Jim Walters (Presenter)
MilliporeSigma / Merck KGaA
Authorship: James J Walters (1), Mark Angeles(1), Pegah Jalili(1), Isil Dilek(2), Uma Sreenivasan(2), Kevin Ray (1)
1 – MilliporeSigma / Merck KGaA, Applied Research and Development, St. Louis MO 2 - MilliporeSigma / Merck KGaA, Cerilliant Corp., Round Rock TX
| Short Abstract With the emergence of mass spectrometry for the clinical measurement of proteins in biological matrices, the development of biological reference materials will continue to grow in importance. CRM's with values assigned by metrologically valid procedures will be critical to minimize and control experimental variations in all steps of the workflow including protein extraction, fractionation, enrichment, proteolysis and analysis. To this end, we have verified sequence fidelity and isotopic incorporation of an SIL full length Thyroglobulin for use as an internal standard in quantitative MS workflows. We have also developed an amino acid analysis (AAA) method traceable to NIST SRM 2389 and validated the method against NIST SRM 927. This AAA method may be used for accurate quantification of proteins to enable development of accuracy-based protein reference reference materials. |
Long Abstract
Introduction:
With the emergence of mass spectrometry for the clinical measurement of proteins in biological matrices, the development of biological reference materials will continue to grow in importance. Certified Reference Materials with values assigned by metrologically valid and traceable procedures will be critical to minimize and control experimental variations in all steps of the workflow including protein extraction, fractionation, enrichment, proteolysis and analysis. To this end, we have verified sequence fidelity and isotopic incorporation of an SIL full length Thyroglobulin for use as an internal standard in quantitative MS workflows. We have also developed an amino acid analysis (AAA) method traceable to NIST SRM 2389 and validated the method against NIST SRM 927. This AAA method may be used for accurate quantification of proteins to enable development of accuracy-based protein reference materials.
Methods
As a first step towards development of accuracy-based protein reference materials, we developed an amino acid analysis (AAA) procedure for absolute protein quantification. Primary reference standard SRM 2389a, Amino Acids in HCl, was used for method calibration. SRM 927, Bovine Serum Albumin 7% Solution as well as Thyroglobulin CRM 457 was used for method validation. SIL-TG was expressed in HEK293 cells using serum-free media enriched with 13C6, 15N4 Arg and 13C6, 15N2 Lys. The purity was determined by HPLC-UV-MS and SDS-PAGE. LC-MS/MS of the digested SIL-Tg protein was used to determine stable isotope incorporation and asses sequence coverage using multiple proteolytic enzymes.
Results
During validation of AAA for protein quantification we measured a mean NIST-BSA concentration of 67.20 ± 0.6 g/L, which agrees well with the NIST certified concentration of 67.38 ± 1.38 g/L. This AAA measurement procedure is thus accurate and metrologically traceable to higher order reference materials. CRM 457 is a primary reference material used to establish calibration of working reference materials employed in IVD assays for serum thyroglobulin. The consensus certified mass concentration of CRM 457 is 324 ± 18 ug/mL as established by the colorimetric Lowry method, using BSA as a calibrator. Using our refined AAA method, we observe ~ 70% recovery of CRM 457, which translates to a mean mass concentration in each vial of approximately 233 µg/mL. This value is consistent with AAA values of 236 ± 33 µg/mL stated in the CRM 457 report. SIL-Tg characterization by multi-enzyme digestion and peptide mapping showed ~95% sequence coverage and SIL incorporation of >98%.
Conclusion
This work demonstrates that amino acid analysis as a reference measurement procedure for accurate quantification of proteins should be considered as an approach to develop accuracy-based protein reference materials. We also characterized and quantified SIL-Tg which can be spiked at the initial stage of the analytical workflow to significantly minimize LC-MS bottom-up assay variations.
References & Acknowledgements:
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