Lamp‐based wavelength‐resolved fluorescence detection for protein capillary electrophoresis: Setup and detector performance

A lamp‐based fluorescence detection (Flu) system for CE was extended with a wavelength‐resolved (WR) detector to allow recording of full protein emission spectra. WRFlu was achieved using a fluorescence cell that employs optical fibres to lead excitation light from a Xe‐Hg lamp to the capillary window and protein fluorescence emission to a spectrograph equipped with a CCD. A 280 nm band pass filter etc. together with a 300 nm short pass cut‐off filter was used for excitation. A capillary cartridge was modified to hold the detection cell in a commercial CE instrument enabling WRFlu in routine CE. The performance of the WRFlu detection was evaluated and optimised using lysozyme as model protein. Based on reference spectral data, a signal‐intensity adjustment was introduced to correct for transmission losses in the detector optics that occurred for lower protein emission wavelengths. CE‐WRFlu of lysozyme was performed using BGEs of 50 mM sodium phosphate (pH 6.5 or 3.0) and a charged‐polymer coated capillary. Using the 3‐D data set, signal averaging over time and emission‐wavelength intervals was carried out to improve the S/N of emission spectra and electropherograms. The detection limit for lysozyme was 21 nM, providing sufficient sensitivity to obtain spectral information on protein impurities.     

Parameters affecting the separation of intact proteins in gradient-elution reversed-phase chromatography using poly(styrene-co-divinylbenzene) monolithic capillary columns

An experimental study was performed to investigate the effects of column parameters and gradient conditions on the separation of intact proteins using styrene-based monolithic columns. The effect of flow rate on peak width was investigated at constant gradient steepness by normalizing the gradient time for the column hold-up time. When operating the column at a temperature of 60 °C a small C-term effect was observed in a flow rate range of 1–4 μL/min. However, the C-term effect on peak width is not as strong as the decrease in peak width due to increasing flow rate. The peak capacity increased according to the square root of the column length. Decreasing the macropore size of the polymer monolith while maintaining the column length constant, resulted in an increase in peak capacity. A trade-off between peak capacity and total analysis time was made for 50, 100, and 250 mm long monolithic columns and a microparticulate column packed with 5 μm porous silica particles while operating at a flow rate of 2 μL/min. The peak capacity per unit time of the 50 mm long monolithic column with small pore size was superior when the total analysis time is below 120 min, yielding a maximum peak capacity of 380. For more demanding separations the 250 mm long monolith provided the highest peak capacity in the shortest possible time frame.     

Synthesis of oxazolo[4,5-c]quinoline TRPV1 antagonists

An efficient synthesis of 2-amino-oxazolo[4,5-c]quinoline TRPV1 antagonists is described via a thiourea formation/carbodiimide cyclization sequence. Synthetic route optimization eliminates intermediate isolations and facilitates the rapid preparation of a series of novel pentacyclic TRPV1 antagonists. From this series, compound (S)-4 was identified as a potent and selective ligand for the TRPV1 ion channel.     

Counting connected graphs asymptotically

We find the asymptotic number of connected graphs with k vertices and k−1+l edges when k,l approach infinity, re-proving a result of Bender, Canfield and McKay. We use the probabilistic method, analyzing breadth-first search on the random graph G(k,p) for an appropriate edge probability p. Central is the analysis of a random walk with fixed beginning and end which is tilted to the left.     

Effects of pollution restrictions on dynamic investment policy of a firm

The purpose of this paper is to determine the effects of different pollution standards on the firm's resource allocation decisions. To do so, a dynamic model of the firm is developed in which it is assumed that production causes pollution as an inevitable byproduct. Concerning its investment policy, we suppose that the firm can choose between investing in productive capital goods and investing in abatement efforts.It is shown that, in some cases, future abatement expenses have a negative impact on the present level of productive investment, even if the pollution standard is not binding at the moment. This implies a really dynamic optimal investment policy for the firm, which cannot be obtained within a comparative static analysis.This research has been made possible by a fellowship of the Royal Netherlands Academy of Arts and Sciences. Comments by Frank van der Duyn Schouten and Piet Verheyen (Tilburg University) and by Raymond Gradus (Dutch Ministry of Finance, The Hague) are gratefully acknowledged.     

Volume flexibility and capacity investment: a real options approach

This paper considers the investment decision of a firm where it has to decide about the timing and capacity. We obtain that in a fast-growing market, right after investment the firm produces below capacity, where the utilization rate (the proportion of capacity that is used for production right after the investment) increases with market uncertainty for a very big market trend, and shows no monotonicity for a moderately large market trend. On the other hand, we get that, for a slowly growing or shrinking market, the firm produces up to capacity right after investment. In the intermediate case, the firm produces up to capacity right after investment when uncertainty is low and below capacity when uncertainty is high, whereas the utilization rate decreases with the market uncertainty.     

Looped structure of flowerlike micelles revealed by 1H NMR relaxometry and light scattering

We present experimental proof that so-called "flowerlike micelles" exist and that they have some distinctly different properties compared to their "starlike" counterparts. Amphiphilic AB diblock and BAB triblock copolymers consisting of poly(ethylene glycol) (PEG) as hydrophilic A block and thermosensitive poly(N-isopropylacrylamide) (pNIPAm) B block(s) were synthesized via atom transfer radical polymerization (ATRP). In aqueous solutions, both block copolymer types form micelles above the cloud point of pNIPAm. Static and dynamic light scattering measurements in combination with NMR relaxation experiments proved the existence of flowerlike micelles based on pNIPAm(16kDa)-PEG(4kDa)-pNIPAm(16kDa) which had a smaller radius and lower mass and aggregation number than starlike micelles based on mPEG(2kDa)-pNIPAm(16kDa). Furthermore, the PEG surface density was much lower for the flowerlike micelles, which we attribute to the looped configuration of the hydrophilic PEG block. (1)H NMR relaxation measurements showed biphasic T(2) relaxation for PEG, indicating rigid PEG segments close to the micelle core and more flexible distal segments. Even the flexible distal segments were shown to have a lower mobility in the flowerlike micelles compared to the starlike micelles, indicating strain due to loop formation. Taken together, it is demonstrated that self-assemblies of BAB triblock copolymers have their hydrophilic block in a looped conformation and thus indeed adopt a flowerlike conformation.     

Altered Mascot search results by changing the <Emphasis Type="Italic">m</Emphasis>/<Emphasis Type="Italic">z</Emphasis> range of MS/MS spectra: analysis and potential applications

The Mascot search algorithm is one of the most commonly used tools for protein identification. Tandem mass spectrometry data searched against a protein sequence database is utilized for identifying peptides and proteins, each reported with a score. Higher Mascot scores are associated with lower chances of random hits. The process of peak selection performed by the search engine prior to the search is a critical aspect of the process. Here, we show that Mascot divides the MS/MS spectrum into fixed m/z regions for peak selection, starting at the lowest m/z value of the peak list. Therefore, modifying the m/z range of the peak lists by insertion of a dummy peak with low m/z value changes the ensemble of peaks used for searching. As a consequence, Mascot peptide scores and search results are altered significantly and a different subset of the peptides present in the sample is identified after processing. We further show that the effect can be exploited and additional proteins and peptides can be identified by repeating the search with a combined set of differently processed files, even when applying identical false-positive rates.