Testing white line strength in the dairy cow

The tensile strength of 576 pieces of white line horn collected over 6 mo from 14 dairy cows restricted to parity 1 or 2 was tested. None of the cows had ever been lame. Seven cows were randomly assigned to receive 20 mg/d biotin supplementation, and 7 were not supplemented. Hoof horn samples were taken from zones 2 and 3 (the more proximal and distal sites of the abaxial white line) of the medial and lateral claws of both hind feet on d 1 and on 5 further occasions over 6 mo. The samples were analyzed at 100% water saturation. Hoof slivers were notched to ensure that tensile strength was measured specifically across the white line region. The tensile stress at failure was measured in MPa and was adjusted for the cross-sectional area of the notch site. Data were analyzed in a multilevel model, which accounted for the repeated measures within cows. All other variables were entered as fixed effects. In the final model, there was considerable variation in strength over time. Tensile strength was significantly higher in medial compared with lateral claws, and zone 2 was significantly stronger than zone 3. Where the white line was visibly damaged the tensile strength was low. Biotin supplementation did not affect the tensile strength of the white line. Results of this study indicate that damage to the white line impairs its tensile strength and that in horn with no visible abnormality the white line is weaker in the lateral hind claw than the medial and in zone 3 compared with zone 2. The biomechanical strength was lowest at zone 3 of the lateral hind claw, which is the most common site of white line disease lameness in cattle.     

Apparatus and initial results for pressurized planar electrochromatography

Pressurized planar electrochromatography (PPEC) is a new planar chromatographic technique in which the mobile phase is driven by electroosmotic flow, while the sorbent layer is pressurized in a manner that allows heat to flow from the layer through an electrically insulating, thermally conducting, sheet of aluminum nitride ceramic. A prototype apparatus for performing PPEC is described. Separation by PPEC is faster than by conventional TLC, and an example is presented of a 24-fold enhancement in the speed of separation. PPEC was performed on both regular and high-performance C18 layers, and the latter yield substantially faster separation. The sorbent layer requires conditioning at elevated temperature before use, and solute migration velocity increases with this temperature. The flow rate increases in a linear manner with increasing voltage and diminishes in a nonlinear manner with increasing pressure. Both electrical current and Joule heating diminish with increasing pressure, and the diminution of flow at high pressure can be compensated by an increase in voltage. PPEC is more efficient than classical TLC. Theoretical plate heights diminish with increasing Rf and are in the range 29-21 and 55-27 microm for the high-performance and regular plates, respectively. PPEC retains the advantages of classical TLC but has the ability to separate a substantially higher number of samples simultaneously. An example is presented on the separation of nine samples in 1 min on a 2.5 cm x 10 cm sorbent layer.     

Ultra-high-efficiency strong cation exchange LC/RPLC/MS/MS for high dynamic range characterization of the human plasma proteome

High-efficiency nanoscale reversed-phase liquid chromatography (chromatographic peak capacities of approximately 1000: Shen, Y.; Zhao, R.; Berger, S. J.; Anderson, G. A.; Rodriguez, N.; Smith, R. D. Anal. Chem. 2002, 74, 4235. Shen, Y.; Moore, R. J.; Zhao, R.; Blonder, J.; Auberry, D. L.; Masselon, C.; Pasa-Tolic, L.; Hixson, K. K.; Auberry, K. J.; Smith, R. D. Anal. Chem. 2003, 75, 3596.) and strong cation exchange LC was used to obtain ultra-high-efficiency separations (combined chromatographic peak capacities of >10(4)) in conjunction with tandem mass spectrometry (MS/MS) for characterization of the human plasma proteome. Using conservative SEQUEST peptide identification criteria (i.e., without considering chymotryptic or elastic peptides) and peptide LC normalized elution time constraints, the separation quality enabled the identification of proteins over a dynamic range of greater than 8 orders of magnitude in relative abundance using ion trap MS/MS instrumentation. Between 800 and 1682 human proteins were identified, depending on the criteria used for identification, from a total of 365 microg of human plasma. The analyses identified relatively low-level (approximately pg/mL) proteins (e.g., cytokines) coexisting with high-abundance proteins (e.g., mg/mL-level serum albumin).     

Using mass spectrometry to study copper-protein binding under native and non-native conditions: beta-2-microglobulin

A method based on metal-catalyzed oxidation (MCO) reactions and mass spectrometry (MS) has been used to determine the Cu(II) binding sites in both native and unfolded conformations of beta-2-microglobulin (beta2m). Recent studies have shown that beta2m is destabilized and can form amyloid fibers in the presence of Cu(II). An increased affinity for Cu in unfolded states compared to that of the native state is suspected to facilitate overall protein destabilization. Cu-binding site information for native beta2m is difficult to obtain using traditional techniques because of its propensity to form amyloid fibers at relatively high protein concentrations in the presence of Cu and because of the nonspecific paramagnetic peak broadening observed in NMR analyses. In addition, Cu-binding information of unfolded beta2m is complicated by the high concentrations of denaturants (e.g., 8 M urea) needed to ensure protein unfolding. The MCO/MS approach has been successfully employed in this work to overcome these difficulties. The sensitivity of MS allowed the Cu-binding site of the native protein to be determined at the low concentrations of beta2m necessary to avoid amyloid fiber formation. Results indicate that the N-terminus of the protein and His31 are responsible for Cu(II) coordination in the native state. The MCO/MS method was also successful at determining the Cu-binding site in the presence of 8 M urea with the N-terminus, His31, His51, and His81 found to be Cu-bound in the unfolded state. This result supports the existence of a well-defined but different coordination structure in the unfolded state, which leads to the greater affinity for Cu(II) observed in the unfolded state of the protein. In general, it appears that the MCO/MS method is capable of providing Cu-binding site information for proteins that are difficult to study by traditional means.     

High-throughput comprehensive analysis of human plasma proteins: a step toward population proteomics

A high-throughput (HT) comprehensive analysis approach was developed for assaying proteins directly from human plasma. Proteins were selectively retrieved, by utilizing antibodies immobilized within affinity pipet tips, and eluted onto enzymatically active mass spectrometer targets for subsequent digestion and structural characterization. Several parameters, including uniform parallel protein elution from 96 affinity pipet tips, proper buffering for on-target digestion, termination of the digestion, and MALDI matrix (re)introduction, were evaluated and optimized. The approach was validated via parallel, high-throughput analysis of transthyretin (TTR) and transferrin (TRFE) from 96 identical plasma samples. The 96 parallel analyses for each protein were completed in less than 90 min, measured from protein extraction to insertion in the mass spectrometer. Virtually identical mass spectra were obtained from the 96 TTR analyses, characterized by the presence of 14 tryptic fragments that allowed TTR sequence mapping with 100% coverage. Database search returned TTR as the best match for all 96 data sets. In regard to the TRFE analyses, database searching using data from the 96 spectra returned TRFE as the best match for all but 1 of the spectra. TRFE was mapped with 47-69% sequence coverage, with gaps in the sequence coverage corresponding to the carbohydrate-containing peptide fragments and large and small trypsin fragments that fell outside the window of mass analysis. Overall, the combined high-throughput affinity capture-protein digestion approach showed high reproducibility and speed and yielded an exceptional level of protein characterization, suggesting its use in future population proteomics endeavors.     

Multiplexed MS/MS in a quadrupole ion trap mass spectrometer

A multiplexing method for performing MS/MS on multiple peptide ions simultaneously in a quadrupole ion trap mass spectrometer (QITMS) has been developed. This method takes advantage of the inherent mass bias associated with ion accumulation in the QITMS to encode the intensity of precursor ions in a way that allows the corresponding product ions to be identified. The intensity encoding scheme utilizes the Gaussian distributions that characterize the relationship between ion intensities and rf trapping voltages during ion accumulation. This straightforward approach uses only two arbitrary waveforms, one for isolation and one for dissociation, to gather product ion spectra from N precursor ions in as little as two product ion spectra. In the example used to illustrate this method, 66% of the product ions from five different precursor peptide ions were correctly correlated using the multiplexing approach. Of the remaining 34% of the product ions, only 6% were misidentified, while 28% of the product ions failed to be identified because either they had too low intensity or they had the same m/z ratio as one of the precursor ions or the same m/z ratio as a product ion from a different precursor ion. This method has the potential to increase sample throughput, reduce total analysis times, and increase signal-to-noise ratios as compared to conventional MS/MS methods.     

Uniqueness in remote sensing of the inherent optical properties of ocean water

We examine the problem of uniqueness in the relationship between the remote-sensing reflectance (Rrs) and the inherent optical properties (IOPs) of ocean water. The results point to the fact that diffuse reflectance of plane irradiance from ocean water is inherently ambiguous. Furthermore, in the 400 < lambda < 750 nm region of the spectrum, Rrs(lambda) also suffers from ambiguity caused by the similarity in wavelength dependence of the coefficients of absorption by particulate matter and of absorption by colored dissolved organic matter. The absorption coefficients have overlapping exponential responses, which lead to the fact that more than one combination of IOPs can produce nearly the same Rrs spectrum. This ambiguity in absorption parameters demands that we identify the regions of the Rrs spectrum where we can isolate the effects that are due only to scattering by particulates and to absorption by pure water. The results indicate that the spectral shape of the absorption coefficient of phytoplankton, a(ph)(lambda), cannot be derived from a multiparameter fit to Rrs(lambda). However, the magnitude and the spectral dependence of the absorption coefficient can be estimated from the difference between the measured Rrs(lambda) and the best fit to Rrs(lambda) in terms of IOPs that exclude a(ph)(lambda).     

Phase calibration of spatially nonuniform spatial light modulators

A new 512 x 512 pixel phase-only spatial light modulator (SLM) has been found to deviate from being flat by several wavelengths. Also, the retardation of the SLM relative to voltage varies across the device by as much as 0.25 wavelength. The birefringence of each pixel as a function of address voltage is measured from the intensity of the SLM between crossed polarizers. To these responses are added a reference spatial phase measured by phase shifting interferometry for a single address voltage. Fits to the measured data facilitate the compensation of the SLM to a root-mean-square wave-front error of 0.06 wavelength. The application of these corrections to flatten the full aperture of the SLM sharpens the focal plane spot and reduces the distortion of computer-designed diffraction patterns.     

Gated fiber-optic-coupled detector for in vivo real-time radiation dosimetry

Gated detection of the output of a fiber-optic-coupled radiation dosimeter effectively eliminated the direct contribution of Cerenkov radiation to the signal. The radiation source was an external beam radiotherapy machine that provided pulses of 6-MeV x rays. Gated detection was used to discriminate the signal collected during the radiation pulses, including Cerenkov interference, from the signal collected between the radiation pulses due only to phosphorescence from the Cu(1+)-doped glass detector. Gated detection of the long-lived phosphorescence of the Cu(1+)-doped glass provided real-time dose measurements that were linear with the absorbed dose and that were accurate for all field sizes studied.