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1.  Comparing Isotope Dilution Methods to Label Free Quantitation Methods For The Analysis of Vaccine Standards and Products 
Determining protein content in biologics is an important part of the production process. An example of interest to public health is the influenza vaccine, where the amount of the major antigenic protein hemagglutinin and the amount of egg proteins from the expression system are regulated. Mass spectrometry has advantages of higher specificity, speed and permits other proteins to be analyzed simultaneously. Here, we present the use of a MRM method for quantitation of hemagglutinin and other vaccine proteins developed in our laboratory and compare this approach to a label free method (MSE) for simultaneous identification and absolute quantitation of virus and egg proteins.
Influenza vaccine samples were tryptically digested using a protocol developed in our laboratory to ensure consistent and reproducible results. Traditional IDMS measurements were made on ThermoFisher Scientific TSQ quantum triple quadrupole platform. Label free methods (LC-MSE) were performed on a Waters qTOF Premier platform and the PLGS software. Both instruments were coupled to an Identical Agilent 1200 LC platform to insure an accurate comparison.
The results of the study illustrated that IDMS remains the gold standard for absolute protein quantitation via mass spectrometry. MSE performed with comparable precision and accuracy in cases where the sample was less complex (monovalent pandemic vaccines vs. seasonal trivalent vaccines). In addition the choice of peptides made by the MSE algorithm and the choice of influenza proteins used in the database also affected the precision and accuracy of the MSE absolute quantitation results.
PMCID: PMC3635396
2.  Extraction and inhibition of enzymatic activity of botulinum neurotoxins /B1, /B2, /B3, /B4, and /B5 by a panel of monoclonal anti-BoNT/B antibodies 
BMC Biochemistry  2011;12:58.
Botulism is caused by botulinum neurotoxins (BoNTs), extremely toxic proteins which can induce respiratory failure leading to long-term intensive care or death. Treatment for botulism includes administration of antitoxins, which must be administered early in the course of the intoxication; therefore, rapid determination of human exposure to BoNT is an important public health goal. In previous work, our laboratory reported on Endopep-MS, a mass spectrometry-based activity method for detecting and differentiating BoNT/A, /B, /E, and /F in clinical samples. We also demonstrated that antibody-capture is effective for purification and concentration of BoNTs from complex matrices such as clinical samples. However, some antibodies inhibit or neutralize the enzymatic activity of BoNT, so the choice of antibody for toxin extraction is critical.
In this work, we evaluated 24 anti-BoNT/B monoclonal antibodies (mAbs) for their ability to inhibit the in vitro activity of BoNT/B1, /B2, /B3, /B4, and /B5 and to extract those toxins. Among the mAbs, there were significant differences in ability to extract BoNT/B subtypes and inhibitory effect on BoNT catalytic activity. Some of the mAbs tested enhanced the in vitro light chain activity of BoNT/B, suggesting that BoNT/B may undergo conformational change upon binding some mAbs.
In addition to determining in vitro inhibition abilities of a panel of mAbs against BoNT/B1-/B5, this work has determined B12.2 and 2B18.2 to be the best mAbs for sample preparation before Endopep-MS. These mAb characterizations also have the potential to assist with mechanistic studies of BoNT/B protection and treatment, which is important for studying alternative therapeutics for botulism.
PMCID: PMC3250939  PMID: 22085466

Results 1-2 (2)