Nano trap column

The LC-MS/MS analysis was performed on an EASY-nLC™ 1000 (Thermo Fisher Scientific) coupled to a Q Exactive™ HF (Thermo Fisher Scientific). The loading of the samples was performed on Nano Trap Column (Thermo Fisher Scientific). The compositions of the mobile phase A and B were 0.1% formic acid (A), 0.1% formic acid and 0.1% acetonitrile (B); 5 μl of the sample was injected. The sample was taken into the analytical column for separation through a 60-min chromatographic gradient. The column was equilibrated for 5 min and the column temperature was set to 35 °C. After flowing out of the chromatographic column, samples passed through the Nano Flex Ion Source under the spray voltage of 2.4 kV. Then the charged sample is sprayed into the mass spectrometer for detection, and the mass range was set from m/z 300 to 1800. Alignment of the spectra, peak picking and further analysis were processed using the Proteome Discoverer software (Thermo Fisher Scientific).

Mass Spectrometry of purified recombinant and native proteins was carried out to reveal their true identity. The bands corresponding to each protein were cut out and processed by in- gel tryptic digestion. The digested peptides were reconstituted in 0.1% formic acid in LC-MS grade water and subjected to nano-LC (Nano Advance, Bruker, Germany) followed by identification by captive spray-Maxis-HD qTOF (Bruker, Germany) mass spectrometer (MS) with high mass accuracy and sensitivity. The peptides were enriched in nano trap column (Thermo Scientific) and eluted on to analytical column (Agilent) using a linear gradient of 5–45% acetonitrile at 400 nL/min over 65 min. Positive ions were generated by electro spray and the qTOF operated in data dependent acquisition mode. Data analysis was performed using MS program Mascot (2.4.1 Matrix Science, UK).