High speed tripletof 5600 mass spectrometer

Five microliters of sample (0.4 µg/µL) was injected into a 2D nano liquid chromatography (nanoLC) ESI-MS/MS system (Eksigent Technologies nanoLC Ultra 1D Plus, AB SCIEX, Foster City, CA, USA) coupled to a High Speed TripleTOF 5600 Mass Spectrometer (SCIEX, Foster City, CA, USA) with a Nanospray III source. The trap column was an Acclaim PepMap 100 (ThermoFisher Scientific, Rockford, IL, USA) with a 5 µm particle diameter and 100 Å pore size, switched online with the analytical column. The loading pump delivered a solution of 0.1% formic acid in water at 2 µL/min. The nanopump provided a flow rate of 300 nL/min and was operated under gradient elution conditions, using 0.1% formic acid in water as mobile phase A and 0.1% formic acid in acetonitrile as mobile phase B. These assays were performed in the Proteomic Laboratory at the National Center for Biotechnology (CNB), Madrid, Spain.

Cleavage assay’s peptides with or without proteases were analyzed with Eksigent 425 NanoLC coupled with Sciex high speed TripleTOF™ 5600+ mass spectrometer using sequential window acquisition of all theoretical mass spectra (SWATH-MS) proteomics. Prior to SWATH analysis method, a relative protein quantification library was built from several data-dependent analysis (DDA) runs with the same LC gradient and instrument settings that were used for SWATH analyses. Before MS runs, samples were subjected to reduction, alkylation, and digestion with calpain protease instead of trypsin as described in detail in Reference [29 (link)]. After digestion, peptides were diluted to 14 µl of sample buffer (2% acetonitrile and 0.1% formic acid) and 1 µl of sample was injected to the triple TOF mass spectrometry. Library was created using Protein Pilot 4.7 (Sciex, Redwood City, CA, USA), and all DDA run spectra were identified against coxsackievirus B3 and calpain (human and rat) UniprotKB/SwissProt protein library added with synthetic CVB1 VP3-VP1 peptide. Quantification was done by Peak Viewer and Marker Viewer (Sciex).