News
Combinatorial approach for screening substrates Combinatorial methods have become popular for rapidly synthesizing and screening numerous substrates. Peptide sequences derivedfromphosphoproteins, such as the insulin receptor and the epidermal growth factor receptor (EGFR), are commonly used in protein tyrosine phosphatase (FTP) substrate specificity studies. Shankar Balasubramanian and co-workers at the University of Cambridge (U.K.) describe a novel combinatorial approach for screening PTP substrates from a resin-bound phosphopeptide library based on the EGFRaoo qq8 scdiicncc The screening strategy is applied to substrate specificity studies of leukocyte antigen receptor (LAR) phosphatase. The phosphopeptide library, synthesized on Kieselguhr resin, is similar to that of the EGFRggg.ggg sequence, with one of eight amino acids substituted for each of the three acidic side chains on the N-terminal side of the phosphorylation site. The result is a 512-member library. Six distinct substrate sequences were discovered for LAR PTP by the screening strategy. It was also found that acidic residues on the ^Vterminal site are important for binding. (J. Am. Chem. Soc. 1997 119 9568-69)
Protein vibrational spectra with neutrons Inelastic neutron scattering (INS) is a technique that complements Raman and IR spectroscopies. However, theoretical calculations are easier with INS, which doesn't require knowledge of charge fluctuations. INS intensity is expressed as a dynamic structure factor, which is a function of the momentum and energy transfers between the neutrons and the vibrational modes of the sample. Until recently, INS has been primarily used for the characterization of low-frequency collective global vibrations in proteins. Using a time-focusing crystal analyzer (TFXA) spectrometer, Jeremy C. Smith and co-workers at SBPM/
DBCM (France), Osaka University Qapan), and Rutherford Appleton Laboratory (U.K.) obtain the vibrational neutron spectrum of staphylococcal nuclease and compare it with the dynamic structure factor calculated in the CHARMM molecular mechanics program using normal mode analysis at 25 K. A numbee ro peaks were present in both the experimental and theoretical spectra, and it is possible to directly compare the intensity from experiment and theory. In the region of 1600-2800 cm"1, the experimental spectrum was dominated by statistical noise. C-H and C-N stretch bands identified in the experimental spectra Other stretch bands were present in the calculated spectrum but they were below the noise threshold in the experimental spectrum
Smoothed experimental and calculated inelastic neutron scattering spectra of staphylococcal nuclease at 25 K.
Near-IR fluorescence detector
Screening strategy: (A) N-terminal Fmocprotected phosphopeptide library; ;B) dephosphorylated peptide; (C) cleaved peptide; (D) fluorescently labeled beads. 712 A
Laser-induced fluorescence (LIF) detection offers high sensitivity, rapid analysis times, and high efficiency for capillary electrophoresis (CE) separations. Excitation sources such as gas-ion lasers are commonly used for detection in the visible region because of the large number of labeling dyes that correspond to the laser lines. Diode lasers typically cost less, are smaller, and have longer lifetimes than gas-ion lasers. Also, diode lasers have particularly stable, high-power signals in the near-IR region. Previous studies have investigated farred diode-based LIF detection for CE,
Analytical Chemistry News & Features, December 1, 1997
The authors note that the agreement between experiment and theory is not perfect. They envision improvements in the theoretical model, such as a reduction in the methyl torsional barrier, the addition of terms in the energy function, and the inclusion of bound water molecules in the normal mode analysis (/. Am Chem Soc 1997 119 9268-73)
with reported detection limits on the order of 10"10 M for various dye-labeled DNA sequencing primers. Steven A. Soper and co-workers at Louisiana State University now report improved sensitivity (detection limits in the low-zmol range) for the separation of tricarbocyanine dyes by CE with LIF detection in the near-IR region. The authors attribute the lower detection limits to fewer fluorescence interferences in the near-IR than in the far-red. The reported near-IR detection system consists of a GaAlAs diode laser and a single-photon avalanche diode. Experiments were performed with four commercially available dyes to establish the limits of detection. Various amino acids were then derivatized with a near-IR la-
