Waters nanoacquity ultra performance lc uplc system

LC–MS/MS analyses were performed on a Thermo Scientific LTQ Orbitrap XL (Thermo Scientific) with a Finnigan Nanospray II electrospray ionization source. Tryptic peptides were injected onto a 75 µm × 150 mm BEH C18 column (particle size 1.7 µm, Waters) and separated using a Waters nanoACQUITY Ultra Performance LC (UPLC) System (Waters, Milford, MA). The LTQ Orbitrap XL was operated in the data-dependent mode using the TOP10 strategy. In brief, each scan cycle was initiated with a full MS scan of high mass accuracy (400–2,000 m/z; acquired by the Orbitrap XL at 6 × 10⁴ resolution setting and automatic gain control (AGC) target of 10⁶) (ref. 5 (link)), which was followed by MS/MS scans (AGC target 5,000; threshold 3,000) in the linear ion trap on the 10 most abundant precursor ions. Selected ions were dynamically excluded for 30 s. Singly charged ions were excluded from MS/MS analysis. MS/MS spectra were searched using the Mascot (Matrix Sciences, Inc.) algorithm against a composite database containing the SwissProt Homo sapiens forward and reverse protein sequences. Search parameters allowed two missed tryptic cleavages, a mass tolerance of ± 10 p.p.m. for precursor ions, a mass tolerance of ± 0.02 Da for product ions, a static modification of 57.02146 Da (carboxyamidomethylation) on cysteine and a dynamic modification of 15.99491 Da (oxidation).