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.     

Die Verwendung von Natriumsuperoxyd

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A novel 14C-postlabeling assay using accelerator mass spectrometry for the detection of O6-methyldeoxy-guanosine adducts

Accelerator mass spectrometry (AMS) is currently one of the most sensitive methods available for the trace detection of DNA adducts and is particularly valuable for measuring adducts in humans or animal models. However, the standard approach requires administration of a radiolabeled compound. As an alternative, we have developed a preliminary 14C-postlabeling assay for detection of the highly mutagenic O6-methyldeoxyguanosine (O6-MedG), by AMS. Procedures were developed for derivatising O6-MedG using unlabeled acetic anhydride. Using conventional liquid chromatography/mass spectrometry (LC/MS) analysis, the limit of detection (LOD) for the major product, triacetylated O6-MedG, was 10 fmol. On reaction of O6-MedG with 14C-acetic anhydride, using a specially designed enclosed system, the predominant product was 14C-di-acetyl O6-MedG. This change in reaction profile was due to a modification of the reaction procedure, introduced as a necessary safety precaution. The LOD for 14C-di-acetyl O6-MedG by AMS was determined as 79 amol, approximately 18,000-fold lower than that achievable by liquid scintillation counting (LSC). Although the assay has so far only been carried out with labeled standards, the degree of sensitivity obtained illustrates the potential of this assay for measuring O6-MedG levels in humans.     

Effect of flow and surface conditions on human lymphocyte isolation using microfluidic chambers

Phenotypically pure subpopulations of lymphocytes can provide valuable insights into the immune response to injury and disease. The isolation of these subpopulations presents unique challenges, particularly when preprocessing incubation to attach fluorescent or antibody tags is to be minimized. This paper examines the separation of T and B lymphocytes from mixtures using microfluidic chambers coated with antibodies, focusing on flow conditions and surface chemistry. The adhesion of both cell types decreases as shear stress increases irrespective of the surface chemistry. The incorporation of poly(ethylene glycol) chains along with the antibodies on the chamber surface is shown to significantly improve the reproducibility of cell adhesion and is thus an important part of the overall system design. Furthermore, this technique is shown to be an effective way of isolating highly pure subpopulations of lymphocytes from model mixtures, even when the target cell concentration is low.     

Simple Zeros of the Riemann Zeta-Function

Assuming the Riemann Hypothesis, Montgomery showed by meansof his pair correlation method that at least two-thirds of thezeros of Riemann's zeta-function are simple. Later he and Taylorimproved this to 67.25 percent and, more recently, Cheer andGoldston increased the percentage to 67.2753. Here we proveby a new method that if the Riemann and Generalized LindelöofHypotheses hold, then at least 70.3704 percent of the zerosare simple and at least 84.5679 percent are distinct. Our methoduses mean value estimates for various functions defined by Dirichletseries sampled at the zeros of the Riemann zeta-function. 1991Mathematics Subject Classification: 11M26.     

The pion radius

The pion radius has been measured by direct scattering of 50 GeV negative pions from stationary electrons in a hydrogen target. We find the square of the radius to be 〈r_π²〉 = (0.61 ± 0.15)fm².     

Die Untersuchung der Grenzfl?che gasf?rmig-fest von Essigs?ure-Kupfer(I)-oxid-Verbindungen unter Verwendung der Reflexions-Absorptions-Spektroskopie

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