Surveyor ms pump plus hplc system

lncRNA CBR3-AS1 interacted proteins performed LC–MS/MS analyzed by Thermo Finnigan LTQ linear ion trap mass spectrometer (Thermo Fisher Corporation, San Jose, CA) in line with a Thermo Finnigan Surveyor MS Pump Plus HPLC system. The mass spectrometry analysis was carried out in a data-dependent mode with an automatic switch between a full MS and an MS/MS scan in the obitrap. Peptide sequences were searched using trypsin specificity and allowing a maximum of two missed cleavages. Sequest was searched with a peptide tolerance of 3.0 Da and a fragment ion tolerance of 1.0 Da. The results of peptide sequences information of CBR3-AS1 interact proteins were offered in Additional file 3: Table S2.

The standard LC-MS/MS and database searching were performed according to the protocol described previously (Sun et al., 2015 (link)). Briefly, LC-MS analysis was carried out using a Surveyor MS Pump Plus HPLC system coupled to a Thermo Fisher Finnigan LTQ linear ion trap mass spectrometer (Thermo Fisher Corporation, San Jose, CA, USA) using nano-electrospray ionization. Tryptic peptides were loaded onto a trap column (300SB-C18, 5 × 0.3 mm, 5 μm particle size; Agilent Technologies, Santa Clara, CA, USA) connected through a zero dead volume union to the self-packed analytical column (C18, 100 × 0.1 mm, 3 μm particle size; SunChrom, Germany). The peptides were then separated by linear gradient elution involving 0–45% B over 55 min followed by 45–100% B over 10 min (B is 80% acetonitrile, 0.1% formic acid) at a flow rate of 500 nL/min. MS data were analyzed using SEQUEST against the National Center for Biotechnology Information (NCBI) human protein database and the results filtered, sorted, and displayed using Bioworks 3.2. Returned protein lists were filtered using the parameters: Peptide Xcorr value >1.90 (for +1 charge), >2.75 (for +2 charge), >3.75 (for +3 charge); peptide Delt CN >0.1; protein probabilities <0.001. At least two unique peptides were required for each identified protein.