MSACL 2023 Abstract

Quantitative Amino Acid Analysis by Isotope Dilution LC-MS/MS for Concentration Assignment of Protein Reference Materials

James Blasberg(1), Kevin Ray(1), Pei Liu(1), Uma Sreenivasan(2)
(1) MilliporeSigma, Analytical Research and Development, St. Louis, MO (2) MilliporeSigma, Reference Materials R&D, Round Rock, TX

Pei Liu, PhD (Presenter) MilliporeSigma

Relevant Financial Disclosures (within past 24 months, reported on Feb 17, 2023) Salary MilliporeSigma Conflict mitigation NOT REQUIRED.

Introduction:
Accurate protein concentration measurement is crucial for the development of protein certified reference materials (CRMs), which will be used in the calibration of a variety of procedures used in nutritional and clinical analyses. Accurate concentration assignment of a chemical analyte is typically based on gravimetry and mass purity. However, this is generally not feasible for proteins, as often there is not enough solid protein standard available. , Amino acid analysis (AAA) has become a crucial technique to assign certified, SI traceable concentration values to protein solutions, with ultraviolet or fluorescent based detection being widely used. However, these methods require pre-column or post-column derivatization steps which are inherently more prone to measurement error. Isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) is considered as the “gold standard” for biomolecule quantification as it can provide high accuracy and precision measurements. Here we present the development of a method for quantitative determination of protein solutions using ID-LC-MS/MS.

Methods:
Primary reference standard NIST SRM 2389a, Amino Acids in HCl, was used for calibration. NIST SRM 927e, Bovine Serum Albumin 7% Solution, was used for method optimization and validation. Proteins were hydrolyzed with vapor-phase HCl for 20-24 hours at 110°C in sealed vials. An internal standard Certified Reference Material mix of stable isotope labeled amino acids (Supelco, 96378) was added to calibrators and hydrolyzed proteins. LC-MS/MS was performed using a SeQuant ZIC-cHILIC column (3µm,100Å 150 x 2.1 mm) with a zwitterionic stationary phase on an Agilent 1290 Flex UPLC connected to an AB Sciex 4000 QTRAP. Data were acquired for select amino acids in MRM mode with segmenting to maximize dwell times for better quantitative measurements.

Results:
A linear regression model was applied to all calibration curves and the associated coefficient of determination (R2) were >0.99 for all amino acid transitions, demonstrating a low variability and good accuracy of analyte concentration. The coefficient of variation (CV) for the injection reproducibility of the LC-MS/MS measurements across all amino acids ranged from 0.92% (threonine) to 2.1% (serine), with an average CV of 1.5%, signifying a high level of measurement precision. Using this ID-LC-MS/MS method, we observed recovery of NIST BSA SRM927e ranged from 96.9% to 101.7%, with an average recovery of 99.9%.

Conclusion:
This work demonstrates that amino acid analysis by isotope dilution LC-MS/MS can provide protein quantification with excellent accuracy and precision. Measurement accuracy and traceability was established through the use of NIST SRMs for calibration and control. This method will provide an improved approach for assignment of concentration values of accuracy-based protein reference materials used for calibration of LC-MS methods, such as those used in clinical diagnostics.