Modification of tricine–SDS–PAGE for online and offline analysis of phosphoproteins by ICP-MS

This study describes a modification to tricine sodium dodecyl sulphate polyacrylamide gel electrophoresis to make it more effective for the separation of low molecular mass proteins and for coupling with inductively coupled plasma mass spectrometry (ICP-MS). The modified method employs low-percentage polyacrylamide gels (7–10%) (w/v) and low reagent concentrations that provide efficient separations, good quantitation and low matrix levels that are compatible with ICP-MS. Using phosphopeptides as a model system, and offline analysis, we obtained recoveries of 73% (w/v) in a 9% gel compared with 55% in a conventional 16% gel. Online coupling was achieved by modification of a standard commercially available gel electroelution apparatus and casting of the gel into a 7.3-cm-long tube. Online separation of a digest of β-casein was demonstrated with recovery of the mono- and tetraphosphopeptides, which were identified by comparison with peptide standards. A mass balance study with the standards yielded recoveries of 95% for tetraphosphopeptides and 48% for monophosphopeptides. The factors affecting the separations and recoveries are discussed in detail. The detection limits for 10-μL samples of the mono- and tetraphosphopeptides were 0.7 μM (7 pmol) and 0.2 μM (2 pmol) respectively.     

‘Fixed charge’ chemical derivatization and data dependant multistage tandem mass spectrometry for mapping protein surface residue accessibility

Protein surface accessible residues play an important role in protein folding, protein-protein interactions and protein-ligand binding. However, a common problem associated with the use of selective chemical labeling methods for mapping protein solvent accessible residues is that when a complicated peptide mixture resulting from a large protein or protein complex is analyzed, the modified peptides may be difficult to identify and characterize amongst the largely unmodified peptide population (i.e., the ‘needle in a haystack’ problem). To address this challenge, we describe here the development of a strategy involving the synthesis and application of a novel ‘fixed charge’ sulfonium ion containing lysine-specific protein modification reagent, S,S′-dimethylthiobutanoylhydroxysuccinimide ester (DMBNHS), coupled with capillary HPLC-ESI-MS, automated CID-MS/MS, and data-dependant neutral loss mode MS³ in an ion trap mass spectrometer, to map the surface accessible lysine residues in a small model protein, cellular retinoic acid binding protein II (CRABP II). After reaction with different reagent:protein ratios and digestion with Glu-C, modified peptides are selectively identified and the number of modifications within each peptide are determined by CID-MS/MS, via the exclusive neutral loss(es) of dimethylsulfide, independently of the amino acid composition and precursor ion charge state (i.e., proton mobility) of the peptide. The observation of these characteristic neutral losses are then used to automatically ‘trigger’ the acquisition of an MS³ spectrum to allow the peptide sequence and the site(s) of modification to be characterized. Using this approach, the experimentally determined relative solvent accessibilities of the lysine residues were found to show good agreement with the known solution structure of CRABP II.     

Observation of the crystallization and supersaturation of mixed component NaNO3-Na2SO4 droplets by FTIR-ATR and Raman spectroscopy

We present here a study of the phase behavior of mixed component NaNO(3)-Na(2)SO(4) (SNS) droplets with NaNO(3) to Na(2)SO(4) molar ratios of 1:1, 3:1, and 10:1, comparing observations with thermodynamic predictions. Measurements are made by Fourier transform infrared attenuated total reflection and micro-Raman spectroscopy for SNS droplets deposited on ZnSe and quartz substrates, respectively. The conventional deliquescence/efflorescence hysteresis in phase behavior is observed. On drying, heterogeneous crystallization leads to phase behavior that is consistent with bulk solution thermodynamics, with the formation of the mixed salt NaNO(3)·Na(2)SO(4)·H(2)O, Na(2)SO(4) (s), and NaNO(3) (s) all observed to form at relative humidities that coincide with predictions by the aerosol inorganics model. However, conditioning of the droplet at high relative humidity prior to drying is observed to lead to quantitative differences between the fractions of different salts formed. When substrate effects do not influence the crystallization process, supersaturated solutions are formed, and this leads to the observation of contact ion pairs. Such measurements of the phase behavior of mixed component droplets are important for testing the reliability of thermodynamic models.     

Effective mass anisotropy of hot electrons in nonparabolic conduction bands of n-doped InGaAs films using ultrafast terahertz pump-probe techniques

The anisotropic effective mass of energetic electrons in an isotropic, nonparabolic conduction band is revealed using ultrafast THz-pump-THz-probe techniques in a n-doped InGaAs semiconductor thin film. A microscopic theory is applied to identify the origin of the observed anisotropy and to show that the self-consistent light-matter coupling contributes significantly to the THz response.     

A new criterion for weak subintegrality 1

In a previous paper the authors, together with Balwant Singh, introduced an elementwisc char­acterization of weak subintegrality for an extension A?B of commutative rings. In the present paper we improve our methods for working with this characterization. This leads us to propose two new types of “elementary weakly subintegral extension”.     

Bestimmung von Kohlenstoff, Wasserstoff und Schwefel in organischen Substanzen

Ohne Zusammenfassung     

Independent edges in bipartite graphs obtained from orientations of graphs

Given a digraph D on vertices v₁, v₂, ?, v_n, we can associate a bipartite graph B(D) on vertices s₁, s₂, ?, s_n, t₁, t₂, ?, t_n, where s_it_j is an edge of B(D) if (v_i, v_j) is an arc in D. Let O_G denote the set of all orientations on the (undirected) graph G. In this paper we will discuss properties of the set S(G) := {β₁ (B(D))) | D ? O_G}, where β₁ is the edge independence number. In the first section we present some background and related concepts. We show that sets of the form S(G) are convex and that max S(G) ≦ 2 min S(G). Furthermore, this completely characterizes such sets. In the second section we discuss some bounds on elements of S(G) in terms of more familiar graphical parameters. The third section deals with extremal problems. We discuss bounds on elements of S(G) if the order and size of G are known, particularly when G is bipartite. In this section we exhibit a relation between max S(G) and the concept of graphical closure. In the fourth and final section we discuss the computational complexity of computing min S(G) and max S(G). We show that the first problem is NP-complete and that the latter is polynomial.     

Coherent synthesis using carrier-envelope phase-controlled pulses from a dual-color femtosecond optical parametric oscillator

By using a dual-color femtosecond optical parametric oscillator (OPO), a coherent waveform was synthesized from two coresonant near-infrared signal pulses whose center wavelengths had a separation of 100 nm. Immediately after the OPO cavity the pulses had independent carrier-envelope phase-slip frequencies, and synthesis was achieved by shifting these frequencies using an acousto-optic modulator driven by an internally generated difference frequency. Soliton self-frequency shifted pulses from a photonic crystal fiber and a cross-correlation frequency-resolved optical gating (XFROG) measurement were used to analyze the result of the synthesis experiment and revealed that the synthesized waveform was a train of high-contrast 30 fs pulses.