A label-free mass spectrometry method for the quantification of protein isotypes

Successful quantitative mass spectrometry (MS) requires strategies to link the mass spectrometer response to the analyte abundance, with the response being dependent on more factors than just analyte abundance. Label-dependent strategies rely on the incorporation of an isotopically labeled internal standard into the sample. Current label-free strategies (performed without internal standards) are useful for analyzing samples that are unsuitable for isotopic labeling but are less accurate. Here we describe a label-free technique applicable to analysis of products from related genes (isotypes). This approach enables the invariant tryptic peptide sequences within the family to serve as “built-in” internal standards and the isotype-specific peptide sequences to report the amount of the various isotypes. A process of elimination segregates reliably trypsin-released standard and reporter peptides from unreliably released peptides. The specific MS response factors for these reporter and standard peptides can be determined using synthetic peptides. Analysis of HeLa tubulin digests revealed peptides from βI-, βII-, βIII-, βIVb-, and βV-tubulin, eight of which were suitable; along with five standard peptides for quantification of the β-tubulin isotypes. To show the utility of this method, we determined that βI-tubulin represented 77% and βIIItubulin represented 3.2% of the total HeLa β-tubulin

Effects of Gangliosides on the Activity of the Plasma Membrane Ca2+-ATPase

Control of intracellular calcium concentrations ([Ca2+]i) is essential for neuronal function, and the plasma membrane Ca2+-ATPase (PMCA) is crucial for the maintenance of low [Ca2+]i. We previously reported on loss of PMCA activity in brain synaptic membranes during aging. Gangliosides are known to modulate Ca2+ homeostasis and signal transduction in neurons. In the present study, we observed age-related changes in the ganglioside composition of synaptic plasma membranes. This led us to hypothesize that alterations in ganglioside species might contribute to the age-associated loss of PMCA activity. To probe the relationship between changes in endogenous ganglioside content or composition and PMCA activity in membranes of cortical neurons, we induced depletion of gangliosides by treating neurons with D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP). This caused a marked decrease in the activity of PMCA, which suggested a direct correlation between ganglioside content and PMCA activity. Neurons treated with neuraminidase exhibited an increase in GM1 content, a loss in poly-sialoganglioside content, and a decrease in PMCA activity that was greater than that produced by D-PDMP treatment. Thus, it appeared that poly-sialogangliosides had a stimulatory effect whereas mono-sialogangliosides had the opposite effect. Our observations add support to previous reports of PMCA regulation by gangliosides by demonstrating that manipulations of endogenous ganglioside content and species affect the activity of PMCA in neuronal membranes. Furthermore, our studies suggest that age-associated loss in PMCA activity may result in part from changes in the lipid environment of this Ca2+ transporter

N-Acetyl-S-(N,N-diethylcarbamoyl) cysteine in rat nucleus accumbens, medial prefrontal cortex, and in RAT and human plasma after disulfiram administration

Disulfiram (DSF), a treatment for alcohol use disorders, has shown some clinical effectiveness in treating addiction to cocaine, nicotine, and pathological gambling. The mechanism of action of DSF for treating these addictions is unclear but it is unlikely to involve the inhibition of liver aldehyde dehydrogenase (ALDH2). DSF is a pro-drug and forms a number of metabolites, one of which is N-acetyl-S-(N,N-diethylcarbamoyl) cysteine (DETC-NAC). Here we describe a LCMS/MS method on a QQQ type instrument to quantify DETC-NAC in plasma and intracellular fluid from mammalian brain. An internal standard, the N,N-di-isopropylcarbamoyl homolog (MIM: 291 > 128) is easily separable from DETC-NAC (MIM: 263 > 100) on C18 RP media with a methanol gradient. The method's linear range is 0.5–500 nM from plasma and dialysate salt solution with all precisions better than 10% RSD. DETC-NAC and internal standards were recovered at better than 95% from all matrices, perchloric acid precipitation (plasma) or formic acid addition (salt) and is stable in plasma or salt at low pH for up to 24 h. Stability is observed through three freeze-thaw cycles per day for 7 days. No HPLC peak area matrix effect was greater than 10%. A human plasma sample from a prior analysis for S-(N,N-diethylcarbamoyl) glutathione (CARB) was found to have DETC NAC as well. In other human plasma samples from 62.5 mg/d and 250mg/d dosing, CARB concentration peaks at 0.3 and 4 nM at 3 h followed by DETC-NAC peaks of 11 and 70 nM 2 h later. Employing microdialysis sampling, DETC-NAC levels in the nucleus accumbens (NAc), medial prefrontal cortex (mPFC), and plasma of rats treated with DSF reached 1.1, 2.5 and 80 nM at 6 h. The correlation between the appearance and long duration of DETC-NAC concentration in rat brain and the persistence of DSF-induced changes in neurotransmitters observed by Faiman et al. (Neuropharmacology, 2013, 75C, 95–105) is discussed

The paclitaxel site in tubulin probed by site-directed mutagenesis of Saccharomyces cerevisiae β-tubulin

AbstractPreviously, we created a paclitaxel-sensitive strain of Saccharomyces cerevisiae by mutating five amino acid residues in β-tubulin in a strain that has a decreased level of the ABC multidrug transporters. We have used site-directed mutagenesis to examine the relative importance of the five residues in determining sensitivity of this strain to paclitaxel. We found that the change at position 19 from K (brain β-tubulin) to A (yeast β-tubulin) and at position 227 from H (brain β-tubulin) to N (yeast β-tubulin) had no effect on the activity of paclitaxel. On the other hand, the changes V23T, D26G and F270Y, drastically reduced sensitivity of AD1-8-tax to paclitaxel. Molecular modeling and computational studies were used to explain the results

Anxiety, depression, and stress as risk factors for atrial fibrillation after cardiac surgery

OBJECTIVE: We sought to determine whether preoperative and postoperative anxiety, depression, and stress symptoms were associated with atrial fibrillation (AF) after cardiac surgery.METHODS: Two hundred and twenty-six cardiac surgery patients completed measures of depression, anxiety, and general stress before surgery, and 222 patients completed these measures after surgery. The outcome variable was new-onset AF, confirmed before the median day of discharge (day 5) after cardiac surgery during the index hospitalization.RESULTS: Fifty-six (24.8%) patients manifested incident AF, and they spent more days in hospital (mean [M], 7.3; standard deviation [SD], 4.6) than patients without AF (M, 5.5; SD, 1.4; P P = .05). This analysis also showed that age was significantly associated with AF (odds ratio, 1.07; 95% confidence interval, 1.03 to 1.12; P CONCLUSION: Anxiety symptoms in the postoperative period were associated with AF. Hospital staff in acute cardiac care and cardiac rehabilitation settings should observe anxiety as related to AF after cardiac surgery. It is not clear how anxious cognitions influence the experience of AF symptoms, and whether symptoms of anxiety commonly precede AF