Nanoacquity uplc beh c18 column

Liquid chromatography–tandem mass spectrometry proteomic analyses of samples were performed with an LTQ-Orbitrap Elite mass spectrometer (ThermoFisher Scientific, Waltham, MA, USA) coupled with a nanoACQUITY (Waters Corporation, Milford, MA, USA) UPLC system. Measurements were conducted in positive polarity mode, with capillary voltage set to 2.5 kV. A sample was initially applied to the nanoACQUITY UPLC Trapping Column (Waters) while water containing 0.1% formic acid was used as a mobile phase. Then, the peptide mixture was transferred to a nanoACQUITY UPLC BEH C18 Column (75 μm inner diameter; 250 mm long; Waters), applying an acetonitrile gradient (5–35% acetonitrile over 160 min) in the presence of 0.1% formic acid with a flow rate of 250 nl/min. Peptides were eluted directly to the ion source of the mass spectrometer. Before each LC run, a blank run was performed to ensure no material was carried-over from a previous analysis.
Higher-energy Collisional Dissociation (HCD) fragmentation was applied. Up to 10 MS/MS events were allowed per MS scan. See the Supplemental Information for additional MS analysis parameters.

12 μM NiV G ectodomain was used to determine sequences of peptides obtained in HDX-MS after pepsin digestion. 5 μM NiV G ectodomain was used as the apo-NiV G HDX-MS sample, and 5 μM NiV G ectodomain mixed with 2x molar excess of His6-ephrinB2-167 was used as the ephrinB2-bound NiV G HDX-MS sample. All protein samples were prepared in deuterated TBS, pD 7.5. Deuterated TBS was prepared by lyophilization of TBS and resuspension in D₂O (Cambridge Isotopes). Triplicate 5 μL protein samples were mixed with 55 μL deuterated TBS at 25 °C, quenched after 0.5, 1, 2, and 5 min with 125 mM sodium phosphate monobasic, pH 2.6, 250 mM TCEP for 1 min, and injected into a Waters G2-Si HDX-MS system (Waters, Corp.) by a LEAP H/DX PAL autosampler. Protein samples were digested on-column with immobilized pepsin (Pierce) at 0 °C, then separated by liquid chromatography on a Waters NanoACQUITY UPLC BEH C18 column with a 7–85% acetonitrile gradient in 0.1% formic acid. Peptides were analyzed by electrospray ionization mass spectrometry with ion mobility separation in a Synapt G2-Si quadrupole time-of-flight mass spectrometer. Peptides were identified with ProteinLynx Global Server (Waters Corp.). Mass spectra were assigned and H/D exchange was determined with DynamX 3.0 (Waters Corp.). The back exchange was < 30% and the data were corrected for this loss.

LC-MS analysis of peptides was performed on a LTQ-Orbitrap Elite mass spectrometer (Thermo Scientific) coupled with a nanoAcquity (Waters Corporation) LC system. Spectrometer parameters were as follows: polarity mode, positive; capillary voltage, 2 kV. A sample was first applied to the nanoAcquity UPLC Trapping Column (Waters) using water containing 0.1 % formic acid as the mobile phase. Next, the peptide mixture was transferred to the nanoAcquity UPLC BEH C18 Column (Waters, 75 μm inner diameter; 250 mm long) and an ACN gradient (5–30 % over 45 min) was applied in the presence of 0.1 % formic acid with a flow rate of 250 nL/min and eluted directly to the ion source of the mass spectrometer. Each LC run was preceded by a blank run to avoid sample carry-over between the analyses.
Qualitative LC-MS/MS analyses were performed on pooled samples in data-dependent acquisition mode and high-energy collision dissociation (HCD) was used for peptide fragmentation. Quantitative analyses of individual samples were performed by using separate survey scan LC-MS runs with resolving power set to 30 000, m/z measurement range of 300–2 000 and the same ACN gradient settings as those used for the LC-MS/MS runs.