Ettan mdlc system

All of the mass analyses were performed using a nano-LC-MS/MS system, which consisted of a nano-HPLC system (the Ettan MDLC system; GE Healthcare, Piscataway, NJ) and a linear trap quadruple (LTQ) mass spectrometer (LTQ VELOS; Thermo Finnigan, San Jose, CA) equipped with a nano-ESI source. A RP trap column (Zorbax 300SB-C18 peptide traps, Agilent Technologies, Wilmington, DE) was used for desalting of samples, and a C18 reverse-phase column (150 μm i.d., 150 mm length, Column Technology Inc., Fremont, CA) was used for separation. Mobile phase A consisted of HPLC-grade water containing 0.1% formic acid (FA), and phase B consisted of 84% HPLC-grade acetonitrile (ACN) containing 0.1% FA. The analytical separation was run at a flow rate of 2 μl/min by using a linear gradient of phase B as follows: 4%-50% for 105 min, 50%-100% for 9 min and 100% for 6 min. The eluent was then introduced into the LTQ mass spectrometer with the ESI spray voltage set at 3.2 kV. For MS survey scans, each scan cycle consisted of one full MS scan, and five MS/MS events were analyzed. The LC-MS/MS analyses were repeated three times for each independent biological sample. Then the LC-MS/MS results were pooled for each biological replicates to reduce technical variation.

Stained electrophoresis gels loaded with Raji cells and EXO_Raji were excised into four parts and submitted for enzymatic digestion. Proteins were first enzymolyzed into peptides. Enzymatic digestion of proteins in the gels was performed as previously described [14 (link)]. Briefly, the Ettan™ MDLC system (GE Healthcare, Shanghai, China) was used to desalt and separate the tryptic peptides mixtures. In this system, samples are desalted on reversed phase (RP) trap columns (Agilent Technologies, Shanghai, China) and subsequently separated on RP columns (Column Technology Inc., Shanghai, China). Mobile phase A (0.1% formic acid in HPLC-grade water) and mobile phase B (0.1% formic acid in acetonitrile) were selected. The tryptic peptide mixture (20 μg) was loaded onto the column, and separation was performed at a flow rate of 2 μl/min with a linear gradient of 4–50% B for 120 min. A Finnigan LTQ™ linear ion trap MS (Thermo Electron, Shanghai, China) equipped with an electrospray interface was connected to the LC setup to detect eluted peptides. Data-dependent MS/MS spectra were obtained simultaneously. Each scan cycle consisted of one full MS scan in the profile mode followed by five MS/MS scans in the centroid mode with the following Dynamic Exclusion™ settings: repeat count: 2; repeat duration: 30 s; exclusion duration: 90 s. Each sample was analyzed in triplicate.