using Melon gel (Thermo Fisher Scientific, Waltham, MA) for the removal of non-IgG proteins from serum, followed by dissociation of the heavy and light chains, isolation of the unique M-protein light chain, and monitoring with liquid chromatography–MS (LC-MS) resulted in a limit of detection down to 0.5 mg/dL.9Mills J.R. Barnidge D.R. Murray D.L. Detecting monoclonal immunoglobulins in human serum using mass spectrometry.Methods. 2015; 81: 56-65Crossref PubMed Scopus (65) Google Scholar Bergen et al10Bergen 3rd, H.R. Dasari S. Dispenzieri A. Mills J.R. Ramirez-Alvarado M. Tschumper R.C. Jelinek D.F. Barnidge D.R. Murray D.L. Clonotypic light chain peptides identified for monitoring minimal residual disease in multiple myeloma without bone marrow aspiration.Clin Chem. 2016; 62: 243-251Crossref PubMed Scopus (43) Google Scholar enriched for the Ig fraction of serum in a κ or λ light chain–specific manner, separated the light chains from the heavy chains by SDS-PAGE, digested the light chains, and detected the patient-specific variable region peptides via LC-MS with a limit of detection of 0.1 mg/dL. Remily-Wood et al11Remily-Wood E.R. Benson K. Baz R.C. Chen Y.A. Hussein M. Hartley-Brown M.A. Sprung R.W. Perez B. Liu R.Z. Yoder S.J. Teer J.K. Eschrich S.A. Koomen J.M. Quantification of peptides from immunoglobulin constant and variable regions by LC-MRM MS for assessment of multiple myeloma patients.Proteomics Clin Appl. 2014; 8: 783-795Crossref PubMed Scopus (25) Google Scholar monitored serum levels of Igs without enrichment with multiple reaction monitoring. However, this procedure had a high level of interassay variability. Recently, Zajec et al12Zajec M. Jacobs J.F.M. Groenen P. de Kat Angelino C.M. Stingl C. Luider T.M. De Rijke Y.B. VanDuijn M.M. Development of a targeted mass-spectrometry serum assay to quantify M-protein in the presence of therapeutic monoclonal antibodies.J Proteome Res. 2018; 17: 1326-1333Crossref PubMed Scopus (25) Google Scholar combined Melon gel purification with protein digestion and targeted peptide mass spectrometry to achieve an increased sensitivity (0.01 to 0.03 mg/dL) and reproducibility for detection of M-protein in serum. This method depended on the synthesis of unique heavy isotope-labeled peptides for each patient assay. Herein, we describe a specific and sensitive method for detection of residual M-protein in patients treated for multiple myeloma. Our method combines immunoaffinity capture of Igs in serum and mass spectrometry–based methods targeting mutated peptides, derived from Ig variable regions, specific to each patient, as determined by rapid amplification of cDNA ends PCR (Figure 1). The use of a heavy-labeled internal standard allows for normalization across samples, enabling relative quantification and estimate of the depth of response. The overall efficiency of the method is highly reproducible, and there is no measurable interference from therapeutic antibodies, even at high concentrations. This method has broad implications for the management of therapy in patients with monoclonal gammopathies. To increase the sensitivity of detection of clonotypic Ig protein sequences and to reduce background serum protein contamination, enrichment for Ig fraction of serum was performed. Ig κ or λ fractions were enriched from serum using the Capture Select Kappa-XL Affinity Matrix or Lambda resins (Thermo Fisher Scientific), respectively, in a Spin-X Costar centrifuge tube filter, with a 45 μm pore size (Corning, St. Louis, MO). Briefly, 5 μL of spiked-in serum was added to 200 μL of phosphate-buffered saline (pH 7.2 to 7.4) in a spin filter with 50 μL of prewashed resin slurry 50% (three times with 400 μL phosphate-buffered saline, centrifuging at 847 × g for 1 minute). The spin filter was rotated end over end for 1 hour at room temperature. The resin was washed with 400 μL of phosphate-buffered saline, pH 7.4, and centrifuged at 847 × g for 1 minute. This procedure was repeated for a total of three washes, and the Igs were eluted with 200 μL of glycine, 100 mmol/L, pH 2.8, into a clean tube containing 20 μL of Tris, 1 mol/L, pH 8.0. All samples were reduced with 5 mmol/L Tris(2-carboxyethyl)phosphine hydrochloride (Thermo Scientific, Rockford, IL), for 15 minutes at 95°C in a thermomixer (Eppendorf, Hauppauge, NY), at 450 rpm, cooled to room temperature, alkylated with 10 mmol/L iodoacetamide (Millipore-Sigma, St. Louis, MO), for 15 minutes in the dark, and digested by addition of 1 μg of trypsin (Promega, Madison, WI) overnight at 37°C, at 450 rpm in a thermomixer. An additional aliquot of 0.5 μg of trypsin was added and incubated for 3 hours under the same conditions. Samples were acidified, and the tryptic peptides were desalted using a C18 micro-spin column (Harvard Apparatus, Holliston, MA), eluted in 70% acetonitrile in 0.1% trifluoroacetic acid, and lyophilized in a SpeedVac concentrator (Thermo Fisher Scientific). To compare the sensitivity of Ig fraction enrichment with the analysis of whole serum, stable isotopically labeled SILuMAb κ (MSQC7; Millipore-Sigma) or λ (MSQC3; Millipore-Sigma) Igs were diluted in human healthy pooled serum (HPS; Fisher Scientific, Waltham, MA) at levels of 0.01, 0.1, 1, and 10 mg/dL. Samples were prepared as described above in triplicate for both SILuMAb κ and λ with enrichment or without any enrichment. Nonenriched samples were prepared in triplicate by diluting 5 μL of each dilution point to 220 μL total volume with 50 mmol/L Tris-HCl, pH 7.0, before reduction, alkylation, and digestion. All other steps were identical to the preparation of the enriched samples. Samples were resuspended in 1% acetonitrile in 0.1% trifluoroacetic acid. Enriched samples were reconstituted in 17.5 μL, and 0.5 μL was injected for analysis. Nonenriched samples were reconstituted in 350 μL, and 1 μL was injected. The 6 × 5 peptide mix (Promega) was added to the samples for monitoring of retention time, mass accuracy, and instrument sensitivity at a total of 100 fmol per injection. The stable isotope labeled IgA peptide (WLQGSQELPR) was spiked into all MM patient samples at a total of 10 fmol per injection. Aliquots were loaded onto an Acclaim PepMap trap column (2 cm × 100 μm) in line with an EASY-Spray analytical column (25 cm × 75 μm inner diameter PepMap C18; 2 μm bead size) using the auto sampler of a Dionex Ultimate 3000 nano LC system (Thermo Fisher Scientific). Solvent A consisted of 2% acetonitrile in 0.1% formic acid, and solvent B consisted of 80% acetonitrile in 0.08% formic acid. Peptides were gradient eluted into a Q Exactive Plus (Thermo Fisher Scientific) mass spectrometer at 300 nL/minute using the following gradient: 2% to 27% solvent B in 75 minutes, 27% to 40.5% in 15 minutes, followed by 40.5% to 100% in 2 minutes. For data-dependent acquisition (DDA), high-resolution full MS spectra were acquired with a resolution of 70,000, an automatic gain control target of 1 × 106, with a maximum ion time of 50 minutes, and scan range of 300 to 1400 m/z. Following each full MS scan, 10 higher-energy collisional dissociation MS/MS spectra were acquired with resolution of 17,500, automatic gain control target of 1 × 105, maximum ion time of 100 minutes, 1.5 m/z isolation window, fixed first mass of 100 m/z, normalized collision energy of 25, and dynamic exclusion of 20 seconds. Lock mass was set for best with masses 445.1200 and 371.1012 m/z. For the parallel reaction monitoring (PRM) experiments, high-resolution full MS spectra were acquired with a resolution of 70,000, an automatic gain control target of 1 × 106, with a maximum ion time of 50 minutes, and scan range of 300 to 1500 m/z. Following each full MS scan, 10 PRM scans were acquired with a resolution of 17,500, automatic gain control target of 1 × 105, maximum ion time of 120 minutes, 1.5 m/z isolation window, fixed first mass of 150 m/z, and normalized collision energy of 27. Lock mass feature was set for best with masses 445.1200 and 371.1012 m/z. The inclusion list contained peptides from the constant region of heavy chain from IgG and IgA, light chains κ and λ, including the heavy isotope counterparts when appropriate, and the unique clonotypic peptides of the SILuMAb antibodies (Supplemental Tables S1 and S2). DDA raw files were searched against a Uniprot human database with the addition of the SILuMAb sequences using Byonic version 2.14.27 (Protein Metrics, Cupertino, CA) within Proteome Discoverer version 2.1 (Thermo Fisher Scientific) using the following settings: trypsin digestion, maximum of two missed cleavages, precursor mass tolerance of 10 ppm, fragment mass tolerance of 20 ppm, and a maximum of two common modifications and two rare modifications. Carbamidomethylation of cysteines was a static modification. Dynamic modifications included oxidation of methionine as common maximum of two, label 13C(6)15N(2) on lysine and label 13C(6)15N(4) on arginine (both as common maximum of three per peptide), and deamidation of asparagine and glutamine rare maximum of two. The search results were filtered to remove any peptide spectral matches with a Byonic score of 10% of abnormal plasma cells), using the Qiagen RNeasy micro kit (Qiagen, Hilden, Germany). Synthesis of cDNA first strand and 5′ PCR was performed with the SMARTer RACE 5′/3′ Kit (Clontech Lab, Mount View, CA), according to the clonally expanded Ig (κ or λ), then 200 ng of DNA was sequenced with 10 pmol of primer in a total volume of 11 μL using Sanger method at Memorial Sloan Kettering Cancer Center Integrated Genomics Operation. Two primers were used for each light chain for 5′ and 3′ ends of κ (IGKC) and λ (IGLC) light chains. The primers were as follows: IGKC_R1: 5′-GGGAGTTACCCGATTGGAGG-3′ (reverse); IGKC_R2: 5′-AAGACAGATGGTGCAGCCAC-3′ (reverse); IGLC_R1: 5′-GTGCTCCCTTCATGCGTGACC-3′ (reverse); and IGLC_R2: 5′-AGTGTGGCCTTGTTGGCTTG-3′ (reverse). For analysis of heavy chain, the following primers were used: IGHG_R1: 5′-GGGGAAGTAGTCCTTGACCAG-3′ (reverse); and IGHA_R1: 5′-GAGGCTCAGCGGGAAGACCTT-3′ (reverse). The mRNA sequences were analyzed by Chromas software version 2.6.2 (Technelysium, South Brisbane, QLD, Australia), and the results were converted to FASTA format. Sequences were compared with the human Ig variable region using IMGT/V-QUEST14Brochet X. Lefranc M.P. Giudicelli V. IMGT/V-QUEST: the highly customized and integrated system for IG and TR standardized V-J and V-D-J sequence analysis.Nucleic Acids Res. 2008; 36: W503-W508Crossref PubMed Scopus (834) Google Scholar online tool for identification of rearrangements. The uniqueness of the mutated peptides was determined by the NextProt online tool.15Schaeffer M. Gateau A. Teixeira D. Michel P.A. Zahn-Zabal M. Lane L. The neXtProt peptide uniqueness checker: a tool for the proteomics community.Bioinformatics. 2017; 33: 3471-3472Crossref PubMed Scopus (45) Google Scholar The κ or λ Ig fractions were enriched from serum samples collected at the time of diagnosis, as described above. The resulting peptides were analyzed using a DDA method, and the raw files were searched in Proteome Discoverer 2.1. Theoretical unique M-protein peptides, predicted from mRNA sequencing, were manually verified; and the suitability of these peptides for targeted LC-MS analysis was determined. At least one best unique peptide from each patient was selected on the basis of peak shape, intensity, fragmentation efficiency, and specificity. Patient serum samples collected at diagnosis were diluted by fivefold with water because of the high concentration of Ig, which would result in overloading of the column, poor chromatography, and saturation of the detector during LC-MS analysis (Supplemental Figure S2). Serum samples collected after treatment were used without dilution. All samples were prepared in triplicate. Serum samples were spiked with the appropriate heavy SILuMAb, depending on the M-protein light chain type, at a final concentration of 1 mg/dL. Samples were then enriched for κ or λ and prepared as described above. Before LC-MS analysis, a heavy isotope-labeled version of an IgA heavy chain constant region peptide (WLQGSQELPR) was added at a final concentration of 10 fmol per injection for normalization across samples (Supplemental Figures S2 and S3). Peptide samples were analyzed via LC-MS using the PRM method, as described above, except for the addition to the inclusion list of the curated clonotypic peptides specific to the patient M-protein and the heavy isotope-labeled IgA peptide. The complete inclusion list included peptides from the constant region of heavy chain type IgG and IgA, light chains κ and λ, the corresponding heavy isotope-labeled counterparts when appropriate, and the unique M-protein peptides for each patient (Supplemental Tables S1 and S2). The total area fragment ion values for endogenous peptides in diagnosis serum samples were multiplied by 5 to correct for the dilution (Supplemental Figure S3). Total fragment ion abundance was normalized across samples using the heavy isotope-labeled IgA heavy chain peptide before any further analysis. Two representative peptides from the constant regions of λ light chain (AAPSVTLFPPSSEELQANK and YAASSYLSLTPEQWK) and κ light chain (SGTASVVCLLNNFYPR and VDNALQSGNSQESVTEQDSK) were used to determine the relative concentration of monoclonal protein in each patient serum sample. These peptides are conserved in all λ or κ light chains and are therefore present in the heavy isotope-labeled Ig used as an internal standard. The ratio of the total fragment ion area of endogenous light chain constant region peptides/the heavy isotope-labeled counterparts (1 mg/dL) was used to determine the relative concentration of Ig in the serum samples at diagnosis. The ratio of the total fragment ion area of the patient's unique clonotypic peptides at diagnosis/the total fragment ion area of the same peptides in the post-treatment samples was determined and represents the fold change in M-protein concentration (Supplemental Figure S3). To test for interference due to the presence of a therapeutic antibody, daratumumab was spiked into each κ light chain patient post-treatment serum sample at a final concentration of 100 mg/dL. The κ light chain–specific fraction was enriched from the serum samples and prepared as described above. Data were analyzed the same way as above in comparison to the diagnosis serum without daratumumab. The current article describes a novel LC-MS–based proteomics method to detect in multiple myeloma patients during and after treatment, using peripheral blood. Our method relies on the detection of clonotypic peptides from the M-protein variable light chain, determined by mRNA sequencing, in serum as a surrogate marker for the presence of neoplastic plasma cells in the bone marrow (Figure 1). The addition of a stable heavy isotope-labeled Ig before any sample preparation serves as a control across the pre-analytical and analytical workflows. It also allows for calculation of the M-protein relative abundance between patient samples collected and processed at different time points without the need for costly and time-consuming synthesis of heavy isotope-labeled peptides specific to each patient M-protein. Enrichment of Ig fractions from serum by κ or λ light chain immunoaffinity capture enhanced sensitivity of detection of clonotypic peptides. Samples were obtained from a cohort of multiple myeloma patients (Supplemental Table S3) for evaluating the method in clinical cases. Conventional diagnostic methods, including protein electrophoresis, immunofixation, flow cytometry, and histologic review, were used for the diagnosis of MM. Supplemental Table S3 includes the subtype of the multiple myeloma clone and MRD status, as determined by the current gold standard method of flow cytometry, alongside with quantification of Igs, free light chains, and M-protein by conventional methods at the time of diagnosis. All patients were in stringent complete response by International Myeloma Working Group response criteria at the end of therapy6Kumar S. Paiva B. Anderson K.C. Durie B. Landgren O. Moreau P. et al.International Myeloma Working Group consensus criteria for response and minimal residual disease assessment in multiple myeloma.Lancet Oncol. 2016; 17: e328-e346Abstract Full Text Full Text PDF PubMed Scopus (1381) Google Scholar; however, three were determined to be MRD positive by multiparameter flow cytometry (MFC). Figure 2 shows the chronology of bone marrow and serum sampling from these patients, from diagnosis to the most recent available medical information. Supplemental Table S4 summarizes the sequences determined by mRNA sequencing. M-protein light chain sequences were determined for all patients, and heavy chain sequences were determined for four patients (Supplemental Table S4). At least one theoretical unique M-protein variable light chain peptide per patient was detected in the diagnosis serum samples and validated for use in the targeted proteomics method. A unique M-protein heavy chain peptide was detected and validated for three of the four patients in whom a variable heavy chain sequence was obtained. Supplemental Table S4 displays the variable chain sequences with mutations in bold and the validated peptides underlined. To determine the improvement in sensitivity from immunoaffinity capture and the limit of detection of our method, a dilution curve experiment was conducted in serum with and without enrichment, using stable isotopically labeled antibodies as an M-protein proxy. The SILuMAb antibodies, κ or λ, were spiked into HPS at five concentrations, ranging from 0.01 to 10 mg/dL. An HPS only sample was also prepared as a negative control. For reference, 10 mg/dL is approximately 100-fold lower than the average concentration of total Ig in the serum of a healthy individual.16Gonzalez-Quintela A. Alende R. Gude F. Campos J. Rey J. Meijide L.M. Fernandez-Merino C. Vidal C. Serum levels of immunoglobulins (IgG, IgA, IgM) in a general adult population and their relationship with alcohol consumption, smoking and common metabolic abnormalities.Clin Exp Immunol. 2008; 151: 42-50Crossref PubMed Scopus (402) Google Scholar Two Ig heavy chain constant region peptides and four peptides from the light chain (two for κ and two for λ) were targeted for relative quantitation. Without enrichment, all peptides were detected down to 1 mg/dL and some peptides were detected at a concentration of 0.1 mg/dL (Figure 3, A and B). However, when κ or λ light chain based enrichment is used, all SILuMAb peptides were detected down to 0.1 mg/dL and at 0.01 mg/dL in some cases (Figure 3, C and D). All SILuMAb peptides had a linear response in this concentration range (R2 ≥ 0.997) with or without enrichment. Endogenous Ig peptide abundance was constant regardless of the concentration of SILuMAb spiked into healthy pooled serum (Figure 3, E and F). The lower limit of detection of each targeted SILuMAb peptide was determined to be 0.01 to 0.15 mg/dL when enriched with a 1.5- to 15-fold increase in lower limit of detection between enriched and unenriched, dependent on the peptide (Table 1).13MacLean B. Tomazela D.M. Shulman N. Chambers M. Finney G.L. Frewen B. Kern R. Tabb D.L. Liebler D.C. MacCoss M.J. Skyline: an open source document editor for creating and analyzing targeted proteomics experiments.Bioinformatics. 2010; 26: 966-968Crossref PubMed Scopus (2983) Google Scholar,17Abbatiello S.E. Schilling B. Mani D.R. Zimmerman L.J. Hall S.C. MacLean B. et al.Large-scale interlaboratory study to develop, analytically validate and apply highly multiplexed, quantitative peptide assays to measure cancer-relevant proteins in plasma.Mol Cell Proteomics. 2015; 14: 2357-2374Crossref PubMed Scopus (135) Google Scholar,18Addona T.A. Abbatiello S.E. Schilling B. Skates S.J. Mani D.R. Bunk D.M. et al.Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma.Nat Biotechnol. 2009; 27: 633-641Crossref PubMed Scopus (865) Google ScholarTable 1Determination of LLoD (in mg
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