Recent atomic clock comparisons at NIST

The record of atomic clock frequency comparisons at NIST over the past half-decade provides one of the tightest constraints of any present-day temporal variations of the fundamental constants. Notably, the 6-year record of increasingly precise measurements of the absolute frequency of the Hg⁺ single-ion optical clock (using the cesium primary frequency standard NIST-F1) constrains the temporal variation of the fine structure constant α to less than 2 · 10⁻⁶yr⁻¹ and offers a Local Position Invariance test in the framework of General Relativity. The most recent measurement of the frequency ratio of the Al⁺ and Hg⁺ optical clocks is reported with a fractional frequency uncertainty of ±5.2 · 10⁻¹⁷. The record of such measurements over the last year sensitively tests for a temporal variation of α and constrains , consistent with zero.     

Effect of Trichoderma reesei Proteinases on the Affinity of an Inorganic-Binding Peptide

An inorganic-binding peptide sequence with high affinity to silica-containing materials was fused to a glycoside hydrolase GH26 mannanase, ManA, from the extremely thermophilic bacterium Dictyoglomus thermophilum. The resulting recombinant enzyme produced in Escherichia coli, ManA-Linker, displayed high binding affinity towards synthetic zeolite while retaining its catalytic activity at 80 °C. ManA-Linker was able to bind to the zeolite at different pH levels, indicating a true pH-independent binding. However, complete degradation of the peptide linker was observed when the recombinant ManA-Linker was exposed to the supernatant from the filamentous fungus Trichoderma reesei. This degradation was caused by extracellular proteinases produced by T. reesei during its growth phase. Several derivatives of ManA-Linker were designed and expressed in E. coli. All the derivatives carrying a single sequence of the linker were still susceptible to T. reesei proteinase degradation. Complete substitution of the linker sequence by (GGGGS)₁₆ resulted in a proteinase-resistant ManA derivative, ManA-Linker-(GGGGS)₁₆, which was able to bind to zeolite in a pH-dependent manner.     

A simplified method for capillary embedment into microfluidic devices - exemplified by sol-gel-based preconcentration

We here describe an alternative method of embedding functionalized capillaries into microdevices fabricated in PDMS. The capillaries have square-shaped outer dimensions and fit into elastic PDMS channel networks of similar dimensions. By modifying the capillary off-chip, the technique makes it possible to integrate a new chip function without risking contamination of already existing chemically patterned areas because of new reagent solutions. Leak-free insertion of these capillaries has earlier been reported, where a thin layer of uncured PDMS bonded the capillary to the microchannel and the lid structure. In this new approach, oxygen plasma is used to bond the square capillary to the PDMS, eliminating the risk of clogging the microsystem with uncured prepolymer. The new embedding technique was exemplified and evaluated by inserting a square capillary piece containing monolithic sol-gel for sample preconcentration application. The assembled microdevice was run with mass spectrometric detection, showing that peptides were preconcentrated without leakage from either the sol-gel itself or around the inserted capillary. Repeated preconcentration runs showed migration times better than 3% for all peptides. We believe that the presented microchip assembling technique greatly simplifies the insertion of functionalized capillary pieces, e.g., an initial preconcentrator to a PDMS device containing other downstream modules.     

Synthesis and electron transfer studies of ruthenium-terpyridine-based dyads attached to nanostructured TiO2

A series of bis(terpyridine)RuII complexes have been prepared, where one of the terpyridines is functionalized in the 4'-position by a phosphonic or carboxylic acid group for attachment to TiO2. The other is functionalized, also in the 4'-position, by a potential electron donor. In complexes 1a, 3a, and 4a,b, this donor is tyrosine or hydrogen-bonded tyrosine, while in 2a it is carotenoic amide. The synthesis and photophysical properties of the complexes are discussed. On irradiation with visible light, the formation of a long-lived charge-separated state was anticipated, via primary electron ejection into the TiO2, followed by secondary electron transfer from the donor to the photogenerated RuIII. However, such a charge-separated state could be observed with certainty only with complex 2a. To explain the result, quantum chemical calculations were performed on the different types of complexes.     

Multivariate comparison between peptide mass fingerprints obtained by liquid chromatography-electrospray ionization-mass spectrometry with different trypsin digestion procedures

Peptide mass fingerprints were obtained for three different proteins using three different digestion procedures in triplicates with liquid chromatography coupled to electrospray ionization mass spectrometry. For each protein the results were compared with multivariate data analysis (cluster analysis, kernel principal component analysis) and pair-wise contrast evaluation. Clear systematic differences between the digestion procedures were established for all the proteins. The visual presentation of the pair-wise differences between procedures could to some extent be related to the protein fragments, although the main objective was to identify m/z and retention regions in the original peptide maps that should be subject to further exploration.     

Synthesis and characterization of 2,6-di(quinolin-8-yl)pyridines. New ligands for bistridentate Ru(II) complexes with microsecond luminescent lifetimes

The synthesis of 4-substituted and 4-aryl-substituted 2,6-di(quinolin-8-yl)pyridines is described. The tridentate ligands were prepared via a palladium-catalyzed Suzuki-Miyaura cross-coupling reaction or via a one-step ring-forming reaction generating the central pyridine ring. X-ray crystal structures and 1H NMR shifts are discussed and compared to the corresponding data for a RuII bistridentate complex. Intramolecular stacking of two quinoline units in the RuII complex is suggested by 1H NMR data and also observed in the X-ray structure.     

A generic stepwise optimization strategy for liquid chromatography electrospray ionization tandem mass spectrometry methods

The feasibility and advantages of using sophisticated chemometric tools in combination with the execution of thoroughly planned experiments to determine experimental conditions for optimal performance of an LC-ESI-MS/MS analysis is demonstrated. A stepwise strategy is proposed, which provides a controlled optimization procedure of the chromatographic quality (in terms of separation among the sample constituents) and maximizes the mass spectrometric signal of the selected product ions. Design of experiments (DOE) and response surface methodology are applied throughout the procedure. The stepwise approach has the advantage of dealing with the different optimization criteria separately, i.e. first ensuring sufficient chromatographic separation, then maximizing the amount of precursor ion entering the mass spectrometer, and finally generating high amounts of selected product ions. The experiments are performed on a linear ion trap mass spectrometer. Retention mapping using the band-tracking model is applied during LC development, which facilitates the optimization of segmented gradients. A set of different siderophores, strong iron chelates, is used as the model substances.     

Precise mass measurement of a double-stranded 500 base-pair (309 kDa) polymerase chain reaction product by negative ion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICRMS) has been used to determine the mass of a double-stranded 500 base-pair (bp) polymerase chain reaction (PCR) product with an average theoretical mass of the blunt-ended (i.e. unadenylated) species of 308 859.35 Da. The PCR product was generated from the linearized bacteriophage Lambda genome which is a double-stranded template. Utilization of ethanol precipitation in tandem with a rapid microdialysis step to purify and desalt the PCR product was crucial to obtain a precise mass measurement. The PCR product (0.8 pmol/μL) was electrosprayed from a solution containing 75% acetonitrile, 25 mM piperidine, and 25 mM imidazole and was infused at a rate of 200 nL/min. The average molecular mass and the corresponding precision were determined using the charge-states ranging from 172 to 235 net negative charges. The experimental mass and corresponding precision (reported as the 95% confidence interval of the mean) was 309 406 +/- 27 Da (87 ppm). The mass accuracy was compromised due to the fact that the PCR generates multiple products when using Taq polymerase due to the non-template directed 3'-adenylation. This results in a mixture of three PCR products with nearly identical mass (i.e. blunt-ended, mono-adenylated and di-adenylated) with unknown relative abundances that were not resolved in the spectrum. Thus, the experimental mass will be a weighted average of the three species which, under our experimental conditions, reflects a nearly equal concentration of the mono- and di-adenylated species. This report demonstrates that precise mass measurements of PCR products up to 309 kDa (500 bp) can be routinely obtained by ESI-FTICR requiring low femtomole amounts. Copyright 1999 John Wiley & Sons, Ltd.