Biomedical subjects

Luke V Schneider

Publications and source records attributed to Luke V Schneider.

5 recordsLinked to original sources

PubMed2005

Stable isotope methods for high-precision proteomics.

Stable isotope tagging methods provide a useful means of determining the relative expression level of individual proteins between samples in a mass spectrometer with high precision (coefficients of variation less than 10%). Because two or more samples tagged with different numbers of stable isotopes can be mixed before any processing steps, sample-to-sample recovery differences are eliminated. Mass spectrometry also allows post-translational modifications, splice variations and mutations (often unnoticed in immunoassays) to be detected and identified, increasing the clinical relevance of the assay and avoiding the issues of non-specific binding and cross-reactivity observed in immunoassays. Several stable isotope tagging methods are available for use in proteomics research. We discuss the advantages and disadvantages of each technique with respect to biomarker discovery, target validation, efficacy and toxicology screening and clinical diagnostic applications.

Isotope Labeling↗

PubMed2005

Rapid separation of protein isoforms by capillary zone electrophoresis with new dynamic coatings.

Many cellular functions are regulated through protein isoforms. Changes in the expression level or regulatory dysfunctions of isoforms often lead to developmental or pathological disorders. Isoforms are traditionally analyzed using techniques such as gel- or capillary-based isoelectric focusing. However, with proper electro-osmotic flow (EOF) control, isoforms with small pI differences can also be analyzed using capillary zone electrophoresis (CZE). Here we demonstrate the ability to quickly resolve isoforms of three model proteins (bovine serum albumin, transferrin, alpha1-antitrypsin) in capillaries coated with novel dynamic coatings. The coatings allow reproducible EOF modulation in the cathodal direction to a level of 10(-9) m2V(-1)s(-1). They also appear to inhibit protein adsorption to the capillary wall, making the isoform separations highly reproducible both in peak areas and apparent mobility. Isoforms of transferrin and alpha1-antitrypsin have been implicated in several human diseases. By coupling the CZE isoform separation with standard affinity capture assays, it may be possible to develop a cost-effective analytical platform for clinical diagnostics.

Animals↗

PubMed2005

Improved CZE capabilities with new dynamic coatings.

Electroosmotic flow (EOF) is one of the fundamental processes affecting both resolution and separation times in capillary zone electrophoresis (CZE). EOF is a function of pH and buffer composition (zeta potential) in bare-silica capillaries at any pH above 3 and runs in the cathodal direction, decreasing the effective separation length for cationic species. On the other hand, the absence of EOF at low pH can significantly increase separation times, particularly for low pl zwitterionic species. This interdependence of pH and EOF limits the ability to design effective separations by capillary zone electrophoresis. The EOTrol family of dynamic coatings (Target Discovery, Inc., Palo Alto, CA, U.S.A.) allows both the magnitude and direction of EOF to be adjusted independently of the buffer composition and pH. Here we report the use of EOTrol in two challenging CZE separations: (1) inorganic cations with small mobility differences, and (2) the rapid separation of organic zwitterions that are only resolvable at low pH.

Cations↗

PubMed2004

Isotope-differentiated binding energy shift tags (IDBEST) for improved targeted biomarker discovery and validation.

Mass spectrometry has proved to be an important tool for protein biomarker discovery, identification and characterization. However, global proteomic profiling strategies often fail to identify known low-abundance biomarkers as a result of the limited dynamic range of mass spectrometry (two to three orders of magnitude) compared with the large dynamic range of protein concentrations in biologic fluids (11 to 12 orders of magnitude for serum). In addition, the number of peptides generated in such methods vastly overwhelms the resolution capacity of mass spectrometers, requiring extensive sample clean-up (e.g., affinity tag, retentate chromatography and/or high-performance liquid chromatography) before mass spectrometry analysis. Baiting and affinity pre-enrichment strategies, which overcome the dynamic range and sample complexity issues of global proteomic strategies, are very difficult to couple to mass spectrometry. This is due to the fact that it is nearly impossible to sort target peptides from those of the bait since there will be many cases of isobaric peptides. IDBEST (Target Discovery, Inc.) is a new tagging strategy that enables such pre-enrichment of specific proteins or protein classes as the resulting tagged peptides are distinguishable from those of the bait by a mass defect shift of approximately 0.1 atomic mass units. The special characteristics of these tags allow: resolution of tagged peptides from untagged peptides through incorporation of a mass defect element; high-precision quantitation of up- and downregulation by using stable isotope versions of the same tag; and potential analysis of protein isoforms through more complete peptide coverage from the proteins of interest.

Biomarkers↗

PubMed2003

"Mass defect" tags for biomolecular mass spectrometry.

We present a new class of "mass defect" tags with utility in biomolecular mass spectrometry. These tags, incorporating element(s) with atomic numbers between 17 (Cl) and 77 (Ir), have a substantially different nuclear binding energy (mass defect) from the elements common to biomolecules. This mass defect yields a readily resolvable mass difference between tagged and untagged species in high-resolution mass spectrometers. We present the use of a subset of these tags in a new protein sequencing application. This sequencing technique has advantages over existing mass spectral protein identification methodologies: intact proteins are quickly sequenced and unambiguously identified using only an inexpensive, robust mass spectrometer. We discuss the potential broader utility of these tags for the sequencing of other biomolecules, differential display applications and combinatorial methods.

Animals↗