Splitless nanolc 2d pump

Capillary LC/FT/MS/MS was performed with a split less nanoLC-2D pump (Eksigent, Livermore, CA, USA), a 50 μm-i.d. column packed with 7 cm of 3 µm-o.d. C18 particles, and a hybrid linear ion trap-Fourier-transform tandem mass spectrometer (LTQ-ELITE; ThermoFisher, San Jose, CA, USA) operated with a lock mass for calibration. The reverse-phase gradient was 2–62% of 0.1% formic acid in acetonitrile over 60 min at 350 nL/min. For unbiased analysis, the top six most abundant eluting ions were fragmented by data-dependent HCD with a mass resolution of 120,000 for MS and 15,000 for MS/MS. For isobaric TMT labeling, probability-based protein database searching of MS/MS spectra against the TrEMBL protein database (release December 29, 2012; 59,862 sequences) was performed with a 10-node MASCOT cluster (v. 2.3.02; Matrix Science, London, UK) with the following search criteria: peak picking with Mascot Distiller; 10 ppm precursor ion mass tolerance, 0.8 Da product ion mass tolerance, three missed cleavages, trypsin, carbamidomethyl cysteines as a static modification, oxidized methionine and deamidated asparagine as variable modifications, an ion score threshold of 20, and TMT-6-plex for quantification.

Capillary LC-FT-MS/MS was performed as described by Raphael et al. (2014) [24 (link)] using a splitless nanoLC-2D pump (Eksigent, CA), a 50 µm-i.d. column packed with 7 cm of 3 µm-o.d. C18 particles, and a hybrid linear ion trap-Fourier-transform tandem mass spectrometer (LTQ-ELITE; Thermo Fisher, CA). The reverse-phase gradient was 2 to 62% of 0.1% formic acid (FA) in acetonitrile over 60 min at 350 nL/min. The top six most abundant eluting ions were fragmented by data-dependent high-energy collision-induced dissociation (HCD). Probability-based and error-tolerant protein database searching of MS/MS spectra against the Trembl protein database (release of 2013) were performed with a 10-node Mascot cluster (version 2.3.02, Matrix Science, London, UK). Search criteria included peak picking with Mascot Distiller, 10 ppm precursor ion mass tolerance, 0.8 Da product ion mass tolerance, 3 missed cleavages, trypsin, carbamidomethyl cysteines and oxidized methionines as variable modifications, an ion score threshold of 20, and TMT-6-plex for quantification.