Randall W. Nelson

Space flight alters bacterial gene expression and virulence and reveals a role for global regulator Hfq

A comprehensive analysis of both the molecular genetic and phenotypic responses of any organism to the space flight environment has never been accomplished because of significant technological and logistical hurdles. Moreover, the effects of space flight on microbial pathogenicity and associated infectious disease risks have not been studied. The bacterial pathogen Salmonella typhimurium was grown aboard Space Shuttle mission STS-115 and compared with identical ground control cultures. Global microarray and proteomic analyses revealed that 167 transcripts and 73 proteins changed expression with the conserved RNA-binding protein Hfq identified as a likely global regulator involved in the response to this environment. Hfq involvement was confirmed with a ground-based microgravity culture model. Space flight samples exhibited enhanced virulence in a murine infection model and extracellular matrix accumulation consistent with a biofilm. Strategies to target Hfq and related regulators could potentially decrease infectious disease risks during space flight missions and provide novel therapeutic options on Earth.

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.

Biosensor chip mass spectrometry: a chip-based proteomics approach.

Rapid advances in genomic sequencing, bioinformatics, and analytical instrumentation have created the field of proteomics, which at present is based largely on two‐dimensional electrophoresis (2‐DE) separation of complex protein mixtures and identification of individual proteins using mass spectrometry. These analyses provide a wealth of data, which upon further evaluation leads to many questions regarding the structure and function of the proteins. The challenge of answering these questions create a need for high‐specificity approaches that may be used in the analysis of biomolecular recognition events and interacting partners, and thereby places great demands on general protein characterization instrumentation and the types of analyses they need to perform. Over the past five years we have been actively involved in interfacing two general, instrumental techniques, surface plasmon resonance‐biomolecular interaction analysis (SPR‐BIA) and matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry, into a single concerted approach for use in the functional and structural characterization of proteins. Reviewed here is the recent progress made using biomolecular interaction analysis ‐ mass spectrometry (BIA‐MS) in the detailed characterization of proteins and protein‐protein interactions and the development of biosensor chip mass spectrometry (BCMS) as a new chip‐based proteomics approach.

Detection of Endogenous B-Type Natriuretic Peptide at Very Low Concentrations in Patients with Heart Failure

Background—The myocardium secretes B-type natriuretic peptide (BNP) in response to stimuli associated with heart failure (HF). However, high immunoreactive-BNP levels in patients with HF are associated with a paradoxical lack of natriuretic response. We hypothesized that commercially available assays for immunoreactive BNP do not reflect the bioactivity of the natriuretic peptide system, because they measure both unprocessed inactive pro-BNP and mature BNP 1-32. We describe an assay for the detection of bioactive BNP 1-32 and confirm very low concentrations in plasma from HF patients. Methods and Results—We developed a quantitative mass spectrometry immunoassay to capture endogenous BNP peptides using high affinity antibodies. Bound BNP and its truncated fragments were detected by matrix assisted laser desorption ionization–time of flight mass spectrometry based on their predicted masses. Mass spectrometry immunoassay revealed rapid in vitro degradation of BNP 1-32 in plasma, which requires plasma collection in the presence of high protease inhibitor concentrations. In 11 of 12 HF patients BNP 1-32 was detectable, ranging from 25 to 43 pg/mL. Several degraded forms of BNP were also detected at similarly low levels. In contrast, parallel measurements of immunoreactive BNP using the Biosite assay ranged from 900 to 5000 pg/mL. Conclusions—Detection of endogenous BNP 1-32 requires special preservation of plasma samples. Mass spectrometry immunoassay technology demonstrates that HF patients have low levels of BNP 1-32. Commercially available immunoreactive-BNP assays overrepresent biological activity of the natriuretic peptide system because they cannot distinguish between active and inactive forms. This observation may, in part, explain the “natriuretic paradox.”

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.

Selected Reaction Monitoring–Mass Spectrometric Immunoassay Responsive to Parathyroid Hormone and Related Variants

BACKGROUND Parathyroid hormone (PTH) assays able to distinguish between full-length PTH (PTH1-84) and N-terminally truncated PTH (PTH7-84) are of increasing significance in the accurate diagnosis of endocrine and osteological diseases. We describe the discovery of new N-terminal and C-terminal PTH variants and the development of selected reaction monitoring (SRM)-based immunoassays specifically designed for the detection of full-length PTH [amino acid (aa)1-84] and 2 N-terminal variants, aa7-84 and aa34-84. METHODS Preparation of mass spectrometric immunoassay pipettor tips and MALDI-TOF mass spectrometric analysis were carried out as previously described. We used novel software to develop SRM assays on a triple-quadrupole mass spectrometer. Heavy isotope-labeled versions of target peptides were used as internal standards. RESULTS Top-down analysis of samples from healthy individuals and renal failure patients revealed numerous PTH variants, including previously unidentified aa28-84, aa48-84, aa34-77, aa37-77, and aa38-77. Quantitative SRM assays were developed for PTH1-84, PTH7-84, and variant aa34-84. Peptides exhibited linear responses (R(2) = 0.90-0.99) relative to recombinant human PTH concentration limits of detection for intact PTH of 8 ng/L and limits of quantification of 16-31 ng/L depending on the peptide. Standard error of analysis for all triplicate measurements was 3%-12% for all peptides, with <5% chromatographic drift between replicates. The CVs of integrated areas under the curve for 54 separate measurements of heavy peptides were 5%-9%. CONCLUSIONS Mass spectrometric immunoassays identified new clinical variants of PTH and provided a quantitative assay for these and previously identified forms of PTH.

Multitoxin biosensor-mass spectrometry analysis : a new approach for rapid, real-time, sensitive analysis of staphylococcal toxins in food

Biomolecular interaction analysis mass spectrometry (BIA-MS) was applied to detection of bacterial toxins in food samples. This two-step approach utilizes surface plasmon resonance (SPR) to detect the binding of the toxin(s) to antibodies immobilized on a surface of a sensor chip. SPR detection is then followed by identification of the bound toxin(s) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Staphylococcal enterotoxin B (SEB) was readily detected in milk and mushroom samples at levels of 1 ng/ml. In addition, non-specific binding of food components to the immobilized antibody and to the sensor chip surface was detected. To evaluate the applicability of BIA-MS in the analysis of materials containing multiple toxic components, sample containing both SEB and toxic-shock syndrome toxin-1 was analyzed. Both toxins were successfully and simultaneously detected through the utilization of multiaffinity sensor chip surfaces.

Surface plasmon resonance mass spectrometry: recent progress and outlooks

The combination of surface plasmon resonance (SPR) and mass spectrometry (MS) has created a unique approach to protein investigations. Surface plasmon resonance is used to quantify interactions between proteins and surface-immobilized ligands, and MS is used to determine the structural features of the bound proteins. Recent progress in SPR-MS includes improved methods and operations, increased limits of detection, multi-protein analysis and protein-complex delineation. With the subsequent design of SPR protein arrays, SPR-MS is expected to enter into the field of high-throughput protein interaction discovery and miniaturized diagnostics.

Rapid development of sensitive, high-throughput, quantitative and highly selective mass spectrometric targeted immunoassays for clinically important proteins in human plasma and serum

OBJECTIVES The aim of this study was to develop high-throughput, quantitative and highly selective mass spectrometric, targeted immunoassays for clinically important proteins in human plasma or serum. DESIGN AND METHODS The described method coupled mass spectrometric immunoassay (MSIA), a previously developed technique for immunoenrichment on a monolithic microcolumn activated with an anti-protein antibody and fixed in a pipette tip, to selected reaction monitoring (SRM) detection and accurate quantification of targeted peptides, including clinically relevant sequence or truncated variants. RESULTS In this report, we demonstrate the rapid development of MSIA-SRM assays for sixteen different target proteins spanning seven different clinically important areas (including neurological, Alzheimers, cardiovascular, endocrine function, cancer and other diseases) and ranging in concentration from pg/mL to mg/mL. The reported MSIA-SRM assays demonstrated high sensitivity (within published clinical ranges), precision, robustness and high-throughput as well as specific detection of clinically relevant isoforms for many of the target proteins. Most of the assays were tested with bona-fide clinical samples. In addition, positive correlations, (R2 0.67-0.87, depending on the target peptide), were demonstrated for MSIA-SRM assay data with clinical analyzer measurements of parathyroid hormone (PTH) and insulin growth factor 1 (IGF1) in clinical sample cohorts. CONCLUSIONS We have presented a practical and scalable method for rapid development and deployment of MS-based SRM assays for clinically relevant proteins and measured levels of the target analytes in bona fide clinical samples. The method permits the specific quantification of individual protein isoforms and addresses the difficult problem of protein heterogeneity in clinical proteomics applications.

Population Proteomics The Concept, Attributes, and Potential for Cancer Biomarker Research

This review outlines the concept of population proteomics and its implication in the discovery and validation of cancer-specific protein modulations. Population proteomics is an applied subdiscipline of proteomics engaging in the investigation of human proteins across and within populations to define and better understand protein diversity. Population proteomics focuses on interrogation of specific proteins from large number of individuals, utilizing top-down, targeted affinity mass spectrometry approaches to probe protein modifications. Deglycosylation, sequence truncations, side-chain residue modifications, and other modifications have been reported for myriad of proteins, yet little is know about their incidence rate in the general population. Such information can be gathered via population proteomics and would greatly aid the biomarker discovery efforts. Discovery of novel protein modifications is also expected from such large scale population proteomics, expanding the protein knowledge database. In regard to cancer protein biomarkers, their validation via population proteomics-based approaches is advantageous as mass spectrometry detection is used both in the discovery and validation process, which is essential for the detection of those structurally modified protein biomarkers.