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CID MS/MS c o m petes with NMR The rate of H/D exchange at individual peptide amide linkages is often used to detect localized structural changes in proteins. NMR is usually considered the only practical method for measuring deuterium at individual peptide linkages. David L. Smith and co-workers at the University of Nebraska-Lincoln provide contradictory evidence, showing that collision-induced dissociation (CID) MS/MS can also do the trick. Using peptide standards from selectively labeled cytochrome c, for which most amide hydrogen-exchange rates have been determined by NMR, ,he researchers were able to validate the accuracy of their CID MS/MS results. Folded cytochrome c was exposed to D20 for a given time, and the protein was subsequently digested with pepsin into peptides. Five representative doubly charged fragments were then analyzed by CID MS/MS. Deuterium concentrations at the peptide linkages were determined based on differences in the m/z values of the CID fragments. The expected levels of deuterium were calculated based on isotopic-exchange rate constants determined by NMR and compared with those determined by CID MS/MS Excellent correlation was found between deuterium levels in the b-series of the CID fragment ions and deuterium levels calculated using NMR data. However, analysis of the /'-series of ions yielded several discrepancies, which the authors attribute to the scrambling of the amide hydrogens during fragmentation. Thus, only the b ions provide a reliable indication oo the extent of hydrogen exchange. MS and NMR are shown to be complementary techniques for measuring hydrogen-exchange rates; however, MS has greater sensitivity, is more amenable to large proteins of moderate solubility, and can measure even the most rapidly exchanging amide hydrogens. (J. Am. Chem. Soc. 1919,12121966-67)
Tilting at rough surfaces Like trees growing along a mountain pass, the orientation of molecules coating a surface can be influenced by the "hills" and "valleys" of that surface. Typically, surface roughness is ignored as a factor in spectroscopic studies of surface molecular orientation. When it is considered, it is the rootmean-square (7?q) roughness parameter, as measured by atomic force microscopy (AFM), that is used. However, Rq values are dependent on the scan size chosen in the AFM experiment. Moreover, roughness features smaller than the AFM tip diameter are essentially lost. To deal with these problems, Kathy L Rowlen and Garth J. Simpson of the University of Colorado have developed a scale-dependent method for determining local surface normal tilt-angle distribution. This new method is based on fractal variance correlation function analysis. The researchers evaluated their approach by collecting tapping-mode data on a fusedsilica surface using scan sizes from 125 nm to 5 um. Good correlations are shown between the measured local surface normal tilt angle distribution and the distributions calculated by the new method from scaledependent roughness. The researchers also claim that their method can be extrap-
Fast LC/MS Increasing sample throughput is a top priority for many pharmaceutical companies. To achieve this goal, Neviile JJ Haskins and co-workers at SmithKline Beecham Pharmaceuticals (U.K.) have turned to fast chromatography. But separating complex mixtures is only half of the story. Accurate identification requires an additional step, such as coupled MS detection. However, it is difficult to obtain accurate mass measurements based on relatively few scans across fast chromatographic peaks. Haskins and his colleagues demonstrate that this is possible by combining a quadrupole orthogonal time-of-flight mass trometer with fast gradient LC.
Measured (solid) and calculated (dashed) local surface normal tilt angle distributions for various lateral separations. As the lateral separation decreases, experimental artifacts yield a measured histogram that is almost certainly not representative of the surface— favoring instead the calculated distribution.
olated to size scales below the AFM instrument's resolution. One key point of this study is that surface roughness can contribute significantly to molecular orientation, even for films that coat optically flat surfaces. (J. Phys. Chem. B .999,103, 1525-31)
This novel combination was used to analyze a series of compounds related to cimetidine. Fast LC separations were completed in 5 min, and accurate mass information was obtained, with errors generally better than 5 ppm. In addition, isobaric species (CnH,0N3O and C10HC)F.,N, each with a nominal mass of 200 Da), which could not be distinguished by low-resolution MS, were easily distinguished by the new approach. According to the authors, the technique is particularly valuable for determining the structures of low levels of unknowns in complex mixtures. (Rapid Commun. Mass Spectrom. 1919 13 216-21)
Analytical Chemistry News & Features, May 1, 1999 3 0 5 A
