Surveyor autosampler

The digestate obtained from the SDS-PAGE gel slices were analyzed by nanoflow liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) using an LTQ-XL ion-trap mass spectrometer (Thermo, Fremont, CA, USA). Reversed phase columns were packed in-house to approximately 7 cm (100 μmi.d.) using 100 Å, 5 μM Zorbax C18 resin (Agilent Technologies, Santa Clara, CA, USA) in a fused silica capillary with an integrated electrospray tip. A 1.8 kV electrospray voltage was applied via a liquid junction upstream of the C18 column. Samples were then injected into the C18 column using a Surveyor autosampler (Thermo, Fremont, CA, USA). The column was washed with buffer A [5% (v/v) ACN, 0.1% (v/v) formic acid] for 10 min at 1 μL/min before each loading. Peptides were subsequently eluted from the C18 column with 0–50% Buffer B [95% (v/v) ACN, 0.1% (v/v) formic acid] over 58 min at 500 nL per min followed by 50–95% Buffer B over 5 min at 500 nL per min. The column eluate was then directed into a nanospray ionization source of the mass spectrometer. Spectra were scanned over the range 400–1500 amu. Automated peak recognition, dynamic exclusion window set to 90s38 (link) and tandem MS of the top six most intense precursor ions at 35% normalization collision energy were performed using Xcalibur™ software (version 2.06) (Thermo, Fremont, CA, USA).

As previously described [65 ], peptides were analyzed by nanoLC-MS/MS using a LTQ-XL ion-trap mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). Reverse phase columns were packed in-house to approximately 7 cm (100 μm i.d.) using 100 Å, 5 μm Zorbax C18 resin (Agilent Technologies, Santa Clara, CA, USA) in a fused silica capillary with an integrated electrospray tip. A 1.8 kV electrospray voltage was applied via a liquid junction up-stream of the C18 column. Samples were injected onto the C18 column using a Surveyor autosampler (Thermo Fisher Scientific), followed by an initial wash with buffer A (5% (v/v) ACN, 0.1% (v/v) formic acid) for 10 min at 1 μL/min. Peptides were eluted from the C18 column with 0%-50% Buffer B (95% (v/v) ACN, 0.1% (v/v) formic acid) over 58 min at 500 nL/min followed by 50–95% Buffer B over 5 min at 500 nL/min. The eluted solution was directed into the nanospray ionization source of the mass spectrometer. Spectra were scanned over the range 400–1500 amu, with automated peak recognition, a dynamic exclusion window set to 90 s and tandem MS of the top 6 most intense precursor ions at 35% normalization collision energy using Xcalibur software (version 2.06; Thermo Fisher Scientific).

Sample separation was carried out using a Surveyor autosampler and an MSPumpPlus (Thermo Fisher Scientific, Runcorn, UK) LC system on a reverse phase Thermo Fisher Scientific Hypersil Gold LC column (1.0 × 100 mm, 5 μm) operated with a Hypersil guard cartridge, suitable for general separations. Mass spectrometry detection was performed using an LCQ ion trap (Thermo Fisher Scientific, Hemel Hempstead, UK) operated using an ESI source in positive mode. Instrument control and data analysis were carried out using Xcalibur 2.0.7 software with data processed using both the Quan Browser Xcalibur tool and Microsoft Excel.