Site-specific detection of S-nitrosylated PKB alpha/Akt1 from rat soleus muscle using CapLC-Q-TOF(micro) mass spectrometry

Protein Kinase Balpha(PKBalpha, or Akt1) is believed to play a crucial role in programmed cell death, cancer progression and the insulin-signaling cascade. The protein is activated by phosphorylation at multiple sites and subsequently phosphorylates and activates eNOS. Free cysteine residues of the protein may capture reactive, endogenously produced nitric oxide (NO) as S-nitrosothiols. Site-specific detection of S-nitrosylated cysteine residues, usually at low stoichiometry, has been a major challenge in proteomic research largely due to the lack of mass marker for S-nitrosothiols that are very labile under physiologic conditions. In this report we describe a sensitive and specific MS method for detection of S-nitrosothiols in PKB alpha/Akt1 in rat soleus muscle. PKB alpha/Akt1 was isolated by immunoprecipitation and 2D-gel electrophoresis, subjected to in-gel tryptic digestion, and cysteinyl nitrosothiols were reacted with iodoacetic acids [2-C(12)/C(13) = 50/50] under ascorbate reduction conditions. This resulted in the production of relatively stable carboxymethylcysteine (CMC) immonium ions (m/z 134.019 and m/z 135.019) within a narrow argon collision energy (CE = 30 +/- 5 V) in the high MS noise region. In addition, free and disulfide-linked cysteine residues were converted to carboxyamidomethylcysteines (CAM). Tryptic S-nitrosylated parent ion was detected with a mass accuracy of 50 mDa for the two CMC immonium ions at the triggered elution time during capillary liquid chromatography (LC) separation. A peptide containing Cys(296) was discriminated from four co-eluting tryptic peptides under lock mass conditions (m/z 785.8426). S-nitrosothiol in the tryptic peptide, ITDFGLBKEGIK (B: CAM, [M + 2H](2+) = 690.86, Found: 690.83), is believed to be present at a very low level, since the threshold for the CMC immonium trigger ions was set at 3 counts/s in the MS survey. The high levels of NO that are produced under stress conditions may result in increased S-nitrosylation of Cys(296) which blocks disulfide bond formation between Cys(296) and Cys(310) and suppresses the biological effects of PKB alpha/Akt1. With the procedures developed here, this process can be studied under physiological and pathological conditions.     

On the generalized Lie structure of associative algebras

We study the structure of Lie algebras in the category ^H MA ofH-comodules for a cotriangular bialgebra (H, 〈|〉) and in particular theH-Lie structure of an algebraA in ^H MA. We show that ifA is a sum of twoH-commutative subrings, then theH-commutator ideal ofA is nilpotent; thus ifA is also semiprime,A isH-commutative. We show an analogous result for arbitraryH-Lie algebras whenH is cocommutative. We next discuss theH-Lie ideal structure ofA. We show that ifA isH-simple andH is cocommutative, then any non-commutativeH-Lie idealU ofA must contain [A, A]. IfU is commutative andH is a group algebra, we show thatU is in the graded center ifA is a graded domain. Dedicated to the memory of S. A. Amitsur Supported by a Fulbright grant. Supported by NSF grant DMS-9203375.     

Copper and iron interactions with angiotensin-converting enzyme inhibitors. A study by fast-atom bombardment tandem mass spectrometry

Fast atom bombardment, combined with high-energy collision-induced tandem mass spectrometry, has been used to investigate gas-phase metal-ion interactions with captopril, enalaprilat and lisinopril, all angiotensin-converting enzyme inhibitors.Suggestions for the location of metal-binding sites are presented. For captopril, metal binding occurs most likely at both the sulphur and the nitrogen atom. For enalaprilat and lisinopril, binding preferably occurs at the amine nitrogen. Copyright 1999 John Wiley & Sons, Ltd.     

Kinetic studies of the reactions between silicon nitride and carbon

The reactions between silicon nitride and carbon take place in two stages, the surface silica of silicon nitride powders reacts with carbon first followed by the decomposition of silicon nitride and the residual silicon reacting with carbon. The kinetics of the two stage reactions has been studied by isothermal thermogravimetric analysis. Physico-geometric models for both of the reaction stages have been proposed, and the kinetic parameters have been calculated. The implications of the kinetic models and parameters are discussed.