Amazon ion trap ms ms

Peptide samples prepared by both the gel-free and gel-based methods were analysed by nLC-ESI-MS/MS. The peptides were solubilised in 20 μl 2% acetonitrile with 0.1% trifluoroacetic acid and separated on a nanoflow uHPLC system (Thermo RSLCnano) before online analysis by electrospray ionisation (ESI) MS on an Amazon ion trap MS/MS (Bruker Daltonics). Peptide separation was performed on a Pepmap C18 reversed phase column (Thermo Scientific™ Acclaim™ PepMap™ 100 C18 LC Column, 3 μm particle size, 75 μm ID, 150 mm length), desalted and concentrated for 4 min on a C18 trap column followed by an acetonitrile gradient (in 0.1% [v/v] formic acid) (3.2 to 32% [v/v] for 4 to 27 min, 32 to 80% [v/v] for 27 to 36 min, held at 80% [v/v] for 36 to 41 min and re-equilibrated at 3.2%) for a total time of 45 min. A fixed solvent flow rate of 0.3 μl/min was used for the analytical column. The trap column solvent flow rate was fixed at 25 μl/min, using 2% acetonitrile with 0.1% (v/v) trifluoroacetic acid. MS analysis was performed using a continuous duty cycle of survey MS scan followed by up to ten MS/MS analyses of the most abundant peptides, choosing the most intense multipli-charged ions with dynamic exclusion for 120 s.

Prior to gel electrophoresis, protein concentration was determined using the Bradford protein assay with bovine serum albumin as standard (BSA; Sigma-Aldrich, USA). Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on 4–15% gradient polyacrylamide gels in a Criterion electrophoresis system (BioRad Ltd, Hemel Hempstead, UK) as previously described.^{33 (link)} Samples of milk taken at each time point were separated by SDS-PAGE. The identity of protein in the SDS-PAGE bands was determined in a reference gel by analysis of milk from a healthy cow and a cow with mastitis run under the identical conditions, followed by LC-MS/MS. Protein bands were excised and processed^{33 (link)} prior to analysis at Glasgow Polyomics on a nanoflow uHPLC system (Thermo RSLCnano) and electrospray ionisation (ESI) mass spectrometry (MS) on an Amazon ion trap MS/MS (Bruker Daltonics). MS data were processed using Data Analysis software (Bruker) and the automated Matrix Science Mascot Daemon server (v2.1.06). Protein identifications were assigned using the Mascot search engine to interrogate protein sequences in the NCBI databases restricting the search to Bos taurus proteins.