C-terminal processing of yeast Spt7 occurs in the absence of functional SAGA complex

Background  

Spt7 is an integral component of the multi-subunit SAGA complex that is required for the expression of ~10% of yeast genes. Two forms of Spt7 have been identified, the second of which is truncated at its C-terminus and found in the SAGA-like (SLIK) complex.     

High surface area of porous silicon drives desorption of intact molecules

The surface structure of porous silicon used in desorption/ionization on porous silicon (DIOS) mass analysis is known to play a primary role in the desorption/ionization (D/I) process. In this study, mass spectrometry and scanning electron microscopy (SEM) are used to examine the correlation between intact ion generation with surface ablation and surface morphology. The DIOS process is found to be highly laser energy dependent and correlates directly with the appearance of surface ions (Si(n)(+) and OSiH(+)). A threshold laser energy for DIOS is observed (10 mJ/cm(2)), which supports that DIOS is driven by surface restructuring and is not a strictly thermal process. In addition, three DIOS regimes are observed that correspond to surface restructuring and melting. These results suggest that higher surface area silicon substrates may enhance DIOS performance. A recent example that fits into this mechanism is the surface of silicon nanowires, which has a high surface energy and concomitantly requires lower laser energy for analyte desorption.     

Phosphonium labeling for increasing metabolomic coverage of neutral lipids using electrospray ionization mass spectrometry

Mass spectrometry has become an indispensable tool for the global study of metabolites (metabolomics), primarily using electrospray ionization mass spectrometry (ESI‐MS). However, many important classes of molecules such as neutral lipids do not ionize well by ESI and go undetected. Chemical derivatization of metabolites can enhance ionization for increased sensitivity and metabolomic coverage. Here we describe the use of tris(2,4,6,‐trimethoxyphenyl)phosphonium acetic acid (TMPP‐AA) to improve liquid chromatography (LC)/ESI‐MS detection of hydroxylated metabolites (i.e. lipids) from serum extracts. Cholesterol which is not normally detected from serum using ESI is observed with attomole sensitivity. This approach was applied to identify four endogenous lipids (hexadecanoyl‐sn‐glycerol, dihydrotachysterol, octadecanol, and alpha‐tocopherol) from human serum. Overall, this approach extends the types of metabolites which can be detected using standard ESI‐MS instrumentation and demonstrates the potential for targeted metabolomics analysis. Published in 2009 by John Wiley & Sons, Ltd.     

A High‐Throughput Mass Spectrometric Enzyme Activity Assay Enabling the Discovery of Cytochrome P450 Biocatalysts

Assaying for enzymatic activity is a persistent bottleneck in biocatalyst and drug development. Existing high‐throughput assays for enzyme activity tend to be applicable only to a narrow range of biochemical transformations, whereas universal enzyme characterization methods usually require chromatography to determine substrate turnover, greatly diminishing throughput. We present an enzyme activity assay that allows the high‐throughput mass‐spectrometric detection of enzyme activity in complex matrices without the need for a chromatographic step. This technology, which we call probing enzymes with click‐assisted NIMS (PECAN), can detect the activity of medically and biocatalytically significant cytochrome P450s in cell lysate, microsomes, and bacteria. Using this approach, a cytochrome P450_BM3 mutant library was successfully screened for the ability to catalyze the oxidation of the sesquiterpene valencene.     

Light-directed movement of polymer microstructures

Light has been used to induce photochemical changes in the surface chemistry of porous polymer microstructures giving rise to a substantial change in volume. When illumination is asymmetric, this results in light-directed motion of the structure. Swellable trimethylolpropane trimethacrylate cross-linked poly(2-hydroxylethyl methacrylate) microstructures were constructed by azo-bis-isobutyronitrile photopolymerization using a 20 x 0.5 NA microscope objective and 365 nm laser excitation. Structures were aminiated with glycine and protected with the photolabile group 4-nitroveratryloxycarbanyl (NVOC). Addition of NVOC resulted in a volume increase >10% when performed in the solvent N,N'-dimethylformamide. Photochemical cleavage of NVOC using asymmetric illumination of a cone-shaped microstructure with a 365 nm laser induced polymer shrinkage in excess of 4% at the base of the cone and resulted in a maximum velocity of 1 mm/s at the tip of the cone. Symmetric illumination gave rise to displacement of solvent from the microstructure due to shrinkage with a velocity in excess of 0.01 mm/s. This system could in principle be used for light-directed movement of micromechanical systems, optical control of microfluidics, or light activated chemical delivery.