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Proteomics and Protein Markers |
Departments of1 Laboratory Medicine and 2 Medicine, University of Washington, Seattle, WA.
a Address correspondence to this author at: 1959 NE Pacific St., Box 357110, Room NW120, University of Washington, Seattle, WA 98195. Fax (206) 598-6189; e-mail ahoof{at}u.washington.edu.
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Abstract |
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Methods: We identified 3 peptides in tryptic digests of thyroglobulin that were detected at low concentrations by tandem mass spectrometry, raised polyclonal antibodies to those peptides, and used the antibodies to extract the 3 corresponding peptides from tryptic digests of human serum. We quantified each endogenous peptide using LC-MS/MS and multiple reaction monitoring with external calibrators.
Results: The detection limit for endogenous thyroglobulin in serum was 2.6 µg/L (4 pmol/L). Direct comparison with immunoassay revealed good correlation (r2 = 0.81).
Conclusions: Immunoaffinity peptide enrichment–tandem mass spectrometry can detect tryptic peptides of thyroglobulin at picomolar concentrations while also digesting the endogenous immunoglobulins that can potentially interfere with traditional immunoassays. Our observations suggest a general analytical strategy for using immunoaffinity isolation together with tandem mass spectrometry to quantify tumor antigens and other low-abundance proteins in human serum.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several studies have meticulously characterized the problems associated with the quantification of serum thyroglobulin, problems that are shared by many other immunoassays. For example, approximately 10% of patients have antibodies to Tg that could potentially interfere with immunoassays (6)(7)(8)(9). The prevalence of Tg autoantibodies increases to 25% in patients with differentiated thyroid carcinoma, although the reasons for this are incompletely understood (10)(11)(12). In addition, 3% of all patients have nonspecific heterophilic interfering antibodies that bind to reagent immunoglobulins, typically causing false-positive results in sandwich immunometric assays. Heterophilic antibody blocking reagents can reduce the number of false positives 30-fold, but a number of subjects continue to have false-positive results (13). Commercial serum thyroglobulin assays also suffer from a lack of concordance across platforms, likely owing to large variability in posttranslational modification of Tg in vivo (14)(15)(16). Even with an international reference material available, results for patient samples are often substantially different with different assays—an issue common to other immunoassays (17)(18)(19).
To test a general approach to detecting low-abundance proteins in serum that avoids the limitations inherent to immunoassays, we took advantage of the power of mass spectrometry (MS) to detect specific peptides in complex mixtures. Previous attempts to use MS to quantify low-abundance endogenous serum proteins include the direct detection of peptides from trypsin-digested serum with (20) and without (21) serum fractionation. However, only nanomolar concentrations of endogenous protein were detectable. We therefore tested a method developed by Anderson et al. (22), termed "stable isotope standards and capture by antipeptide antibodies," that combines immunoaffinity peptide purification with tandem mass spectrometry. As a test protein, we selected thyroglobulin.
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Materials and Methods |
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internal standard peptide
Stable isotope–labeled internal standard peptide (VIFDANAPV*AVR, where V* represents 13C5,14N-labeled valine) was synthesized and analyzed for purity and amino acid content (AnaSpec). We based subsequent calculations of concentration on the purity assessment and amino acid analysis. Isotopic purity of the peptide was 99.9%.
quantification of international tg reference material
After reduction and alkylation, triplicate dilution series of a certified reference preparation of Tg (BCR-457; Sigma) were digested for 2 h at 37 °C using 0.5 µg sequencing-grade modified trypsin in 0.1% Rapigest (Waters Corp.). Peptides were then lyophilized and reconstituted in 5% acetonitrile, 0.1% formic acid in water. Suspended peptides were mixed with an equal volume of isotope-labeled peptide (15 nmol/L) and analyzed using LC-MS/MS. To determine the concentration of BCR-457, we fitted data from 3 independent experiments using the relation y = Axn, which was used to calculate the dilution necessary to achieve a response of 1.0 [i.e., solving for the dilution (x) where the response (y) equals 1.0].
antiserum preparation
Based on the MS/MS analysis of tryptic digests of Tg (see Results), we selected 3 peptides for antiserum production (VIFDANAPVAVR, peptide 1; LGDQEFIK, peptide 2; and FPLGESFLVAK, peptide 3) (EZBiolab). We conjugated each peptide (2 mg) to 10 mg keyhole limpet hemocyanin (KLH) (Calbiochem) using the glutaraldehyde method. We also conjugated peptide 1 to KLH using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDAC) (Sigma), according to the manufacturer’s instructions. The efficiency of conjugation was >65% for each reaction, as assessed by MS/MS quantification of peptide that passed through a 10-kDa MWCO Microcon filter (Millipore). The peptide-KLH conjugates were combined and used to immunize 2 New Zealand White rabbits (standard protocol; Pacific Immunology).
peptide-conjugate beads, affinity purification of antibody, and antipeptide paramagnetic beads
We conjugated the peptides to BSA using the same procedures as for KLH. Peptide-BSA conjugates were then covalently bound to CNBr-activated Sepharose 4B (0.32 g) (GE Lifesciences) according to the manufacturer’s instructions. Analysis of the protein concentration of the supernatant confirmed >63% conjugate binding to the beads (Coomassie Plus; Pierce). We incubated the peptide-BSA-Sepharose conjugate with 10 mL rabbit serum (16 h, 4 °C), washed the beads with PBS, and eluted the antibodies in 870-µL fractions with 200 mmol/L glycine, pH 2.80, into tubes containing 27 µL 3 mol/L Tris, pH 8.6, and 100 µL 3 mol/L KCl. Peak fractions were pooled. The yield was 2–6 mg per 10 mL rabbit serum. The affinity-purified antibody was covalently bound to tosyl-activated Dynal M-280 beads (Invitrogen) according to the manufacturer’s instructions. Reactions resulted in 2.3 (0.12) [mean (SD)] pmol binding sites/µL paramagnetic beads (2 x 109 beads/µL).
affinity purification of thyroglobulin peptides
Serum (100 µL) was diluted with 400 µL 100 mmol/L NH4HCO3, 0.005% Tween 20, and reduced with 5 mmol/L dithiothreitol (60 min, 37 °C, with rotation). Samples were alkylated in the dark with 15 mmol/L iodoacetamide (15–120 min) and digested in 2 steps: first with 4.5 µg trypsin (4 h, 37 °C, with rotation) and second with 4.5 µg trypsin (16 h, 37 °C, with rotation). We added 20 µL beads (46 pmol peptide binding sites) and 10 µL internal standard peptide (15 nmol/L), followed by incubation for 16 h at 4 °C. The beads were then washed twice with 500 µL 100 mmol/L ammonium acetate and twice with 500 µL water. Bound peptides were eluted with 15 µL 2% acetic acid (1–2 h, room temperature, with rotation). We processed human serum calibrators with known Tg concentrations in parallel with clinical specimens (0, 2, 5, and 20 µg/L).
liquid chromatography-tandem mass spectrometry
Complete method details are provided in the online Supplemental Methods. Peptides were loaded onto a peptide-trapping column and washed with 2% acetonitrile, 0.1% formic acid in water before being eluted onto a 0.15-mm-diameter C18 analytical column with a linear gradient to 34.3% acetonitrile, 0.1% formic acid over 8 min at 1 µL/min. We analyzed the peptides in multiple reaction monitoring (MRM) mode using an Applied Biosystems API 4000 QTRAP with the m/z transitions 636.4/1059.6 (precursor+2/y10), 636.4/541.3 (precursor+2/y5) for peptide 1, 475.3/836.4 (precursor+2/y7), 475.3/779.4 (precursor+2/y6) for peptide 2, 604.3/850.5 (precursor+2/y8), 604.3/963.5 (precursor+2/y9) for peptide 3, and 639.8/1065.6 (precursor+2/y10), 639.4/547.3 (precursor+2/y5) for the internal standard peptide. Endogenous peptide peak areas were normalized to the peak area of the internal standard peptide and averaged for duplicate injections. Normalized endogenous peptide peak areas were then converted to Tg concentrations using a calibration curve generated from external human serum calibrators as illustrated in the online Supplemental Fig. 1.
human samples
All human samples were used in accordance with guidelines established by the Human Subjects Committee of the University of Washington.
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Results |
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isotope dilution quantification of thyroglobulin in the certified reference material
The international certified reference material BCR- 457, which is used to provide traceable commercial immunoassays, was generated in 1992 from pooled human thyroglobulin from cadaveric thyroid tissue (23). However, the concentration of thyroglobulin was quantified using the Lowry method, which has been shown to be less precise than direct detection (24). We therefore quantified the amount of thyroglobulin in the preparation by isotope dilution, using a stable isotope–labeled synthetic peptide (peptide 1). Linear dilutions of a thyroglobulin–internal standard peptide mixture were digested, and the peptide mixture was supplemented with known amounts of internal standard peptide (Fig. 1
). This approach indicated that the concentration of thyroglobulin in BCR-457 was 317 (7) mg/L (n = 3), which is very similar to the previously assigned value of 324 (18) mg/L.
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optimization of digestion for quantification by lc-ms/ms
Thyroglobulin is a large macromolecule consisting of two 330-kDa monomers. We first attempted to detect Tg after using size-exclusion chromatography or ultrafiltration to separate it from other serum proteins before trypsin digestion. Enrichment of Tg using size separation was ineffective, resulting in a limit of detection more than 1000-fold higher than the clinically desired limit of detection. We therefore optimized the digestion of whole serum for the immunoaffinity enrichment of Tg peptides. One important source of variability in proteomic analyses is partial proteolytic digestion of complex biological material. High concentrations of chaotropic salts are commonly used to denature proteins and help facilitate more complete tryptic digestion (25). Although the denaturants are generally removed by solid-phase extraction after digestion, their use could increase variability owing to differential recovery of peptide and additional specimen manipulation. As an alternate approach, we tested different concentrations of Tween 20, a nonionic detergent that helps denature proteins and may also improve stringency in the immunoaffinity purification step. Reduction and alkylation of 20% serum made the solution cloudy, but adding trypsin always cleared it when Tween 20 was present (0.005%, wt/vol). To promote proteolysis, we therefore included Tween rather than chaotropic salts in the serum digestion step. Sequencing-grade modified trypsin, which is usually used at much higher concentrations than in our studies, is expensive. We therefore investigated other commercial preparations of modified trypsin at 135–17 000 units of enzyme-specific activity per sample. However, only sequencing-grade trypsin was able to clarify the 10 human samples tested. Importantly, at the relatively low concentrations of trypsin used, it is unlikely that these clarified samples were completely digested.
optimizing tg quantification by immunoaffinity peptide enrichment and lc-ms/ms
We incubated tryptic digests of reduced and alkylated serum with anti-Tg peptide antibodies bound to paramagnetic beads. After washing, bound peptides were eluted from the beads and bound to a C18 trapping column. The column was then washed, and the peptides were eluted and resolved on an analytical column with an acetonitrile gradient. Fig. 2
illustrates the ion current chromatogram for the internal standard and endogenous peptide MRM transitions. Peptides 1 and 2 elute earlier than peptide 3 and have a narrower peak width. Tandem mass spectra were collected in parallel with the MRM transitions, using the linear ion trap of the 4000 Q-TRAP mass spectrometer (Fig. 2
, right panels). Interpretable MS/MS spectra were acquired for each peptide in samples containing as little as 100 µg/L endogenous Tg.
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To determine which of the 3 peptides performed best in the assay, we tested human serum containing 319 µg/L endogenous thyroglobulin diluted serially with serum lacking Tg. The 3 peptides exhibited different behaviors (Fig. 3
). Each of the peptides had a marked background signal resulting from peptide that populated antigen-binding sites after affinity purification of the antibody. Peptide 2 performed best, however, with a minimal detectable concentration of 3.9 µg/L in this experiment. Peptide 1 had a higher background signal and had a minimal detectable concentration of 35 µg/L. The peak area of peptide 3 demonstrated greater variability in its signal-to-noise ratio, and the minimal detectable concentration of Tg was 319 µg/L. After digestion, we added internal standard peptide to control for variability in the volume of immunoglobulin-coated beads added, variability in sample volume injected, and mass spectrometer performance. After normalization to the internal standard peptide, the responses of peptides 1 and 2 had characteristics similar to their respective ion current peak areas, but the response of peptide 3 was still more erratic (Fig. 3
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Because of its superior performance in the LC-ESI-MS/MS analysis, we used peptide 2 to quantify Tg in subsequent human serum specimens. We calculated peptide response as the peak area of analyte divided by the peak area of the surrogate internal standard isotope–labeled peptide, which was chemically identical to peptide 1 rather than peptide 2. We analyzed external human serum calibrators (0, 2, 5, and 20 µg/L) in parallel with other specimens. Performance characteristics of the assay are presented in Table 1
. Intraassay imprecision was 13.7% at 22.3 µg/L, 22.5% at 5.5 µg/L, and 21.4% at 3.8 µg/L (6 pmol/L). Interassay precision was 17.4% at 6.0 µg/L. The lower limit of detection, defined as the concentration corresponding to mean + 2 SDs of the zero calibrator, was 2.6 µg/L (4 pmol/L), which is equivalent to 0.8 femtomoles of peptide injected on column. At 3.8 µg/L, the mean signal-to-noise ratio was 13.8. Recovery of Tg added at 21.6 µg/L to 5 different human serum samples lacking Tg ranged from 85.0% to 99.6% [mean (SD), 93.0% (5.4%)]. Calculated using peak area, carryover was 0.8% (0.8%).
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correlation of tg quantification by immunoassay and mass spectrometry
To determine how well the results of the LC-ESI-MS/MS assay correlated with those from a clinical immunoassay for Tg, we analyzed 33 samples previously quantified by the Beckman Access 2 assay. The range of Tg concentrations determined by the immunoassay was 3.0–33.2 µg/L. The Deming regression equation had a slope (95% CI) of 1.03 (0.85–1.22) and an intercept of –1.84 (–4.93 to 1.26). The correlation (r2) was 0.81, and the standard error of the residuals (Sy|x) was 3.91 (Fig. 4
).
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Discussion |
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2 glycoprotein (21), which was detected with a limit of detection of 90 nmol/L (approximately 3 mg/L). A study that fractionated serum to quantify C-reactive protein had a limit of detection of 7 nmol/L (20). Whiteaker et al. (28) used stable isotope standards and capture by antipeptide antibodies to detect endogenous fibulin-2 in mouse plasma with a limit of detection of 380 pmol/L. Quantifying Tg by peptide capture and LC-MS/MS performed well in comparison with the immunoassay we used, even in the low picomolar range in serum. Peptide immunoaffinity purification/mass spectrometry (22) has potential advantages over immunoassays. For example, it is in principle straightforward to multiplex the assay by including additional antipeptide antibodies and isotope-labeled peptides. Moreover, because of the power of MS/MS and MRM to detect peptides in complex mixtures, it is not necessary to develop highly specific antibodies that react only with the peptides or proteins of interest. Indeed, in this experiment, the rabbit antiserum used contained antibodies directed against all 3 Tg peptides. Finally, because our protocol digests endogenous immunoglobulins that can potentially interfere with immunometric measurements, it should avoid many potential limitations of conventional immunoassays (8)(13)(26).
The peptide immunoaffinity enrichment strategy offers other advantages. For example, many investigators have attempted to decrease the limit of detection of MS analysis by immunodepleting abundant proteins. However, removing immunoglobulins could also deplete the analyte of interest in patients with autoantibodies. It is well known that many proteins strongly interact with albumin (29), so removal of this single-most-abundant serum protein could also produce erroneously low measurements. By directly detecting analyte in reference to a stable isotope–labeled internal standard peptide, mass spectrometry has the advantage of being more easily standardized across laboratories (18), provided a reference preparation is available, as is the case for Tg (23). The improvements in low-abundance serum protein quantification presented here could help provide an alternative diagnostic approach to the methods that are traditionally plagued by interference and lack of standardization (7)(19)(26).
It is important to note several limitations of our studies. For example, 4 µg affinity purified polyclonal antibody was required for each sample and calibrator, which is 2–3 orders of magnitude more than what is commonly used in sandwich ELISA protocols (30). Additionally, small amounts of the peptides were noncovalently associated with the albumin-peptide conjugate and bound directly to the CNBr-activated beads used for affinity purification. Those peptides were slowly hydrolyzed from the solid phase, which led to a background signal. As a result, only peptide 2 of the 3 peptides tested allowed sufficient detection of endogenous thyroglobulin, owing to its lower background signal. In addition, it appears that peptide 3 elicited a weaker antibody response than the other peptides, as evidenced by its relatively high limit of detection. Using other strategies to generate affinity-purified, high-avidity polyclonal antibodies might alleviate these problems, which could also be avoided by using monoclonal antibodies that do not require affinity purification. Even in the face of difficulties with 2 of the 3 peptides, however, we were able to demonstrate good intraassay imprecision for peptide 2 at a concentration of 3.8 µg/L (6 pmol/L) in human serum.
Despite these limitations, this peptide affinity isolation strategy offers major advantages. It is relatively straightforward to generate polyclonal antibodies to candidate biomarkers, and cross-reacting antibodies can be used. Also, an internal standard automatically corrects for differences in analyte recovery and detection during sample work-up and analysis.
In conclusion, we employed Tg, a protein that is present at low concentrations in serum and commonly associated with interfering antibodies, to demonstrate the feasibility of using peptide affinity capture and MS/MS to quantify a well-characterized tumor marker. Further studies of this approach have the potential to provide new benchmark methods for quantifying proteins in clinical samples.
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Acknowledgments |
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Authors’ Disclosures of Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:
Employment or Leadership: None declared.
Consultant or Advisory Role: J. W. Heinecke: Merck and Novartis and Insilicos.
Stock Ownership: None declared.
Honoraria: J. W. Heinecke: Merck and Novartis.
Research Funding: This research was supported in part by grants from the NIH (P30DK017047, P30ES07083, PO1HL030086) to J. W. Heinecke. A. N. Hoofnagle was supported by a Pilot and Feasibility Award from the Clinical Nutrition Research Unit (P30DK035816), which also provided additional support (M. H. Wener, J. O. Becker). Mass spectrometric experiments were performed at the Clinical Mass Spectrometry Center, Department of Laboratory Medicine, and the Mass Spectrometry Core, Diabetes and Endocrinology Research Center, University of Washington. The University of Washington has submitted a patent application using this methodology to quantify thyroglobulin in human serum.
Expert Testimony: None declared.
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.
Acknowledgments: The authors thank Matthew Champion, Jeff Whiteaker, and Mike MacCoss for helpful discussions.
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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