Jennifer R. Krone

Mass spectrometric immunoassay.

A new, general method of immunoassay is demonstrated. The approach is based on the microscale immunoaffinity capture of target antigens followed by mass-specific identification and quantitation using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Immunoaffinity capture of antigens effectively overcomes signal suppression effects typically encountered during traditional matrix-assisted laser desorption/ionization analysis of complex biological mixtures while simultaneously concentrating the analyte into a small volume. Mass spectrometric detection of antigens is unambiguous, as antigen signals are observed at characteristic mass-to-charge values in the mass spectrum, offering a high level of immunity to artifacts due to nonbiospecific retention of mixture components. However, the most important aspect of such mass-specific detection is the ability to use a single assay to screen biological systems for the presence of multiple, mass-resolved antigens. Analyte quantitation is possible by using a single antibody to capture both the antigen and an antigen variant which has been chemically modified to have a different mass. With proper calibration, the relative signal intensities of the two species in the mass spectrum can be used to determine the antigen concentration. Sample incubation and processing methods were such that a typical analysis could be performed in less than 1 h while subnanomolar sensitivities were maintained. The technique has been used for the rapid, selective, and quantitative screening of human blood for the presence of myotoxin a, and Mojave toxin form the venoms of the prairie rattlesnakes, Crotalus viridis viridis, and and the Mojave rattlesnake, Crotalus scutulatus scutulatus.

Mass spectrometric immunoassay

A general technique linking micro-scale affinity capture with matrix-assisted laser desorption/ionization (MALDI) mass spectrometric detection has been developed for the rapid, sensitive and accurate determination of antigens present in biological fluids. Strategies for the qualitative and quantitative determination of single and/or multiple antigens are presented.

Interfacing biomolecular interaction analysis with mass spectrometry and the use of Bioreactive mass spectrometer probe tips in protein characterization

Publisher Summary Biomolecular interaction analysis (BIA) and matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF) are capable of the accurate characterization of biomolecules with extreme speed and sensitivity. Although the two analytical approaches operate on mutually exclusive detection principles (either surface plasmon resonance detection of a refractive index change or the physical determination the molecular mass of a gas-phase ion), they can share a common denominator—the use of affinity interactions in selecting the analyte. Interfacing of the two, thereby creates a unique approach for the investigation of the kinetic parameters of biomolecular interaction (using BIA), and the unambiguous confirmation of the presence of targeted affinity ligands by direct mass analysis. In more recent times, new mass spectrometric approaches for the rapid, sensitive, and accurate characterization of proteins are in development. This chapter discusses some of the findings on the interfacing of biomolecular interaction analysis with mass spectrometry, and the use of enzymatically active—or bioreactive—mass spectrometer probe tips in the characterization of analytes. The use of bioreactive mass spectrometer probe tips to serially digest myoglobin is discussed in the chapter. The object of the serial digestion is to simultaneously view the relative stability of molecule fragments of myoglobin (generated during an initial, limited digestion of the myoglobin under denaturing conditions using pepsin-active tips at low pH), by exposing the fragment set to extensive digestion (using trypsin tips) under renaturing conditions.

Mass Spectrometric Methods for Biomolecular Characterization

Mass spectrometry has long been viewed to possess the ability to readily contribute to the rapid, sensitive and accurate characterization of biomolecules. However, it has been only within the last decade, with the advent of electrospray ionization (ESI) [1] and matrix-assisted laser desorption/ionization (MALDI) [2] mass spectrometries, that routine biomolecular analyses at physiological levels have become a reality. Indeed, these techniques have opened the door to the regular practice of biological mass spectrometry (at the bench level), and in doing so, have paved the way for a number of mass spectrometric-based methods directed at the intricate investigation of biomolecular structure and function. Generally, these methods revolve around the combination of other analytical techniques or methodologies (e.g. chromatography, electrophoresis, enzymatic/chemical treatment of analyte) with mass spectrometry. In order to achieve the highest levels of analytical performance, the integration of techniques must be performed in manners which minimize sample losses, maintain a desired rapidity, and preserve a high level of accuracy.

Rapid protein characterization using bioreactive mass spectrometer probe tips

Methods have been developed for the rapid and sensitive mass spectrometric characterization of peptides. The approach uses bioreactive mass spectrometer probe tips which are capable of modifying proteins for analytical purpose. In the demonstrated case, enzymatic proteolysis is initiated upon application of analyte to the probe tips, time allowed for digestion, and the results analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The probe tips have been used for proteolytic mapping and partial sequence determination of picomol quantities of peptide while maintaining analysis times of approximately 5 minutes.