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Biomedical subjects

K A Tubbs

Publications and source records attributed to K A Tubbs.

5 recordsLinked to original sources
PubMed2001

Determination of beta-2 microglobulin levels in plasma using a high-throughput mass spectrometric immunoassay system.

A high-throughput mass spectrometric immunoassay (MSIA) system for the analysis of proteins directly from biological fluids is reported. A 96-well-format robotic workstation equipped with antibody-derivatized affinity pipet tips was used for the parallel extraction of specific proteins from samples and subsequent deposition onto 96-well arrayed matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) targets. Interferences from nonspecifically bound proteins were minimized through choice of appropriate affinity pipet tip derivatization chemistries. Sample preparation for MALDI-TOFMS was enhanced through the use of hydrophobic/hydrophilic contrasting targets, which also presented functionalities found to promote matrix/analyte crystal growth. Automated mass spectrometry was used in the unattended acquisition of data, resulting in an analysis rate of approximately 100 samples/h (biological fluid-->data). The quantitative MSIA of beta2m levels present in human plasma samples is given as illustration.

Adult↗
PubMed2001

Detection and quantification of beta-2-microglobulin using mass spectrometric immunoassay.

The use of mass spectrometric immunoassay (MSIA) in analyzing beta-2-microglobulin (beta(2)m) present in human biological fluids (tears, saliva, plasma, and urine) is described. Pipettor tips containing porous affinity frits, derivatized with polyclonal anti-beta(2)m immunoglobulin, were manufactured and used to selectively isolate and concentrate beta(2)m from the biofluids, after which matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to detect beta(2)m unambiguously at its characteristic molecular mass. The affinity tips were found rapid to use, requiring approximately 15 min per analysis, and exhibited low nonspecific binding properties that yielded essentially interference-free analyses. The beta(2)m MSIA was made quantitative by inclusion of an internal standard into the analysis for signal normalization. The resulting assay had a Linear dynamic range (R(2) = 0.983) covering a beta(2)m concentration range of 0.010-1.0 mg/L with a standard error of approximately 5%. In application, urine samples from healthy individuals were screened and compared with sample from an individual suffering from renal infection. Results indicated an approximately 30-fold increase in beta(2)m levels in samples taken from the infected individual. During the screening, MSIA was able to distinguish between wild-type and glycosylated forms of beta(2)m, which made possible the accurate quantification of wild-type beta(2)m without interference from glycosylated versions of the protein. These results demonstrate a new approach to the rapid and accurate detection/quantification of beta(2)m present in biological fluids.

Adult↗
PubMed2000

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.

Biosensing Techniques↗
PubMed1999

BIA/MS of epitope-tagged peptides directly from E. coli lysate: multiplex detection and protein identification at low-femtomole to subfemtomole levels.

The use of biomolecular interaction analysis mass spectrometry to selectively isolate, detect, and characterize epitope-tagged peptides present in total cell lysates is demonstrated. Epitope-tagged tryptic peptides were captured via affinity interactions with either chelated Ni2+ or monoclonal antibodies and detected using surface plasmon resonance biomolecular interaction analysis (SPR-BIA). After SPR-BIA the tagged peptides were either eluted from the biosensor chips for mass spectrometric analysis or analyzed directly from the biosensor chip using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). Protein database searches were performed using the masses of the tagged tryptic peptides, resulting in identification of the protein into which the epitope tag was inserted. Detection limits for both SPR-BIA and MALDI-TOF were at the low-femtomole to subfemtomole level. The approach represents a (multiplexed) high-sensitivity chip-based technique capable of identifying epitope-tagged proteins as they are present in complex mixtures.

Amino Acid Sequence↗