Novel Interface for High-Throughput Analysis of Biotherapeutics by Electrospray Mass Spectrometry.

With the rapid rise of therapeutic antibodies and antibody-drug conjugates, significant investments have been made in developing workflows that utilize mass spectrometry to detect these intact molecules, the large fragments generated by their selective digestion, and the peptides generated by traditional proteomics workflows. The resultant data is used to gain insight into a wide range of parameters, including primary sequence, disulfide bonding, glycosylation patterns, biotransformation, and more. However, many of the technologies utilized to couple these workflows to mass spectrometers have significant limitations that force nonoptimal modifications to upstream sample preparation steps, limit the throughput of high-volume workflows, and prevent the harmonization of diverse experiments onto a single hardware platform.

Usefulness of multilocus polymerase chain reaction followed by electrospray ionization mass spectrometry to identify a diverse panel of bacterial isolates.

Polymerase chain reaction electrospray ionization mass spectrometry (PCR/ESI-MS) was tested for its ability to accurately identify a blinded panel of 156 diverse bacterial isolates, mostly human and/or animal pathogens. Here, 142/156 (91%) isolates were correctly identified to the genus level and 115/156 (74%) were correctly identified to the species level. Only 9% were misidentified.

Selective ion filtering by digital thresholding: a method to unwind complex ESI-mass spectra and eliminate signals from low molecular weight chemical noise.

In this work, we present a simple method by which to preferentially detect either high molecular weight or low molecular weight ions generated by electrospray ionization. This approach, termed selective ion filtering by digital thresholding (SIFdT) is demonstrated on a commercial ESI-TOF instrument that employs a fast digitizer coupled to a microchannel plate detector. The digital representation of each individual scan is digitally filtered prior to spectral coaddition.

Base composition analysis of human mitochondrial DNA using electrospray ionization mass spectrometry: a novel tool for the identification and differentiation of humans.

In traditional approaches, mitochondrial DNA (mtDNA) variation is exploited for forensic identity testing by sequencing the two hypervariable regions of the human mtDNA control region. To reduce time and labor, single nucleotide polymorphism (SNP) assays are being sought to possibly replace sequencing. However, most SNP assays capture only a portion of the total variation within the desired regions, require a priori knowledge of the position of the SNP in the genome, and are generally not quantitative.

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.

Rapid identification of emerging pathogens: coronavirus.

We describe a new approach for infectious disease surveillance that facilitates rapid identification of known and emerging pathogens. The process uses broad-range polymerase chain reaction (PCR) to amplify nucleic acid targets from large groupings of organisms, electrospray ionization mass spectrometry for accurate mass measurements of PCR products, and base composition signature analysis to identify organisms in a sample. We demonstrate this principle by using 14 isolates of 9 diverse Coronavirus spp.

Mass spectrometry provides accurate characterization of two genetic marker types in Bacillus anthracis.

Epidemiological and forensic analyses of bioterrorism events involving Bacillus anthracis could be improved if both variable number tandem repeats (VNTRs) and single nucleotide polymorphisms (SNPs) could be combined on a single analysis platform. Here we present the use of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) to characterize 24 alleles from 6 VNTR loci and 11 alleles from 7 SNP loci in B. anthracis.

Alternative approaches to infrared multiphoton dissociation in an external ion reservoir.

In this work we present variations on in-hexapole infrared multiphoton dissociation (IRMPD) for the characterization of modified oligonucleotides using an ESI-FTICR spectrometer. We demonstrate that IRMPD in the external ion reservoir provides a comprehensive series of fragments allowing thorough characterization of a wide range of oligonucleotides containing alternative backbones and 2' substitutions. An alternative pulse sequence is presented that allows alternating MS and IRMPD MS/MS spectra to be acquired on a chromatographic timescale without loss in ionization duty cycle.

Infrared multiphoton dissociation with a hollow fiber waveguide.

A novel scheme for performing infrared multiphoton dissociation (IRMPD) is presented in which a hollow fiber waveguide (HFWG) is used to transmit IR radiation into the ion storage region of a mass spectrometer. Efficient dissociation of oligonucleotide and protein ions is demonstrated on an ESI-FTICR instrument in which IRMPD is performed in the external ion reservoir and on a quadrupole ion trap. Using a simple optical scheme consisting of a single focusing lens and an x, y translator, the 10.

Multitarget affinity/specificity screening of natural products: finding and characterizing high-affinity ligands from complex mixtures by using high-performance mass spectrometry.

In this work we describe a high-throughput screening approach based on electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR) that rapidly interrogates the noncovalent interaction between RNA-based drug targets and components derived from a bacterial natural product library. The screening process detects molecules present in the natural product library that bind to a synthetic RNA target that mimics the prokaryotic 16S rRNA A-site, while simultaneously measuring specificity for the synthetic A-site target using a control RNA target that lacks the critical structural element of the A-site construct. This screening approach known as multitarget affinity/specificity screening (MASS) demonstrated the expected binding of paromomycin from a fractionated natural product library derived from Streptomyces rimosus sp.

Human RNase H1 activity is regulated by a unique redox switch formed between adjacent cysteines.

Human RNase H1 is active only under reduced conditions. Oxidation as well as N-ethylmaleimide (NEM) treatment of human RNase H1 ablates the cleavage activity. The oxidized and NEM alkylated forms of human RNase H1 exhibited binding affinities for the heteroduplex substrate comparable with the reduced form of the enzyme.