Tracie L. Williams

Quantification of influenza virus hemagglutinins in complex mixtures using isotope dilution tandem mass spectrometry

Influenza vaccination is the primary method for preventing influenza and its severe complications. Licensed inactivated vaccines for seasonal or pandemic influenza are formulated to contain a preset amount of hemagglutinin (HA), the critical antigen to elicit protection. Current methods to establish the HA concentration of vaccines rely on indirect measurements that are subject to considerable experimental variability. We present a liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for the absolute quantification of viral proteins in a complex mixture. Through use of an isotope dilution approach, HA from viral subtypes H1, H3, H5, and B was determined both directly and rapidly. This method can be applied to purified virus preparations, to monovalent bulk concentrates, or to trivalent inactivated influenza vaccines with improved speed, sensitivity, precision, and accuracy. This LC/MS/MS approach may substantially increase the reliability of methods used to quantitate the amount of antigen in seasonal and pandemic influenza vaccines and reduce the time and effort to deliver influenza vaccines for public health use during the next influenza pandemic.

Optimization of digestion parameters for protein quantification

We present a rapid and efficient in-solution enzymatic digestion protocol suitable for mass spectrometry-based absolute protein quantification techniques. The digestion method employs RapiGest SF (an acid-labile surfactant), an excess amount of modified trypsin (enzyme-to-substrate ratio of 2.5:1), and an incubation time of 2 h. No reduction/alkylation reagents are used. Digestion parameters were varied systematically to monitor their effect on rate and completeness of digestion. To demonstrate the general applicability of the method, the optimization was done using a viral hemagglutinin (HA) as a model protein and then applied to ricin, a potent protein toxin extracted from the castor bean (Ricinus communis). The parameters that were optimized included incubation time, concentration of RapiGest SF, enzyme-to-substrate ratio, and incubation temperature. The optimization was done by comparing the yields from two protein-specific peptides originating from two different sites of the HA protein. The analysis was performed by liquid chromatography-tandem mass spectrometry in multiple reaction monitoring mode using isotopically labeled peptide standards for quantification.

Simultaneous quantification of hemagglutinin and neuraminidase of influenza virus using isotope dilution mass spectrometry

Influenza vaccination is the primary method for preventing influenza and its severe complications. Licensed inactivated vaccines for seasonal or pandemic influenza are formulated to contain a preset amount of hemagglutinin (HA), the critical antigen to elicit protection. There is currently no regulatory method that quantifies neuraminidase (NA), the other major membrane-bound protein thought to have protective capability. This is primarily due to the limitations both in sensitivity and in selectivity of current means to quantify these antigens. Current methods to establish the HA concentration of vaccines rely on indirect measurements that are subject to considerable experimental variability. We present a liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for the absolute quantification of viral proteins in a complex mixture. Through use of an isotope dilution approach, HA and NA from viral subtypes H1N1, H3N2, and B were determined both directly and rapidly. Three peptides of each subtype were used in the analysis of HA to ensure complete digestion of the protein and accuracy of the measurement. This method has been applied to purified virus preparations, to monovalent bulk concentrates, to trivalent inactivated influenza vaccines, and even crude allantoic fluid with improved speed, sensitivity, precision, and accuracy. Detection of 1 μg/mL of protein is easily obtained using this method. The sensitivity of the method covers the range expected in vaccine preparations, including adjuvant-based vaccine. This LC/MS/MS approach substantially increases the selectivity, accuracy and precision used to quantify the amount of viral proteins in seasonal and pandemic influenza vaccines and reduce the time and effort to deliver influenza vaccines for public health use during the next influenza pandemic.

Ultra performance liquid chromatography isotope dilution tandem mass spectrometry for the absolute quantification of proteins and peptides.

A selective, rapid, and sensitive 12.7-min ultra performance liquid chromatography-isotope dilution tandem mass spectrometry (UPLC-ID/MS/MS) method was developed and compared to conventional high-performance liquid chromatography-isotope dilution tandem mass spectrometry (HPLC-ID/MS/MS) for the absolute quantitative determination of multiple proteins from complex matrixes. The UPLC analysis was carried out on an Acquity UPLC ethylene-bridged hybrid (BEH) C18 reversed-phase column (50 x 2.1 mm i.d., 1.7-microm particle size) with gradient elution at a flow rate of 300 microL/min. For the HPLC separation, a similar gradient profile on a reversed-phase C18 column with dimensions of 150 x 1.0 mm at a flow rate of 30 microL/min was utilized. The aqueous and organic mobile phases were 0.1% formic acid in water and acetonitrile, respectively. Detection was performed on a triple-quadrupole mass spectrometer operated in the multiple reaction monitoring mode. Linear calibration curves were obtained in the concentration range of 10-90 fmol/microL. Relative standard deviation values equal to or less than 6.5% were obtained by the UPLC-ID/MS/MS method, thus demonstrating performance equivalent to conventional HPLC-ID/MS/MS for isotope dilution quantification of peptides and proteins. UPLC provides additional dimensions of rapid analysis time and high-sample throughput, which expands laboratory emergency response capabilities over conventional HPLC.

Targeted N-Linked Glycosylation Analysis of H5N1 Influenza Hemagglutinin by Selective Sample Preparation and Liquid Chromatography/Tandem Mass Spectrometry

Using liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis of deglycosylated and intact glycopeptides from tryptic digests of whole influenza virus, we determined that the six predicted N-linked glycosylation sites within the N-terminal ectodomain of hemagglutinin (HA) from three selected H5N1 strains are occupied. The use of selective sample preparation strategies, including solid-phase extraction (SPE) of glycopeptides via hydrazide capture chemistry as well as hydrophilic interaction liquid chromatography (HILIC), sufficiently reduced sample complexity to allow determination of occupied glycosylation sites. The specific amino acid sequence of the tryptic glycopeptides for the identified sites varied slightly among strains, but the overall locations of the occupied glycosylation sites were conserved in the protein sequence. We used this knowledge of glycosylation site occupation to examine the glycans attached to these occupied sites on HA for a reassortant H5N1 strain grown in embryonated chicken eggs. By applying mass spectrometry-based methodologies for examining glycosylation to the study of influenza virus proteins, we can better understand the effect that this post-translational modification has upon the virulence and antigenicity of emerging strains.

Quantification of immunoreactive viral influenza proteins by immunoaffinity capture and isotope-dilution liquid chromatography-tandem mass spectrometry.

An immunocapture isotope dilution mass spectrometry (IC-IDMS) method was developed to quantify antibody-bound influenza hemagglutinins (HA) in trivalent influenza vaccines (TIV). Currently, regulatory potency requirements for TIV require HA quantification based on the single radial immunodiffusion (SRID) assay, which is time-consuming, laborious, and requires production of large quantities of reagents globally. In IC-IDMS, antiserum to the HA of interest captured viral proteins that were in the correct conformation to be recognized by the antibodies. The captured proteins were digested, and evolutionarily conserved tryptic peptides were quantified using isotope-dilution liquid chromatography-tandem mass spectrometry. IC-IDMS relies on antibody-antigen binding similar to SRID but incorporates the accuracy and precision of IDMS. Polyclonal antibodies (pAb-H3) prepared by injection of sheep with purified H3 HA captured 82.9% (55.26 fmol/μL) of the total H3 HA (66.69 fmol/μL) from the commercial TIV and 93.6% (57.23 fmol/μL) of the total H3 HA (61.14 fmol/μL) in purified virus. While other HA (H1, B), neuraminidase (N1, N2, NB), viral matrix proteins, and nucleoproteins were also captured by this antiserum, our results were not affected due to the specificity of the mass spectrometer. IC-IDMS is an accurate, precise, sensitive, and selective method to measure antibody-bound HA in purified virus and commercial vaccines.

Amino Acid Analysis of Peptides Using Isobaric-Tagged Isotope Dilution LC−MS/MS

Protein quantification using stable isotope dilution mass spectrometry requires the quantification of specific peptides unique to the protein of interest. Since these peptides are used as calibration standards, accurate and precise measurement of these target peptides is critical. This peptide measurement has typically been made by amino acid analysis (AAA) using absorbance or fluorescence detection methods. This approach can be limited to only a few amino acids, is often not traceable to high-quality reference standards, and not uncommonly has coefficients of variation (CVs) that exceed 10%. We report here an isobaric-tagged isotope dilution mass spectrometry method for AAA that provides excellent sensitivity, specificity, and precision; utilizes a broad range of amino acids; and uses U.S. National Institute of Standards and Technology (NIST) amino acid standards for an accuracy base. The average CV for the method applied to three different peptides with measurements on 7 different days was 3.57% (range 2.72-4.20%). We applied this method to the quantification of three NIST standard peptides and hemagglutinin, an influenza virus surface protein.

Development of influenza A(H7N9) candidate vaccine viruses with improved hemagglutinin antigen yield in eggs.

The emergence of avian influenza A(H7N9) virus in poultry causing zoonotic human infections was reported on March 31, 2013. Development of A(H7N9) candidate vaccine viruses (CVV) for pandemic preparedness purposes was initiated without delay. Candidate vaccine viruses were derived by reverse genetics using the internal genes of A/Puerto/Rico/8/34 (PR8). The resulting A(H7N9) CVVs needed improvement because they had titers and antigen yields that were suboptimal for vaccine manufacturing in eggs, especially in a pandemic situation.

Quantification of Viral Proteins of the Avian H7 Subtype of Influenza Virus—An Isotope Dilution Mass Spectrometry Method Applicable for Producing more Rapid Vaccines in the Case of an Influenza Pandemic

Vaccination is the most effective means to prevent influenza and its serious complications. Influenza viral strains undergo rapid mutations of the surface proteins hemagglutinin (HA) and neuraminidase (NA) requiring vaccines to be frequently updated to include current circulating strains. It is nearly impossible to predict which strains will be circulating in the next influenza season. It is, therefore, imperative that the process of producing a vaccine be streamlined and as swift as possible. We have developed an isotope dilution mass spectrometry (IDMS) method to quantify HA and NA in H7N7, H7N2, and H7N9 influenza. The IDMS method involves enzymatic digestion of viral proteins and the specific detection of evolutionarily conserved target peptides. The four target peptides that were initially chosen for analysis of the HA protein of H7N2 and H7N7 subtypes were conserved and available for analysis of the H7N9 subtype that circulated in China in the spring of 2013. Thus, rapid response to the potential pandemic was realized. Quantification of a protein is performed by employing multiple peptides to ensure that the enzymatic digestion of the protein is efficient in the region of the target peptides, verify the accuracy of the measurement, and provide flexibility in the case of amino acid changes among newly emerging strains. The IDMS method is an accurate, sensitive, and selective method to quantify the amount of HA and NA antigens in primary liquid standards, crude allantoic fluid, purified virus samples, and final vaccine presentations.

Identification of Influenza A/PR/8/34 Donor Viruses Imparting High Hemagglutinin Yields to Candidate Vaccine Viruses in Eggs

One of the important lessons learned from the 2009 H1N1 pandemic is that a high yield influenza vaccine virus is essential for efficient and timely production of pandemic vaccines in eggs. The current seasonal and pre-pandemic vaccine viruses are generated either by classical reassortment or reverse genetics. Both approaches utilize a high growth virus, generally A/Puerto Rico/8/1934 (PR8), as the donor of all or most of the internal genes, and the wild type virus recommended for inclusion in the vaccine to contribute the hemagglutinin (HA) and neuraminidase (NA) genes encoding the surface glycoproteins. As a result of extensive adaptation through sequential egg passaging, PR8 viruses with different gene sequences and high growth properties have been selected at different laboratories in past decades. The effect of these related but distinct internal PR8 genes on the growth of vaccine viruses in eggs has not been examined previously. Here, we use reverse genetics to analyze systematically the growth and HA antigen yield of reassortant viruses with 3 different PR8 backbones. A panel of 9 different HA/NA gene pairs in combination with each of the 3 different lineages of PR8 internal genes (27 reassortant viruses) was generated to evaluate their performance. Virus and HA yield assays showed that the PR8 internal genes influence HA yields in most subtypes. Although no single PR8 internal gene set outperformed the others in all candidate vaccine viruses, a combination of specific PR8 backbone with individual HA/NA pairs demonstrated improved HA yield and consequently the speed of vaccine production. These findings may be important both for production of seasonal vaccines and for a rapid global vaccine response during a pandemic.