Nanoacquity nano hplc system

We evaluated the peptide sequences of goat milk casein hydrolysates using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a nanoAcquity nano HPLC system (Waters, Milford, MA) and a Q Exactive mass spectrometer (Thermo Scientific) as reported previously (Forde et al., 2015; (link)Song et al., 2017a) . Fraction 1 was injected into the trap column (20 mm × 100; Polymicro, Phoenix, AZ) packed with an Aqua C18 column (5 μm, 125 Å; Phenomenex, Torrance, CA) and separated on a micro-analytical column (50 μm × 10 cm; Polymicro) packed with an Aqua C18 column (3 μm, 125 Å; Phenomenex). The eluting program included solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile); the linear gradient was 1 to 40% solvent B for 40 min. The flow rate was 0.2 μL/min. The eluate was injected directly into the MS system, and electrospray ionization mode was carried out with full mass spectrum scanning at 100 to 2500 m/z. All identified peptide sequences of goat milk casein hydrolysates were confirmed by comparing them with Capra hircus milk casein from the UniProt (http: / / www .uniprot .org/ ) and National Center for Biotechnology Information (http: / / www .ncbi .nlm .nih .gov) databases.

After confirming the detection of the polymorphic peptide (ETLDPSAPK) using global proteomics (Table S2), targeted allele-specific quantification was carried out to assess the levels of the two variants.
The QconCAT sequence included heavy isotope-labeled versions of the two surrogate peptides (ETLDPSAPK, ETLDPSAPR). Microsomal material (0.6-1.3 µg) from 24 liver samples was analyzed using scheduled multiple reaction monitoring (MRM) on a nanoACQUITY nano-HPLC system (Waters, UK) coupled to a TSQ Vantage triple quadrupole mass spectrometer (Thermo Fisher Scientific, USA) operated using Xcalibur v2.0.6 (Thermo Fisher). Peptides were eluted from a Waters HSS T3 C18 analytical column (1.8 µm, 75 µm × 150 mm) at a flow rate of 300 nl min -1 with a gradient of 3% to 50% acetonitrile over 40 min. Skyline v1.4 was used to design the transitions (Table 1) and Skyline v4.2 was used to analyze the data. Samples were interspersed by blanks, which were analyzed to assess carry-over. For quantitative assay development, a pooled sample from 50 livers (30 µg) was prepared and analyzed using the designed assay to assess technical, analytical, intra-day and inter-day variability. Linearity was evaluated by analyzing a range of 0.08-2.31 µg of microsomal pool protein.