Analyst qs software

Histone H1 expression was analyzed in control and VPA-treated mESCs by reverse-phase HPLC and Mass-Spec as described previously [37 ]. Briefly, cells were harvested and snap-frozen before nuclei extraction using 0.5% Nonidet P-40 in RSB (10 mM NaCl, 3 mM MgCl2, 10 mM TrisHCl, pH 7.5, protease inhibitors) and in a Dounce homogenizer at 4ºC. Released nuclei were then pelleted and resuspended in RSB buffer before extraction of chromatin and histone proteins as described previously [90 (link)]. 50–100 mg of total histone preparations was injected into a C18 reverse-phase HPLC column (Vydac) on an A¨ KTA UPC10 system (GE Healthcare). The effluent from the column was monitored at 214 nm (A214), and the peaks areas were recorded and determined with AKTA UNICORN 5.11 software. Relative amounts of total H1s were determined by ratio of the total A214 of all H1 peaks to half of the A214 of H2B peak. The A214 values of the H1 and H2B peaks were adjusted to account for the differences in the number of peptide bonds in each H1 subtype and H2B. Fractions corresponding to the H1d/H1e peak from HPLC analysis were collected and subjected to mass spectrometry analysis on a Qstar XL MS/MS system (Applied Biosystems) with electrospray ionization (ESI) as the ionization method. Analyst QS software (Applied Biosystems) was used for data acquisition and analysis.

Initially, mitochondrial and cytosolic fractions were separated using a commercially available kit (ProteoExtract Cytosol/Mitochondria Fractionation Kit, Merck, USA) according to manufacturer’s instructions. Mito-Esc was quantified in the mitochondrial and cytosolic fractions obtained from HAEC and aortas of ApoE^−/− mice of different treatment groups as mentioned in the Animal Experiments section. Electrospray ionization (ESI)-mass spectrometry (MS) measurements (positive mode) were performed using a quadrupole time-of-flight mass spectrometer (QSTAR XL, Applied Biosystems/MDS Sciex, Foster City, CA). The data acquisition was under the control of Analyst QS software (Applied Biosystems). For the CID (collision-induced dissociation) experiments, the precursor ions were selected using the quadrupole analyzer and the product ions were analyzed using the TOF analyzer47 (link).
The mitochondrial/cytosolic extracts (50 μl) were diluted with 50 μl of methanol and introduced into the ESI source (injection volume 20 μl) using methanol: water (80:20, v/v) as a mobile phase gradient with flow rate 600 μl/min. Stock solution (0.5 mM) of Mito-Esc was made in methanol: water (50:50, v/v). For spiking experiments, appropriate volumes of standard solutions (1–50 μM) were added to the mitochondrial/cytosolic extracts of untreated samples.

Native His₆-MDH (2 μM, 400 μl), thermally aggregated His₆-MDH or sHsp/His₆-MDH complexes (both formed for 30 min at 47 °C) in buffer C (50 mM HEPES pH 7.6, 50 mM KCl, 5 mM MgCl₂, 2 mM DTT) were incubated with 50 μl MagneHis Ni-Particles (Promega) for 15 min at room temperature. His₆-MDH (aggregated or sHsp-bound) was isolated by placing the reaction in a magnetic reack. The supernatant was subsequently removed and the beads were washed once with buffer C. D₂O-based buffer C was added to initiate amide proton-deuteron exchange. After 30 s the exchange reaction was quenched by adding ice-cold low pH quench buffer (500 mM K-phosphate buffer, pH 2.2) containing pepsin (25 μg ml⁻¹, Roche). Protein was digested from the Ni-Particles for 1 min on ice. Quenched, digested samples were injected into the HPLC setup, with online peptic digest, and analysed on an electrospray ionization quadrupole time-of-flight mass spectrometer (QSTAR Pulsar, Applied Biosystems) as described in ref. 53 (link). Analysis of deuteron incorporation into peptides was performed by using AnalystQS software (Applied Biosystems/MDS SCIEX, Germany). The assignment of the isotope peaks and the selection of the peptides presented were done manually.