Biomedical subjects

Allan L Bieber

Publications and source records attributed to Allan L Bieber.

4 recordsLinked to original sources

PubMed2006

Development of recombinant-based mass spectrometric immunoassay with application to resistin expression profiling.

This report addresses the need for additional assays for human resistin (hRES) by developing a rational progression of the mass spectrometric immunoassay to incorporate recombinant proteins. The recombinant-based hRES mass spectrometric immunoassay (RES-MSIA) was initially developed for the qualitative analysis of the human resistin homodimer from normal (healthy) plasma samples. The method involved selective extraction and detection of both endogenous and recombinant resistant proteins. RES-MSIA was then applied to the rigorous quantification of resistin. The resistin standard addition curve was constructed from serially diluted concentrations of rhRES using endogenous hRES, inherent in the human plasma, as the internal reference standard (IRS). The roles of endogenous and recombinant resistin were subsequently reversed, using rhRES as the IRS during RES-MSIA quantification. Concurrently, the relative ratio of hRES to rhRES was used as an ancillary technique to rapidly determine the relative concentration of hRES in each of plasma samples. Overall, normal hRES levels determined by RES-MSIA were found to be comparable to those selected and determined by ELISA. With regard to gender, female donor samples were slightly elevated over males. Four single cardiac samples were analyzed and found to have hRES concentrations approximately three times that of the normal. The recombinant-based RES-MSIA is rapid and is amendable to parallel high-throughput robotic processing of resistin related disease cohorts.

Gene Expression Profiling↗

PubMed2005

High-throughput MS-based protein phenotyping: application to haptoglobin.

A high-throughput affinity capture and reduction approach was developed for phenotype and post-translational modification analysis of a complexed globular protein, haptoglobin (Hp), directly from human plasma. Hp was selectively retrieved utilizing anti-Hp antibodies immobilized onto affinity pipette tips, eluted onto a formatted mass spectrometer target for reduction of Hp alpha-chains (Hpalpha1 and Hpalpha2) and subjected to subsequent MALDI-MS analysis. The affinity capture and reduction approach was originally developed from a pre-extraction reduction methodology that was optimized to an affinity capture post-reduction technique for intact Hp alpha-chain variant analysis, phenotype classification and ensuing post-translational variant detection. Three common Hp phenotypes (1-1, 2-1 and 2-2) were assigned according to detection of Hpalpha1 and/or Hpalpha2 reduced intact chain(s) average mass(es). The affinity capture post-reduction approach was scaled for high-throughput Hp alpha-chain phenotype analysis from a normal plasma cohort. The entire sample cohort was successfully analyzed and phenotyped using the developed approach. Additionally, Hp post-translational variants were detected and assigned via accurate MS analyses. The results of this study suggest use of the methodology in future analyses of other similarly complexed proteins and in normal versus disease cohort population proteomics studies.

Haptoglobins↗

PubMed2004

Metal ligand affinity pipettes and bioreactive alkaline phosphatase probes: tools for characterization of phosphorylated proteins and peptides.

An alkaline phosphatase-bioreactive probe, in which the enzyme is covalently bound to the mass spectrometry target, has been developed for studies of phosphoproteins. The bioreactive probe was used in combination with affinity capture and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to study hydrolysis of several phosphoproteins found in human saliva. Human salivary proteins were extracted from diluted human saliva with immobilized metal-affinity pipettes, which under defined conditions bound the phosphoproteins of interest preferentially over histatins. Phosphoproteins were eluted directly from the affinity pipettes to the bioreactive probe with diluted ammonium hydroxide, which provided conditions appropriate for hydrolysis by the alkaline phosphatase covalently bound to the probe surface. Results indicate the combination of metal-affinity pipette extraction, alkaline phosphatase-bioreactive probes, and matrix-assisted laser desorption/ionization mass spectrometry is an effective way to find and characterize phosphoproteins, known and unknown, in complex mixtures. Facile hydrolysis of human salivary phosphoproteins by the bioreactive probes was readily observed.

Alkaline Phosphatase↗

PubMed2002

Mass spectrometry of nicotinic acetylcholine receptors and associated proteins as models for complex transmembrane proteins.

Studies were conducted to optimize matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry (MALDI TOF MS) in analyzing the composition of nicotinic acetylcholine receptors (nAChR) from Torpedo californica electric tissue in their membrane-bound, detergent-solubilized, and affinity-purified states. Mass spectra obtained from nAChR-rich membrane fractions gave reasonably good representations of protein compositions indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of those same samples. Efficiency of extraction of nAChR from membranes was not markedly different for most detergents, but quality and signal size of mass spectra were clearly influenced by detergent composition and concentration, protein concentration, and MALDI matrix composition. The best spectra, allowing detection and accurate size determinations for samples containing as little as 10 fmol of pure nAChR, were obtained for samples solubilized in Triton X-100 and assayed by use of a sinapinic acid matrix. Although informative spectra could be obtained for nAChR affinity purified on alpha-cobratoxin (Naja naja siamensis) columns and extracted using sinapinic acid, superior spectra with much higher signal:noise were obtained if extraction media contained Triton X-100 or sodium dodecyl sulfate. nAChR subunit masses determined were similar regardless of the membrane-associated, detergent-solubilized, or affinity-purified state of the preparation. These studies illustrate how masses can be determined for nAChR subunits and for other protein components in Torpedo membrane preparations, such as RAPsyn and Na(+)-K(+)-ATPase alpha and beta subunits. They also provide an underpinning for streamlined analysis of the composition of complex transmembrane proteins using MALDI TOF MS.

Animals↗