July 1, 2009 (Vol. 29, No. 13)

Meeting Served as a Launchpad for Novel Solutions to Expedite Biomarker Research

Describing the new RapidFire® 300 platform as an automated in vitro ADME solution for drug discovery research, BioTrove introduced the label-free, high-throughput, MS-based system for screening and analysis of in vitro ADME data at ASMS.

The instrument can process 3,000–4,000 MS samples in an eight-hour shift, with 6–8 second total processing time per sample, according to the company. BioTrove developed the RapidFire 300 instrument in response to customer demand for a system that could go beyond plate-based high-throughput screening and be used for applications such as cytochrome P450 inhibition and induction assays, metabolic stability and P-glycoprotein inhibition studies, and permeability and plasma protein binding assays. Compared to the RapidFire 200, the new instrument offers enhanced automation to enable sequence-based rather than plate-based sampling, allowing users to cherry-pick samples of interest for a particular assay.

At the meeting, BioTrove scientists presented a poster in which they compared triple quadrupole (QqQ)-MS to time-of-flight (TOF)-MS for measuring cytochrome P450 inhibition assays. The assays were monitored using the RapidFire interfaced to an Agilent 6220 Accurate-Mass TOF-MS and an Applied Biosystems/Sciex (Life Technologies and MDS Analytical) API4000 QqQ-MZS, and the two approaches yielded similar results. The authors concluded that RapidFire in conjunction with TOF-MS may facilitate a range of in vitro assays in which MS method development is a bottleneck.

Another poster presented by Anthony Paiva and colleagues from Bristol-Myers Squibb explored the feasibility of using RapidFire-MS (RF-MS) technology for compound-specific analysis of in vitro ADME samples generated from a Caco-2 assay using model compounds. A comparison of the RF-MS method and conventional LC-MS “showed similar sensitivity, linearity, robustness, and carry-over,” and the two methods yielded similar concentration and permeability results, they said. The researchers describe an 8x speed advantage of RF-MS over LC-MS.

Genedata recently launched Expressionist 5.2 with capabilities designed for MS biomarker discovery. The release focuses on the Refiner MS and Analyst modules that are able to process and analyze metabolomic and proteomic raw MS data. A poster presented by Jens Hoefkens and colleagues from Genedata and the University of California Davis demonstrated an automated workflow for rapid alignment and identification of lipid biomarkers obtained from chip-based direct infusion nanoelectrospray tandem MS. The system processed 70 infusion experiments in about 20 seconds. The results documented changes in lipid patterns associated with exposure to tobacco smoke in rats.

Cell Biosciences showcased the CB1000 automated capillary-based nano-immunoassay protein analysis system, which was launched in April. The instrument separates proteins based on isoelectric focusing (IEF) within 5-cm long, 100-micron diameter glass capillaries. Exposure of the capillaries to ultraviolet light then activates a coating on the interior surface that covalently binds the proteins to the capillary wall.

The immobilized proteins are probed with primary and labeled secondary antibodies to generate a chemiluminescent signal that can be used to quantify the amount of protein present along the length of the capillary. IEF separation can discriminate multiple phosphorylation states and detect shifts in phospho-isoform distribution. Cell Biosciences designed the system to facilitate direct characterization and quantification of signal transduction proteins in assays that require as few as 25 cells.

“Our customers are characterizing signaling pathways in samples that simply are too small for traditional protein analysis techniques,” stated Walter Ausserer, vp of marketing at Cell Biosciences. “The CB1000’s ability to generate detailed profiles of signaling proteins in primary cells, sorted rare cell populations, and tissue micro-biopsies is generating a lot of excitement.

Protein Discovery introduced a programmable molecular weight fractionation system called the GELFREE™ 8100 Fractionation System, which partitions protein mixtures into liquid-phase fractions based on molecular weight. The Gel Elution Liquid Fraction Entrapment Electrophoresis system uses SDS-PAGE and can be combined with IEF and reverse-phase HPLC techniques. The GELFREE 8100’s cartridges are composed of eight independent channels, each containing a 1-cm gel column surrounded by sample-loading and fraction-collection chambers. The instrument contains a 3.5 kDa ion-permeable trapping membrane that repels negatively charged proteins and serves a molecular weight cut-off function.

“All the previous methods of molecular weight-based fractionation on gels and columns have been low-capacity, low-recovery, too slow, or too limited to a narrow size range,” according to Jeremy Norris, Ph.D., vp of R&D. “The difference in the GELFREE 8100 System is that it gives high and reproducible recoveries of any fraction from 3.5 kDa to 150 kDA, and you can load each cartridge with up to 8 mg of sample, so you can study low-abundance proteins.”

GlaxoSmithKline (GSK) is using GE Healthcare’s Whatman FTA® technology on a broad scale in the development of its oral drug compounds. Christopher Evans, Ph.D., section manager, worldwide bioanalysis and systems management at GSK, cited multiple advantages for the collection and storage of blood samples on FTA DMPK Cards.

The workflow is straightforward and allows users simply to punch out a portion of the blood spot, extract the sample, and perform LC/MC to determine a drug’s concentration, he said. In the future, GSK hopes to extend this approach to biomarker detection.

With its acquisition of Whatman, GE Healthcare now markets the FTA DMPK Cards, which contain a chemically treated fiber matrix that provides long-term protection of the DNA and protein contained in the blood samples from degradation due to chemical and environmental factors. FTA DMPK Cards are available with collection sites for 1, 2, 3, or 4 samples and, depending on the card configuration, they can store up to 500 µL of whole blood. Collection, transport, and storage of blood samples on FTA DMPK Cards do not require refrigeration or freezing.

Eliminating the need for cold storage for blood sample collection opens the door to “different countries where you can perform studies,” noted Chet Bowen, principal scientist, worldwide bioanalysis and systems management at GSK. Bowen emphasized the benefits of evaluating anti-malarial and antiviral agents, for example, in the countries where the diseases are endemic.

The ability to sample small (15 µL) aliquots of blood from the subject allows for lower volume blood draws and serial sampling in a single test animal over time, a notable advantage in rodent studies, thereby reducing the number of animals required for safety-assessment studies, providing ethical and cost advantages, explained Dr. Evans.

Furthermore, “the ability to do an entire pharmacokinetic (PK) or toxicokinetic study in a single animal yields substantial improvement in data quality,” Dr. Evans said. GSK has used FTA technology to evaluate PK parameters in more than 70 small molecule compounds in preclinical development. In addition to evaluating drug and metabolite levels, dried blood samples could also be used for biomarker analysis for pharmacogenomic studies. Dr. Evans predicted that the industry will begin to move from the collection of plasma to dried blood samples on a broad basis in the near future.

The cHiPLC™ Nanoflex system from Eksigent is a docking station that can accommodate up to three microfluidic chips containing a nanoLC or trap column. It can be used in combination with any nanospray MC system. A fluidic jumper chip routes the fluids through the instrument, which features a dead volume free connection of less than 1 nL. Eksigent also showcased its NanoLC-Ultra™ HPLC system for proteomics research. Key features include a self-priming/self-purging design, flow rate resolution >1 nL/min, and a maximum operating pressure of 10,000 psi.

Scientists from Eksigent presented a poster at ASMS in which they described LC-MS analysis using the Eksigent NanoLC 20 Ultra Plus LC-MS system, the cHiPLC Nanoflex microfluidic platform, a Thermo Scientific (Thermo Fisher Scientific) LTQ MS system, and the PicoView® nanospray source (New Objective).

For these experiments, the cHiPLC Nanoflex contained two 15-cm column chips packed with the company’s ChromXP C18 3 µm 120 Å support material. The results demonstrated that the advantages of greater peak capacity achieved using serial columns—in terms of improved sequence coverage and more peptides identified—are greater with increasing complexity of the sample. The two-column setup identified about 2.5 times more peptides in the water-soluble fraction of an E. coli cell lysate digest.

AQUA™ technology from Sigma-Aldrich for MS quantification of protein biomarkers is based on the AQUA peptide, a synthetic tryptic peptide that corresponds to a peptide of interest and incorporates one stable isotope-labeled amino acid. The AQUA Peptide Library currently consists of more than 700 synthetic peptides, with custom peptide synthesis available.

Phytronix Technologies demonstrated its LDTD™ laser diode thermal desorption ionization source for MS of dried samples at the ASMS conference. The quantitative, solvent-free system offers an alternative to liquid chromatography and can reportedly process up to 960 samples with run times as low as 4.5 seconds per sample. The instrument utilizes atmospheric pressure chemical ionization, in positive or negative mode, to process samples for MS analysis.

CEM’s microwave-based instruments may be best known for their advantages in chemical and peptide synthesis and analytical sample preparation, but the low frequency energy generated by microwave irradiation can also facilitate proteomics sample prep and protein hydrolysis upstream of mass spectrometric analysis.

Using the company’s Discover manual peptide synthesis single-mode microwave platform, researchers can accelerate enzymatic and chemical digestion of protein samples, the company reported. The Discover can digest up to 20 samples (in gel or in solution), ranging in size from 30 µL to 1 mL, in 15 minutes, and perform complete deglycosylation in less than one hour.

The ProteoMiner™ protein enrichment technology from Bio-Rad Laboratories removes a large proportion of the high-abundance proteins in a complex mixture upstream of LC/MS analysis, facilitating detection of low-abundance protein biomarkers. This sample-prep tool compresses the dynamic range of protein concentrations without relying on immunodepletion methods.

“ProteoMiner differs from immunodepletion in that it does not use antibodies, and therefore, is not limited to specific sample types or species,” said Kate Smith, product manager for expression proteomics at Bio-Rad. “It has been used successfully with serum, plasma, urine, cerebrospinal fluid, bile, and other body fluids.” Users mix their sample with a diverse library of hexapeptides bound to chromatographic beads. The bead capacity limits the amount of protein that can bind to the support material.

High-abundance proteins will rapidly saturate their hexapeptide ligands and excess protein can then be washed away, while low-abundance proteins will remain bound to their ligands and concentrated on the chromatographic support. ProteoMiner kits are available in two capacities: large can accommodate 50 mg of protein, while the smaller capacity is suitable for 10 mg of protein.

Sricharan Bandhakavi, Matthew Stone, and Timothy Griffin presented a poster detailing research in which they incubated a clarified saliva sample supplemented with protease inhibitors with two hexapeptide libraries—one the standard ProteoMiner library and the second a carboxylated version of the library—and employed two different protein fractionation strategies, 2-D fractionation with LC-MS/MS analysis, or 3-D fractionation, which included an intermediate step of strong cation exchange fractionation between IEF and reverse-phase LC-MS/MS analysis. They performed MS analysis using the Thermo LTQ-Orbitrap.

In April, NextGen introduced a protein biomarker assay methodology that allows for quantitative measurement of protein expression in FFPE tissue samples. This technique employs the Liquid Tissue® sample-prep reagents and protocols developed by Expression Pathology, and assays the solubilized proteins using multiple reaction monitoring (MRM) MS.

NextGen scientists collaborated on several posters presented at the conference. One poster described the development of an LC-MRM/MS assay for three putative biomarkers of osteoarthritis in synovial fluid (dermicidin, afamin, and proteoglycan4), and use of the assay to analyze 200 clinical samples.

Another presented a study of the effects of PIF, an embryo-derived peptide that appears to have a role in initiating maternal immune-tolerance shortly after fertilization, on gene and protein expression in the spinal cord of a mouse model of multiple sclerosis. One objective was to identify new candidate protein biomarkers for monitoring and predicting disease progression and assessing the efficacy of drug treatment. The results demonstrated significant changes in hundreds of proteins following PIF administration, including reduced levels of inflammatory markers (cytokines) and apoptosis promoters, and enhanced expression of proteins involved in neural function.

The digital Proteome Chip™ (dPC) from Protein Forest fractionates complex protein samples such as cell lysates based on pH. The chip contains 41 polymerized gel plugs with a 0.05 pH resolution between adjacent plugs. Users reduce alkylate and desalt their samples before adding them to the dPC running buffer. Separation by parallel isoelectric focusing takes about 30 minutes. The separated samples can then be analyzed using in-gel tryptic digestion followed by mass spec identification.

Cerno Bioscience’s MassWorks™ sCLIPS™ (self Calibrated Line-shape Isotope Profile Search) provides a method for MS calibration to enhance formula identification. Even with MS techniques that can achieve 1 ppm mass accuracy, the user will be left with several dozen to hundreds of formula candidates to choose from, according to the company. sCLIPS works by calibrating the MS line-shape to a mathematically defined function and comparing calibrated and theoretical spectra to yield spectral accuracy values that can be used to identify an unknown ion formula.

Biocrates recently introduced its AbsoluteIDQ™ mass spec assay prep kit for metabolite identification and quantification in biomarker discovery research to the U.S. market. Optimized for use with the Applied Biosystems API 4000™ and 4000 Q TRAP®, the system processes 10-µL samples and covers more than 160 metabolites in four main classes.

The RapidFire® system simultaneously quantifies multiple analytes in complex mixtures presented by biological samples, according to BioTrove.

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