Proteomic identification of Listeria monocytogenes surface-associated proteins.

This study aimed to identify proteins exposed on the surface of Listeria monocytogenes cells for diagnostic reagent development. Brief trypsin treatment of L. monocytogenes cells followed by peptide separation and identification by nano-LC and online-MS/MS was performed. In parallel, as a negative control, proteins secreted into the digest buffer as well as proteins from cell lysis were identified. One hundred and seventy-four proteins were identified in at least two of three trials in either the negative control or during cell digest. Nineteen surface, 21 extracellularly secreted, 132 cytoplasmic, and two phage proteins were identified. Immunofluorescence microscopy of L. monocytogenes cells revealed the surface localization of two potential candidates for L. monocytogenes isolation and detection: lipoprotein LMOf2365_0546 and PBPD1 (LMOf2365_2742). In this report, we present the first data set of surface-exposed L. monocytogenes proteins currently available. The data have been deposited to the ProteomeXchange Consortium with identifier PXD000035.

Strain identification of commercial influenza vaccines by mass spectrometry.

Current influenza vaccine manufacturing and testing timelines require that the constituent hemagglutinin (HA) and neuraminidase (NA) strains be selected each year approximately 10 months before the vaccine becomes available. The threat of a pandemic influenza outbreak requires that more rapid testing methods be found. We have developed a specialized on-filter sample preparation method that uses both trypsin and chymotrypsin to enzymatically digest peptide-N-glycosidase F (PNGase F)-deglycosylated proteins in vaccines. In tandem with replicate liquid chromatography-mass spectrometry (LC-MS) analyses, this approach yields sufficient protein sequencing data (>85% sequence coverage on average) for strain identification of HA and NA components. This has allowed the confirmation, and in some cases the correction, of the identity of the influenza strains in recent commercial vaccines as well as the correction of some ambiguous HA sequence annotations in available databases. This method also allows the identification of low-level contaminant egg proteins produced during the manufacturing process.

Simultaneous quantification of the viral antigens hemagglutinin and neuraminidase in influenza vaccines by LC-MSE.

Current methods for quality control of inactivated influenza vaccines prior to regulatory approval include determining the hemagglutinin (HA) content by single radial immunodiffusion (SRID), verifying neuraminidase (NA) enzymatic activity, and demonstrating that the levels of the contaminant protein ovalbumin are below a set threshold of 1 µg/dose. The SRID assays require the availability of strain-specific reference HA antigens and antibodies, the production of which is a potential rate-limiting step in vaccine development and release, particularly during a pandemic. Immune responses induced by neuraminidase also contribute to protection from infection; however, the amounts of NA antigen in influenza vaccines are currently not quantified or standardized. Here, we report a method for vaccine analysis that yields simultaneous quantification of HA and NA levels much more rapidly than conventional HA quantification techniques, while providing additional valuable information on the total protein content. Enzymatically digested vaccine proteins were analyzed by LC-MS(E), a mass spectrometric technology that allows absolute quantification of analytes, including the HA and NA antigens, other structural influenza proteins and chicken egg proteins associated with the manufacturing process. This method has potential application for increasing the accuracy of reference antigen standards and for validating label claims for HA content in formulated vaccines. It can also be used to monitor NA and chicken egg protein content in order to monitor manufacturing consistency. While this is a useful methodology with potential for broad application, we also discuss herein some of the inherent limitations of this approach and the care and caution that must be taken in its use as a tool for absolute protein quantification. The variations in HA, NA and chicken egg protein concentrations in the vaccines analyzed in this study are indicative of the challenges associated with the current manufacturing and quality control testing procedures.