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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.

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.

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) [C.sub.18] reversed-phase column (50 x 2.1 mm i.d., 1.7-[micro]m particle size) with gradient elution at a flow rate of 300 [micro]L/min. For the HPLC separation, a similar gradient profile on a reversed-phase [C.sub.l8] column with dimensions of 150 x 1.0 mm at a flow rate of 30 [micro]L/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/[micro]L. 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.

The lethai toxin produced during Bacillus anthracis infection is a complex of protective antigen, which localizes the toxin to the cell receptor, and lethal factor (LF), a zinc-dependent endoproteinase whose known targets include five members of the mitogen-activated protein kinase kinase (MAPKK) family of response regulators. We have developed a method for detecting functional LF in serum. Anti-LF murine monoclonal antibodies immobilized on magnetic protein G beads were used to capture and concentrate the LF from serum. The captured LF was exposed to ah optimized MAPKK-based peptide substrate, which it hydrolyzed into two smaller peptides. The LF cleavage products were then analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) and quantified by isotope dilution-MS. The entire analytical method can be performed in less than 4 h with detection of LF levels as low as 0.05 ng/mL. The method was used to quantify LF levels in serum from rhesus macaques infected with B. anthracis. Serum samples obtained at day 2 postinfection contained 30250 ng/mL LF and illustrated the clear potential to detect LF earlier in the infection cycle. This method represents a highly specific and rapid diagnostic tool for early anthrax and has a potential additional role as a research tool for understanding toxemia and effects of medical countermeasures for anthrax.

Botulinum neurotoxin (BoNT) causes the disease botulism, which can be lethal if untreated. Rapid determination of exposure to BoNT is an important public health goal. Previous work in our laboratory focused on the development of Endopep-MS, a mass spectrometry-based endopeptidase method for detecting and differentiating BoNT in buffer. This method can rapidly determine the presence of BoNT in a sample and differentiate the toxin type of BoNT present but does not yield additional information about the subtype. We now describe here the application of Endopep-MS to detect BoNT A in a spiked milk sample. This work also describes subtype identification achieved through mass spectrometric analysis of the protein toxin itself and does not require the presence of DNA from the toxin-producing bacteria. Tryptic digests of A1 and A2 subtypes of BoNT were analyzed by mass spectrometry, and peptides unique to either the A1 or A2 subtype were subjected to tandem mass spectrometry analysis to confirm their identities. Finally, subtype identification through mass spectrometric analysis was performed on BoNT A isolated from spiked milk. In its entirety, this method would allow for analysis of BoNT with toxin type identification in a few hours and subtype identification within 24 h.

The composite multivariate quality control (CMQC) system was capable of isolating unacceptable errors in one or more variables without rejecting entire runs that contain such errors. Each of the components of the CMQC system readily detected errors and alterations which were normally seen with multivariate analytical measurements systems. Also, the presence of univariate, bivariate and multivariate quality control rules enhanced the detection of systmatic errors. In addition, the multivariate quality control limit could only be approximated through simulated runs.

Biological monitoring is an important method of assessing human exposure to toxic chemicals and biological toxins. To provide a means of assessing exposure, we have developed methods to measure protein toxins in human clinical samples. These methods include the Endopep-MS method, which can detect and differentiate botulinum neurotoxins (BoNTs) in food and clinical samples. The Endopep-MS method has three levels of selectivity that include extracting the toxin with high-affinity antibodies that are selective for only one of the BoNT serotypes; cleavage of synthetic peptides according to the specific enzymatic activity for each of the BoNT serotypes; and selectively detecting the toxin-dependent peptides by using high resolution matrix-assisted laser-desorption ionization time-of-flight mass spectrometry. The Endopep-MS method is specific for detecting each of the BoNT serotypes. Since botulism in humans is generally caused by BoNT A, B, E, or F, these serotypes are the focus of this report. The method is very sensitive, detecting less than 0.5 mouse LD50 of BoNT A in serum and 0.1 mouse LD50 of BoNT B, E, and F in human serum samples. The method has been applied to selected food matrices and serum and stool samples from individuals diagnosed with botulism. [Copyright &y& Elsevier]