Milk Protein Identification by SDS-PAGE and MS

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.