Bioanalytical Development Tools - - BioPharm International

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Bioanalytical Development Tools


BioPharm International


Background

"Protein-protein, protein-DNA and protein-RNA interactions are of central importance in biological systems. Quadrapole Time-of-flight (Q-TOF) mass spectrometry is a sensitive, promising tool for studying these interactions. By combining this technique with chemical crosslinking, it is possible to identify the sites of interactions within these complexes. Due to the complexities of the mass spectrometric data of crosslinked proteins, new software is required to analyze the resulting products of these studies.

Result

We designed a Cross-Linked Peptide Mapping (CLPM) algorithm that takes advantage of all of the information available in the experiment including the amino acid sequence from each protein, the identity of the crosslinker, the identity of the digesting enzyme, the level of missed cleavage, and possible chemical modifications. The algorithm does in silico digestion and crosslinking, calculates all possible mass values and matches the theoretical data to the actual experimental data provided by the mass spectrometry analysis to identify the crosslinked peptides.

Conclusion

Identifying peptides by their masses can be an efficient starting point for direct sequence confirmation. The CLPM algorithm provides a powerful tool in identifying these potential interaction sites in combination with chemical crosslinking and mass spectrometry. Through this cost-effective approach, subsequent efforts can quickly focus attention on investigating these specific interaction sites."

Polymerase Chain Reaction

Polymerase chain reaction exponentially amplifies a specific region of DNA, making it easier to study by other analytical techniques. It is turning out to be a very good test method for disease. Two related articles have appeared in Advanstar publications.

A PCR-based assay of antenatal maternal serum can determine fetal Rh status in over 99 percent of cases with 100 percent accuracy, according to a recently published French study involving 285 pregnant women. Researchers were able to determine fetal RhD status in all but two cases. (In those two cases, the RhD-negative phenotype of the mother was not the result of a complete RHD gene deletion.) No false-positive or false-negative results occurred; all sera from women carrying an RhD-positive fetus had positive results for RHD gene detection, and all sera from women carrying an RhD-negative fetus had negative results.28

Methylation-specific polymerase chain reaction (M-PCR) is a sensitive, specific, fast, and relatively inexpensive method of diagnosing Klinefelter syndrome, according to data presented by Cornell University researchers. M-PCR may serve as an adjunct to karyotype and Y chromosome microdeletion assay, both of which are widely recommended forms of screening for men with low sperm production.

Of equal importance, the M-PCR test using the FMR1 and XIST genes was found to be 100 percent sensitive and 100 percent specific for the diagnosis of Klinefelter syndrome. The investigators could detect a minimum of one percent of XX/XY mosaicism using methylated-to-unmethylated FMR1 PCR product ratio. Total material cost per test, excluding labor and overhead, was an inexpensive $5.49, with a turnaround time of 48 hours.29

Surface Plasmon Resonance

SPR arises from propagation of electromagentic waves (plasmons) along the surface of a thin metal layer. Optical sensors pick up the reflection of these waves. Correct matching of the medium next to the metal and the angle of the incoming light goes into resonance and there is no reflection. That data is used for analysis.

To increase the sensitivity and specificity of the binding assays, particularly in the case of antibody binding, users can take advantage of an extension of SPR when surface plasmon fluorescence spectroscopy (SPFS) is applied simultaneously with SPR.30

The electromagnetic field intensity at the gold surface is strongly related to the reflectivity of the system. Detecting the fluorescence intensity of the labeled molecule in addition to the SPS reflectivity, improves the sensitivity of our device by at least one order of magnitude. Adsorption of dye-labeled molecules on the metal film enhances the fluorescence emission.30

Surface plasmons on gold substrates cannot be efficiently excited using a wavelength lower than 500nm due to the increasing absorption of the gold related to interband transitions. To obtain a well-defined surface plasmon mode with the corresponding electro-magnetic field enhancement an excitation wavelength above 600 nm should be chosen. This fact limits the number of suitable dyes to the long wavelength absorbing molecules.30


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