Mass Spectrometric ITEM-FOUR Analysis Reveals Coding Single-Nucleotide Polymorphisms in Human Cardiac Troponin T That Evade Detection by Sandwich ELISAs Which Use Monoclonal Antibodies M7 and M11.7 from the Elecsys Troponin T® Assay
Abstract
1. Introduction
2. Results
2.1. Characterization of Antibodies and Epitope Peptides
2.2. Binding Strength Analysis by ITEM-FOUR Mass Spectrometry
2.3. Binding Motif Deduction
3. Discussion
4. Materials and Methods
4.1. Preparation of Solutions with Peptides, Antibodies, and Antibody–Peptide Complexes
4.2. Protein and Peptide Concentration Determination
4.3. SDS-PAGE Analysis of the hcTnT Antigen
4.4. Western Blot Analysis of the Anti-hcTnT Antibodies
4.5. Mass Calibration of Mass Spectrometry Instruments
4.6. Preparation of Nano-ESI-MS Emitters, Filling and Mounting
4.7. Q-ToF 2 Instrument Settings and Data Acquisition
4.8. Synapt G2S Instrument Settings and Data Acquisition
4.9. ITEM-FOUR Spectral Data Analysis
4.10. ITEM-FOUR Calculations of Apparent Kinetic and Apparent Thermodynamic Values
4.11. Molecular Modeling of Protein and Peptide Structures
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
List of Abbreviations
Å | Ångström |
ACC | American College of Cardiology |
anti-TNFα | anti-tumor necrosis factor alpha |
BSA | bovine serum albumin |
cSNP | coding single-nucleotide polymorphism |
cTnT | cardiac troponin T |
DCM | dilative cardiomyopathy |
dbSNP | single-nucleotide polymorphism database |
ELISA | enzyme-linked immunosorbent assay |
ESC | European Society for Cardiology |
ESI | electrospray ionization |
HCM | hypertrophic cardiomyopathy |
hcTNT | human cardiac troponin T |
hs | high-sensitivity |
ITEM | intact transition epitope mapping |
ITEM FOUR | intact transition epitope mapping—force differences between original and unusual residues |
kDa | kiloDalton |
MI | myocardial infarction |
m/z | mass-to-charge ratio |
nano-ESI-MS | nano-electrospray ionization–mass spectrometry |
PBS | phosphate-buffered saline |
POC | point of care |
PRIDE | PRoteomics IDEntification |
PVDF | polyvinylidene fluoride |
Q-ToF | quadrupole time of flight |
RCM | restrictive cardiomyopathy |
rpm | revolutions per minute |
RIPA | radioimmunoprecipitation assay |
SDC | sodium deoxycholate |
SDS | sodium dodecyl sulfate |
SDS-PAGE | sodium dodecyl sulfate–polyacrylamide gel electrophoresis |
SNP | single-nucleotide polymorphism |
tif | tagged image file |
Tn | troponin |
TnI | troponin I |
TNNT2 | troponin T gene |
TnT | troponin T |
TRIS | tris(hydroxymethyl)aminomethane |
ΔCV | voltage difference in collision cell |
3D | three-dimensional |
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Peptide No. (wt or SAP) (a,b) | Amino Acid Sequence (wt or SAP) (a,b) | SNP Entry (c) | SNP (c) | Cardiomyopathy Association (c) | Atom No. | Mr (Mono) (d) | MM (Exp.) (e) |
---|---|---|---|---|---|---|---|
11 (wt) | LVSLKDRIERRRAER | n.a. | n.a. | n.a. | 278 | 1896.14 | 1896.91 |
12 (E138K) | LVSLKDRIKRRRAER | rs 730881100 | G > A | hypertrophic | 283 | 1896.27 | 1896.44 |
13 (R140C) | LVSLKDRIERCRAER | rs 397516463 | C > T | hypertrophic/familial restrictive | 264 | 1842.03 | 1842.09 |
14 (R141W) | LVSLKDRIERRWAER | rs 74315380 | C > T | dilated | 279 | 1926.11 | 1926.36 |
15 (R141G) | LVSLKDRIERRGAER | rs 74315380 | C > G | not provided | 262 | 1797.07 | 1797.19 |
16 (R141P) | LVSLKDRIERRPAER | rs 397516464 | G > C | dilated | 269 | 1837.08 | 1837.43 |
17 (R141Q) | LVSLKDRIERRQAER | rs 397516464 | G > A | dilated | 272 | 1868.09 | 1868.18 |
21 (wt) | AEQQRIRNEREKERQ | n.a. | n.a. | n.a. | 274 | 1969.04 | 1969.67 |
22 (E146Q) | AQQQRIRNEREKERQ | rs 371142225 | G > C | dilated | 275 | 1968.07 | 1968.78 |
23 (E146K) | AKQQRIRNEREKERQ | rs 371142225 | G > A | dilated | 279 | 1968.10 | 1969.02 |
24 (Q148R) | AEQRRIRNEREKERQ | rs 730880232 | A > G | not provided | 280 | 1997.08 | 1997.43 |
25 (R149C) | AEQQCIRNEREKERQ | rs 397516465 | C > T | familial dilated | 261 | 1914.94 | 1915.46 |
26 (R149S) | AEQQSIRNEREKERQ | rs 397516465 | C > A | not provided | 262 | 1899.96 | 1899.56 |
27 (R149H) | AEQQHIRNEREKERQ | rs 397516466 | G > A | dilated | 268 | 1950.00 | 1951.24 |
28 (R151W) | AEQQRIWNEREKERQ | rs 74315379 | C > T | dilated | 275 | 1999.03 | 2000.02 |
29 (R151Q) | AEQQRIQNEREKERQ | rs 730881101 | G > A | familial restrictive | 268 | 1941.00 | 1941.99 |
30 (R154W) | AEQQRIRNEWEKERQ | rs 483352832 | C > T | dilated | 275 | 1999.03 | 2000.95 |
31 (R154Q) | AEQQRIRNEQEKERQ | rs 745632066 | G > A | familial restrictive | 268 | 1941.00 | 1941.90 |
32 (E155K) | AEQQRIRNERKKERQ | rs 984218824 | G > A | familial restrictive | 279 | 1968.10 | 1969.03 |
Complex (a) | Peptide Sequence | Mean Charge ± SD. (b,c) | Initial (%) (b,c,d) | Final (%) (b,c,e) | Δ (% pts) | ΔCV50 (V) (b) | dx (V) (b) | Slope (%/V) (b) | R2 (b,c) |
---|---|---|---|---|---|---|---|---|---|
M7 + P11 | LVSLKDIERRRAER | 24.1 ± 0.23 | 56.43 | 84.36 | 27.93 | 25.27 | 6.69 | 1.04 | 0.995 |
M7 + P12 | LVSLKDIKRRRAER | 24.5 ± 0.67 | 89.38 | 92.28 | 2.90 | n.a. | n.a. | n.a. | n.a. |
M7 + P13 | LVSLKDIERCRAER | 24.3 ± 0.29 | 40.36 | 83.40 | 43.04 | 23.30 | 6.55 | 1.64 | 0.999 |
M7 + P14 | LVSLKDIERRWAER | 24.1 ± 0.22 | 54.37 | 93.86 | 39.49 | 18.02 | 4.33 | 2.28 | 0.997 |
M7 + P15 | LVSLKDIERRGAER | 24.1 ± 0.01 | 70.69 | 84.98 | 14.29 | 24.89 | 6.09 | 0.59 | 0.995 |
M7 + P16 | LVSLKDIERRPAER | 24.2 ± 0.15 | 55.34 | 96.90 | 41.56 | 15.83 | 5.05 | 2.06 | 0.993 |
M7 + P17 | LVSLKDIERRQAER | 24.0 ± 0.01 | 56.18 | 82.69 | 26.51 | 24.61 | 6.46 | 1.03 | 0.999 |
M11.7 + P21 | AEQQRIRNEREKERQ | 23.6 ± 0.11 | 56.91 | 96.29 | 39.38 | 12.77 | 5.77 | 1.71 | 0.996 |
M11.7 + P22 | AQQQRIRNEREKERQ | 23.7 ± 0.10 | 87.08 | 97.12 | 10.04 | 25.18 | 7.70 | 0.33 | 0.970 |
M11.7 + P23 | AKQQRIRNEREKERQ | 23.3 ± 0.01 | 67.00 | 89.77 | 22.77 | 22.47 | 7.29 | 0.78 | 0.994 |
M11.7 + P24 | AEQRRIRNEREKERQ | 23.3 ± 0.06 | 90.25 | 93.57 | 3.32 | n.a. | n.a. | n.a. | n.a. |
M11.7 + P25 | AEQQCIRNEREKERQ | 23.2 ± 0.17 | 80.75 | 93.33 | 12.58 | 10.06 | 6.00 | 0.52 | 0.962 |
M11.7 + P26 | AEQQSIRNEREKERQ | 22.9 ± 0.00 | 80.48 | 86.48 | 6.00 | 26.67 | 6.49 | 0.25 | 0.996 |
M11.7 + P27 | AEQQHIRNEREKERQ | 23.1 ± 0.07 | 80.22 | 93.49 | 13.27 | 14.04 | 7.60 | 0.44 | 0.987 |
M11.7 + P28 | AEQQRIWNEREKERQ | 23.4 ± 0.01 | 86.91 | 93.90 | 6.99 | 13.80 | 6.90 | 0.25 | 0.955 |
M11.7 + P29 | AEQQRIQNEREKERQ | 23.2 ± 0.15 | 84.39 | 93.92 | 9.53 | 9.96 | 5.17 | 0.46 | 0.981 |
M11.7 + P30 | AEQQRIRNEWEKERQ | 23.5 ± 0.09 | 90.48 | 94.03 | 3.55 | n.a. | n.a. | n.a. | n.a. |
M11.7 + P31 | AEQQRIRNEQEKERQ | 23.2 ± 0.02 | 93.60 | 93.39 | −0.21 | n.a. | n.a. | n.a. | n.a. |
M11.7 + P32 | AEQQRIRNERKKERQ | 23.1 ± 0.06 | 69.09 | 96.03 | 26.94 | 17.63 | 7.32 | 0.92 | 0.992 |
Complex (a) | Peptide Sequence | (b) [1/s] | (b,c) [Ø] | (b) [kJ/mol] | (b) [kJ/mol] | (b) [kJ/mol] | Binding Type |
---|---|---|---|---|---|---|---|
M7 + P11 | LVSLKDIERRRAER | 3.69∙1012 | 0.59 | 1.28 | 40.25 | 38.96 | orthodox |
M7 + P12 | LVSLKDIKRRRAER | n.a. | n.a. | n.a. | n.a. | n.a. | non-binding |
M7 + P13 | LVSLKDIERCRAER | 1.72∙1012 | 0.28 | 3.17 | 55.16 | 51.99 | orthodox |
M7 + P14 | LVSLKDIERRWAER | 1.75∙1012 | 0.28 | 3.13 | 92.09 | 88.96 | orthodox |
M7 + P15 | LVSLKDIERRGAER | n.a. | n.a. | n.a. | n.a. | n.a. | unorthodox |
M7 + P16 | LVSLKDIERRPAER | 2.95∙1012 | 0.48 | 1.84 | 85.61 | 83.77 | orthodox |
M7 + P17 | LVSLKDIERRQAER | 3.82∙1012 | 0.62 | 1.14 | 38.06 | 36.92 | orthodox |
M11.7 + P21 | AEQQRIRNEREKERQ | 5.70∙1012 | 0.92 | 0.21 | 72.53 | 72.32 | orthodox |
M11.7 + P22 | AQQQRIRNEREKERQ | n.a. | n.a. | n.a. | n.a. | n.a. | unorthodox |
M11.7 + P23 | AKQQRIRNEREKERQ | 7.33∙1012 | 1.18 | −0.42 | 37.41 | 37.81 | orthodox |
M11.7 + P24 | AEQRRIRNEREKERQ | n.a. | n.a. | n.a. | n.a. | n.a. | non-binding |
M11.7 + P25 | AEQQCIRNEREKERQ | n.a. | n.a. | n.a. | n.a. | n.a. | unorthodox |
M11.7 + P26 | AEQQSIRNEREKERQ | n.a. | n.a. | n.a. | n.a. | n.a. | unorthodox |
M11.7 + P27 | AEQQHIRNEREKERQ | n.a. | n.a. | n.a. | n.a. | n.a. | unorthodox |
M11.7 + P28 | AEQQRIWNEREKERQ | n.a. | n.a. | n.a. | n.a. | n.a. | unorthodox |
M11.7 + P29 | AEQQRIQNEREKERQ | n.a. | n.a. | n.a. | n.a. | n.a. | unorthodox |
M11.7 + P30 | AEQQRIRNEWEKERQ | n.a. | n.a. | n.a. | n.a. | n.a. | non-binding |
M11.7 + P31 | AEQQRIRNEQEKERQ | n.a. | n.a. | n.a. | n.a. | n.a. | non-binding |
M11.7 + P32 | AEQQRIRNERKKERQ | 8.85∙1012 | 1.43 | −0.88 | 50.35 | 51.23 | orthodox |
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Kormann, K.; Ruß, M.; Röwer, C.; Koy, C.; Glocker, M.O. Mass Spectrometric ITEM-FOUR Analysis Reveals Coding Single-Nucleotide Polymorphisms in Human Cardiac Troponin T That Evade Detection by Sandwich ELISAs Which Use Monoclonal Antibodies M7 and M11.7 from the Elecsys Troponin T® Assay. Int. J. Mol. Sci. 2025, 26, 4892. https://doi.org/10.3390/ijms26104892
Kormann K, Ruß M, Röwer C, Koy C, Glocker MO. Mass Spectrometric ITEM-FOUR Analysis Reveals Coding Single-Nucleotide Polymorphisms in Human Cardiac Troponin T That Evade Detection by Sandwich ELISAs Which Use Monoclonal Antibodies M7 and M11.7 from the Elecsys Troponin T® Assay. International Journal of Molecular Sciences. 2025; 26(10):4892. https://doi.org/10.3390/ijms26104892
Chicago/Turabian StyleKormann, Kristjan, Manuela Ruß, Claudia Röwer, Cornelia Koy, and Michael O. Glocker. 2025. "Mass Spectrometric ITEM-FOUR Analysis Reveals Coding Single-Nucleotide Polymorphisms in Human Cardiac Troponin T That Evade Detection by Sandwich ELISAs Which Use Monoclonal Antibodies M7 and M11.7 from the Elecsys Troponin T® Assay" International Journal of Molecular Sciences 26, no. 10: 4892. https://doi.org/10.3390/ijms26104892
APA StyleKormann, K., Ruß, M., Röwer, C., Koy, C., & Glocker, M. O. (2025). Mass Spectrometric ITEM-FOUR Analysis Reveals Coding Single-Nucleotide Polymorphisms in Human Cardiac Troponin T That Evade Detection by Sandwich ELISAs Which Use Monoclonal Antibodies M7 and M11.7 from the Elecsys Troponin T® Assay. International Journal of Molecular Sciences, 26(10), 4892. https://doi.org/10.3390/ijms26104892