Enhanced Universal Quantification of Biomolecules Using Element MS and Generic Standards: Application to Intact Protein and Phosphoprotein Determination.

Tools that provide absolute quantification of biomolecules, particularly of proteins and their post-translational modifications, without needing suitable specific standards, are urgently demanded nowadays. To this end, we have significantly improved the recently introduced strategy based on CH4 addition to the plasma for absolute quantification of biomolecules using HPLC-ICP-MS. Addition of CO2 has been optimized and finally selected as a safer, more efficient quantitative strategy that is able to provide constant (<6% error) signal response factor for the six elements assayed (S, P, As, Se, Br, I) under compromised conditions. In the particular case of absolute protein quantification, accuracy and precision attainable for S-based absolute determination of intact proteins using internal and external S-generic standards were compared. Potential for real sample analysis was demonstrated by the high-sensitivity analysis of toxins present in snake venoms. Finally, multielemental speciation capabilities of the approach have been also demonstrated through P and S simultaneous analysis in phosphoproteomics. Simultaneous accurate determination of both absolute protein amount and corresponding phosphorylation degree for intact ß-casein, and even impurity traces of κ and α-s1 isoforms present, has been successfully achieved using a simple mixture of inorganic P and S standards. The lowest detection limits (<1 fmol protein) ever published for S- and P-based intact protein quantification with ICP-MS are reported.

Effect of mold temperatures on interface between primary and secondary castings of cast-on method for precision metal frameworks.

PURPOSE: The purpose of the present study was to obtain fundamental data for application of the cast-on method by evaluating the effect of mold temperatures on the interface between primary and secondary castings in detail. METHODS: Silver-palladium-gold alloy (Ag-Pd-Au), type-4 gold alloy (Type4), and chromium-cobalt alloy (Cr-Co) were used in the present study. A polished flat, square metal plate, 10.0 mm x10.0 mm x 1.0 mm, was used as the primary casting. A wax pattern, 2.0 mm in diameter and 2.0 mm thick, was prepared for the secondary casting and invested together with the primary casting. The mold was heated at 600, 700 and 800 degrees C for Ag-Pd-Au and Type4, and 700, 800 and 900 degrees C for Cr-Co. After casting, the mold was embedded and sectioned. The cross-section was observed using a scanning electron microscope (SEM) and analyzed using energy dispersive X-ray spectroscopy (EDS). The gaps between the primary and secondary castings were analyzed for each alloy by analysis of variance and Tukey's honestly significant difference test. The significance level was set at 0.05. The heated primary casting without the secondary casting was examined using a thin film X-ray diffractometer (XRD). RESULTS: Gaps were observed between the primary and secondary castings in all examined conditions. The primary casting surface was covered with oxide layers such as CuO and Cr(2)O(3), and became rough with an increase of the mold temperature. CONCLUSIONS: The results suggested that the cast-on method was influenced by the mold temperature.