Nano chiplc trap column

The peptide samples were analyzed on an AB SCIEX TripleTOF 5600 system (AB SCIEX, Framingham, MA) coupled to an Eksigent NanoLC Ultra system (Eksigent, Dublin, CA). The samples (2 μl) were injected using an autosampler. Samples were loaded onto a Nano cHiPLC Trap column 200 lm × 0.5 mm ChromXP C18-CL 3 lm 120 Å (Eksigent) at a flow rate of 2 μl/min for 10 min. The peptides were separated on a Nano cHiPLC column 75 μm × 15 cm ChromXP C18-CL 3 lm 120 Å (Eksigent) using a 120-min linear gradient of 5–35% ACN in 0.1% formic acid at a flow rate of 300 nl/min. MS1 settings: mass range of m/z 400–1250 and accumulation time 0.5 s. MS2 settings: mass range of m/z 100–1800, accumulation time 0.05 s, high sensitivity mode, charge state 2–5, selecting anything over 100 cps, maximal number of candidate/cycle 50, and excluding former targets for 12 s after each occurrence.

Peptides in the digestion mix were identified by in house mass spectrometry. Equal amounts of peptide degradation samples were injected into a Nano-HPLC (Eksigent) and online nanosprayed into an Orbitrap mass spectrometer (LTQ Orbitrap Discovery, Thermo) with a flow rate of 400nL/min. A Nano cHiPLC trap column (200μm x 0.5mm ChromXP c18-CL 5μm 120Å; Eksigent) was used to remove salts in the sample buffer. Peptides were separated in a Nano cHiPLC column (75μm x 15cm ChromXP c18-CL 5μm 300Å; Eksigent) over a gradient of 2% to 40% buffer B (buffer A: 0.1% FA in water; buffer B: 0.1% FA in acetonitrile) and mass spectra were recorded in the range of 370 to 2000Daltons. In tandem MS/MS mode, the eight most intense peaks were selected with a window of 1Da and fragmented. The collision gas was helium, and the collision voltage was 35V. Masses in the mass spectra were searched against source peptide databases with Proteome Discoverer (Thermo Scientific). The integrated area under a peptide peak is proportional to its abundance. Each sample was run on the mass spectrometer at least twice.