Symmetry 300 c18 uplc trap column

Peptide separation was performed on a nanoACQUITY UPLC System (Waters) on-line connected to an LTQ Orbitrap XL mass spectrometer (Thermo Electron). An aliquot of each sample was loaded onto a Symmetry 300 C18 UPLC Trap column (180 μm × 20 mm, 5 μm, Waters). The precolumn was connected to a BEH130 C18 column (75 μm × 200 mm, 1.7 μm, Waters), and equilibrated in 3% acetonitrile and 0.1% FA. Peptides were eluted directly into the LTQ Orbitrap XL mass spectrometer through a nanoelectrospray capillary source (Proxeon Biosystems), at 300 nl/min and using a 120 min linear gradient of 3–40% acetonitrile, followed up by an increase to 40% acetonitrile for the next 30 min. The mass spectrometer automatically switched between MS and MS/MS acquisition in DDA mode. Full MS scan survey spectra (m/z 400–2000) were acquired in the Orbitrap with mass resolution of 30000 at m/z 400. After each survey scan, the six most intense ions above 1000 counts were sequentially subjected to collision-induced dissociation (CID) in the linear ion trap. Precursors with charge states of 2 and 3 were specifically selected for CID. Peptides were excluded from further analysis during 60 s using the dynamic exclusion feature.

A nanoUPLC system (nanoAcquity, Waters Corp., Milford, MA) coupled to a LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, San Jose, CA) via a nanoelectrospray ion source (Proxeon Biosystems) was used for reversed-phase LC-MS/MS analysis. Peptides were loaded onto a trapping column (Symmetry 300 C18 UPLC Trap Column, 180 μm × 20 mm, 5 μm, Waters Corp.). Peptides were separated on a BEH130 C18 column 75 μm × 200 mm, 1.7 μm (Waters Corp.) equilibrated in 3% ACN and 0.1% formic acid with a linear gradient of 3% to 50% ACN at a flow rate of 300 nL/min over 140 min for in-gel digested proteins (naive B-cell, CB, CC, memory B-cell samples), and 170 min for in-solution digested proteins (PC samples). The nUPLC- LTQ Orbitrap XL was operated in the positive ion mode by applying a data-dependent automatic switch between survey MS scan and tandem mass spectra (MS/MS) acquisition. Survey scans were acquired in the mass range of m/z 400 to 2000 with a 30 000 resolution at m/z 400. The 6 most intense ions with states of 2 and 3 were selected in the ion trap for fragmentation by collision-induced dissociation with normalized energy. Dynamic exclusion was enabled for 30 s.