Amazon etd ion trap mass spectrometer

Approximately 2 μg of purified peptide samples were injected onto a peptide L-trap column (Chemicals Evaluation and Research Institute, Tokyo, Japan) using an HTS PAL autosampler (CTC Analytics, Zwingen, Switzerland) and further separated thorough an Advance-nano UHPLC (AMR Inc., Tokyo, Japan) using a reverse-phase C18-column (Zaplous column α, 3-μm diameter gel particles and 100 Å pore size, 0.1 × 150 mm; AMR). The mobile phase consisted of solution A (0.1 % formic acid in water) and solution B (acetonitrile). The flow rate was 500 nL/min, with a concentration gradient of acetonitrile from 5 % B to 35 % B over 120 min. Gradient-eluted peptides were analyzed using an amaZon ETD ion-trap mass spectrometer (Bruker Daltonics, Billerica, MA, USA). Data were acquired in a data-dependent manner, in which MS/MS fragmentation was performed on the ten most intense peaks of every full MS scan.
All MS/MS spectra data were searched against the SwissProt Homo sapiens database with Mascot (version_2.3.01; Matrix Science, London, UK). Search criteria were as follows: enzyme, trypsin; allowance of up to two missed cleavage peptides; mass tolerance ±0.5 Da and MS/MS tolerance ±0.5 Da; and modifications of cysteine carbamidomethylation, methionine oxidation, and N-formylation including formyl (K), formyl (R), and formyl (N terminus).

LC–MS measurements for homocysteine and cystathionine levels in samples from the CBS enzyme activity assay were performed by a selected reaction monitoring assay on an amaZon ETD IonTrap Mass spectrometer (Bruker Daltonics, GmbH, Bremen, Germany) coupled to an Ultimate HPLC (Dionex) system, for more details (see Additional files 3 and 4). In addition, the method was used to measure plasma homocysteine for validation of the commercial kit as described below.

To achieve base-line separation, the UHPLC method for SLs detection was adapted based on the method described elsewhere^{14 (link)}. For UHPLC separation C8 BEH column (1.7 µm, 2.1 × 150mm) was applied using a gradient of A:5 millimolar ammonium acetate/0.1% acetic acid in water and B:acetonitrile. We started with 65% B with an initial time of 3 min, and then it was increased to 100% B within 13 min and 99% B was held for 1 min, with reconditioning for 65% B of 2 min and a pre-runtime of 3 min. Flow rate was 0.250 mL/min and column temperature was 40 °C. Injection volume was 5 µL for OSP pellet. Cooling temperature of sample manager was +4 °C. Extracted pellet and collected fractions were measured with this method. Detection of SLs was performed by amaZon ETD iontrap mass spectrometer (Bruker Daltonics GmbH, Bremen, Germany) in negative ESI mode. Samples were introduced into ESI source at a nitrogen flow rate of 8 L/min (250 °C) with a nebulizer gas pressure of 2.0 bar, capillary voltage of 4500 V and acquisition rate of 52 Hz.