Analysis of autophosphorylation sites in the recombinant catalytic subunit alpha of cAMP-dependent kinase by nano-UPLC–ESI–MS/MS

The catalytic subunit of recombinant wild-type cyclic adenosine monophosphate-dependent protein kinase A (PKA) has been analyzed by a combination of 1D gel electrophoresis, in-gel digestion by trypsin, chymotrypsin, or endoproteinase AspN, and nano-ultraperformance liquid chromatography–MS/MS. The MS/MS spectra were annotated by MASCOT and the annotations were manually controlled. Using Ga(III)-immobilized metal ion affinity chromatography (IMAC), in addition to the four established autophosphorylation sites of the catalytic subunit of recombinant PKA, pSer10, pSer139, pThr197, and pSer338, six new phosphorylated residues have been characterized–pSer14, pThr48, pSer53, pSer212, pSer259, and pSer325. The established phosphorylation sites all are part of a PKA consensus motif and were found to be almost completely modified. In contrast, the newly detected sites were only partially phosphorylated. For estimation of their degree of phosphorylation, a method based on signal intensity measurements was used. For this purpose, signal intensities of all phospho- and non-phosphopeptides containing a particular site were added for estimation of site-specific phosphorylation degrees. This addition was performed over all peptides observed in the different digestion experiments, including their different charge states. pThr48 and pSer259 are located within PKA consensus motifs and were observed to be phosphorylated at 20% and 24%, respectively. pSer14 and pSer53 are located within inverted PKA consensus motifs and were found to be phosphorylated around 10% and 15%, respectively. The sequence environments of pSer212 and pSer325 have no similarity to the PKA consensus motif at all and were observed to be phosphorylated at about 5% or lower. All newly observed phosphorylation sites are located at the surface of the native protein structure of the PKA catalytic subunit. The results add new information on the theme of site-specific (auto)phosphorylation by PKA.      

Quantitative pupil analysis in stimulated emission depletion microscopy using phase retrieval

The resolution attainable with stimulated emission depletion (STED) microscopy greatly depends on the quality of the STED laser focus. So far, visual inspection of a measured STED focus has been the only convenient means of gauging the source of aberrations. Here we describe a method, requiring no instrument modifications, for obtaining an equivalent to the complex pupil function at the back aperture of the objective and show that it provides quantitative information about aberration sources (including aberrations induced by the objective or sample). We show the accuracy of this field representation to be sufficient for reconstructing the STED focus in three dimensions and determining corrective steps.     

ChemInform Abstract: ScFe5P3, ScRu6P4, U2Mo30P19, and Other Ternary Phosphides with a Metal to Phosphorus Ratio Close to 2:1.

The new ternary phosphides ScFe₅P₃ (I), Sc(Ta_0.18Fe_0.82)Fe₄P₃ (II), ZrFe₅P₃ (III), ScRu₆P₄ (IV), ErRu₆P₄ (V), ZrCr₆P₄ (VI), Nd₂Ru₁₂P₇ (VII), and U₂Mo₃₀P₁₉ (VIII) are prepared from mixtures of the elements in most cases with elemental Sn as a flux (alumina crucibles, 650—950 °C, 7—10 d).     

ChemInform Abstract: Structure and Bonding in the Aluminum Radical Species Al×NH3, HAlNH2, HAlNH2×NH3, and Al(NH2)2 Studied by Means of Matrix IR Spectroscopy and Quantum Chemical Calculations.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.     

Application of sensitive hydrogels in chemical and pH sensors

In the present work, pH-sensitive poly(vinyl alcohol)/poly(acrylic acid) (PVA/PAA) blends as well as hydrogels based on poly(N-isopropylacrylamide) (PNIPAAm), which are sensitive to organic solvent concentration in aqueous solutions, were used in silicon micromachined sensors. A sensitivity of approximately 15 mV/pH was obtained for a pH sensor with a 50 μm thick PVA/PAA hydrogel layer in a pH range above the acid exponent of acrylic acid (pK_a=4.7). The output voltage versus pH-value characteristics and the long-term signal stability of hydrogel-based sensors were investigated and the measurement conditions necessary for high signal reproducibility were determined. The influence of the preparation conditions of the hydrogel films on the sensitivity and response time of the chemical and pH sensors is discussed.     

ChemInform Abstract: How Strong Is the GalliumGallium Triple Bond? Theoretical Compliance Matrices as a Probe for Intrinsic Bond Strengths.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.     

ChemInform Abstract: The Extended Stability Range of Phosphorus Allotropes.

A complete stability range of known and new crystalline allotropes of phosphorus including recently discovered tubular modifications and not‐yet‐known crystal structures of [P₁₂] nanorods, and not‐yet‐isolated [P₁₄] nanorods is given using quantum chemical DFT calculations.