Kristin A. Sannes-Lowery

Rapid identification and strain-typing of respiratory pathogens for epidemic surveillance

Epidemic respiratory infections are responsible for extensive morbidity and mortality within both military and civilian populations. We describe a high-throughput method to simultaneously identify and genotype species of bacteria from complex mixtures in respiratory samples. The process uses electrospray ionization mass spectrometry and base composition analysis of PCR amplification products from highly conserved genomic regions to identify and determine the relative quantity of pathogenic bacteria present in the sample. High-resolution genotyping of specific species is achieved by using additional primers targeted to highly variable regions of specific bacterial genomes. This method was used to examine samples taken from military recruits during respiratory disease outbreaks and for follow up surveillance at several military training facilities. Analysis of respiratory samples revealed high concentrations of pathogenic respiratory species, including Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pyogenes. When S. pyogenes was identified in samples from the epidemic site, the identical genotype was found in almost all recruits. This analysis method will provide information fundamental to understanding the polymicrobial nature of explosive epidemics of respiratory disease.

Applications of ESI-MS in drug discovery: interrogation of noncovalent complexes

For many years, analytical mass spectrometry has had numerous supporting roles in the drug development process, including the assessment of compound purity; quantitation of absorption, distribution, metabolism and excretion; and compound-specific pharmacokinetic analyses. More recently, mass spectrometry has emerged as an effective technique for identifying lead compounds on the basis of the characterization of noncovalent ligand–macromolecular target interactions. This approach offers several attractive properties for screening applications in drug discovery compared with other strategies, including the small quantities of target and ligands required, and the capacity to study ligands or targets without having to label them. Here, we review the application of electrospray ionization mass spectrometry to the interrogation of noncovalent complexes, highlighting examples from drug discovery efforts aimed at a range of target classes.

Characterization of multipole storage assisted dissociation: implications for electrospray ionization mass spectrometry characterization of biomolecules.

Ions accumulated in an rf-only multipole for extended periods of time prior to mass analysis can experience a significant degree of fragmentation and produce mass spectra which do not reflect the true nature of the intact analyte(s). This phenomenon, termed multipole storage assisted dissociation (MSAD), places constraints on the maximum number of ions which can be accumulated in the multipole storage device as a result of its finite space charge limit. This phenomenon can be exploited to produce dissociation spectra that are dominated by fragment ions providing important sequence/structure information. In this work we further explore MSAD and characterize parameters including accumulation time, source pressure, and the electrostatic configuration of the multipole storage device, which mediate the phenomenon. Operating parameters are identified that can either enhance or eliminate the phenomenon.

Sequence confirmation of modified oligonucleotides using IRMPD in the external ion reservoir of an electrospray ionization fourier transform ion cyclotron mass spectrometer

Modified oligonucleotides continue to play an important role as antisense compounds that inhibit the expression of genes associated with metabolic disorders, cancer, and infectious diseases. Because the majority of modifications render these molecules refractory to standard enzymatic sequencing techniques, alternative sequencing methods which are fast and reliable are needed. In this work we explore how sugar and backbone modifications affect fragmentation patterns observed from oligonucleotides which are fragmented by infrared multiple photon dissociation in the external reservoir of an electrospray ionization Fourier transform ion cyclotron mass spectrometer. The modifications influence which fragment types (i.e., an-B versus cn) dominate and the ease with which the oligonucleotides are fragmented. General observations for confirming the sequence of oligonucleotides are described.