Massprep robotic protein handling system

Protein bands were excised manually from Coomassie- or silver-stained gels and in-gel digested using a MassPREP robotic protein-handling system (Waters, Millford, MA, USA). Gel pieces were destained twice with 100 μl 50 mM ammonium bicarbonate (Ambic) containing 50% acetonitrile at 40°C for 10 min. Proteins were then reduced with 10 mM DTT in 100 mM Ambic for 30 min at 40°C and alkylated with 55 mM iodoacetamide in 100 mM Ambic for 20 min at 40°C followed by digestion with 0.3 μg trypsin (sequence grade, Promega, Madison, WI) in 50 mM Ambic for 5 h at 40°C. The tryptic peptides were extracted with 1% formic acid in 2% acetonitrile, followed by 50% acetonitrile twice. The liquid was evaporated to dryness and the peptides were separated on an EASY-spray column connected to an EASY-nLC 1000 system (Thermo Scientific). The peptides were eluted in a 60 min gradient (from 5–26% of buffer B (2% acetonitrile, 0.1% formic acid) in 55 min and up to 95% of buffer B in 5 min) at a flow rate of 300 nL/min and analysed on a Fusion Orbitrap mass spectrometer (Thermo Scientific). The spectra were analysed using the Mascot search engine v.2.5.1 (Matrix Science Ltd., UK).

Protein bands were excised manually from a bis-tris 4%–12% gel, using western blotting as a location reference, and in-gel digested using a MassPREP robotic protein-handling system (Waters). Gel pieces were destained twice with 100 μl 50 mM ammonium bicarbonate (Ambic) containing 50% acetonitrile at 40°C for 10 min. Proteins were reduced with 10 mM DTT in 100 mM Ambic for 30 min at 40°C and alkylated with 55 mM iodoacetamide in 100 mM Ambic for 20 min at 40°C followed by in-gel digestion with 0.3 μg Trypsin (Sequence grade, Promega) in 50 mM Ambic for 5 h at 40°C. The tryptic peptides were extracted with 1% formic acid in 2% acetonitrile, followed by 50% acetonitrile twice. The liquid was evaporated to dryness and the peptides were separated on an EASY-spray column connected to an EASY-nLC 1000 system (Thermo Scientific). The peptides were eluted in a 60 min gradient (from 5% to 26% of buffer B (2% acetonitrile, 0.1% formic acid) in 55 min and up to 95% of buffer B in 5 min) at a flow rate of 300 nL/min and analyzed on a Fusion Orbitrap mass spectrometer (Thermo Scientific). The spectra were analyzed using the Mascot search engine v.2.4 (Matrix Science Limited).

Protein bands of interest were excised from SDS-PAGE gels and tryptically digested using the manufacturer's recommended protocol on the MassPrep robotic protein handling system (Waters). The extracted peptides from each sample were analyzed by means of nanoLC-ESI-MS/MS using the NanoAcquity/Q-ToFUltima Global instrumentation (Waters) using a 45-min LC gradient. All MS data were corrected for mass drift using reference data collected from the [Glu¹]-Fibrinopeptide B (human—F3261 Sigma) sampled each minute of data collection. The data were then used to interrogate a database made up of the predicted protein sequences from RLP1 or RPP1 appended with the common Repository of Adventitious Proteins sequences (http://www.thegpm.org/cRAP/index.html) using ProteinLynx Global Server v2.3. All protein identification was carried out in the in-house Biological Mass Spectrometry and Proteomics Facility of the School of Life Sciences at the University of Warwick.