Proteomic analysis of ubiqutiin binding proteins

2.1 Laboratory component 2.1.1 Materials used Coupling solution: • 100mM NaHCO3 • 0.5M NaCl • pH8.3 Blocking solution: • 1M ethanolamine • pH8 Column buffer • 10mM Tris • 150mM NaCl • 0.01% sodium azide • pH7.5 Sample buffer • 1% v/v Triton X100 detergent • 50mM Tris HCl 2.1.2 Conjugation of bait to sepharose beads 5ml of coupling solution was run through each sepharose bead column, containing 0.3ml of beads each. A 5ml solution of coupling buffer with an additional 0.6mg/ml ubiquitin was mixed with one column for a period of two hours, the control contained only coupling solution. Each column was then rinsed with blocking solution and stored in the blocking solution at -20oC. Upon thawing columns were given 3 cycles of 0.1M sodium acetate (pH4) / coupling buffer rinses to remove residual ethanolamide, the columns were stored in column buffer at 4oC and a small aliquot was taken to confirm ubiquitin binding by boiling followed by SDS PAGE analysis (standard protocols). 2.1.3 Tissue preparation and crude cell extract analysis. ~16g of porcine brain tissue was hand dissected, then processed in ~4g batches, each batch was placed in 20ml ice cold sample buffer with * protease inhibitor cocktail. The solutions were homogenised using both a an electric and dounce homogeniser, followed by centrifugation at 20,000rpm for 30 minutes at 4oC. The sample supernatants were then filtered through glass wool and a 10µl aliquot was taken for SDS PAGE analysis (12.5% acrylamide gels, by standard protocols). Sample solutions were stored at -80oC prior to Ub-sepharose analysis. 2.2 Affinity purification of UBPs In addition to the two prepared Ub and control columns, two extra columns were provided by demonstrators, one containing the quadruple ubiquitin mutant at the same protein:bead concentration (10mg/ml). Columns were run dry and rinsed with sample buffer to equilibrate the columns. 20ml of cell extract was then run through each column and the flow through recycled three times. Each column was then washed with sample buffer five times in order to remove unbound proteins, and then allowed to run dry. For the Ub-sepharose and a control, a 900µl of sample buffer was used to re-suspend the beads, and 200µl aliquots were taken (~50µl of beads in each), which were sent off for external LC MS.MS analysis.* 2.2 Data analysis 2.2.1 Mass spectroscopy of SDS PAGE bands Mass spectroscopy data were analysed using Mascot, a peptide mass fingerprint (PMF) analysis service available from ExPASy (Expert Protein Analysis System). Bands from between the 1-2kD range were inputted, with the following parameters; taxonomy-human, enzyme-trypsin, fixed modification-carboxymethyl, peptide tolerance 1Da and mass values – MH+. Returned results were then searched firstly for those with known Ub interactions, and then by known mass and checked against approximate mass estimated from the SDS PAGE gel. 2.2.2 Tandem mass spectroscopy By refining the mascot search a data set was formed containing all of the control hits, all hits containing unique peptide peaks (+RBR) and the top 300 results from the default algorithm which does not require unique peptides(-RBR). Control hits were removed from the dataset, in addition duplicates of +RBR found in the less stringent –RBR set. Likely contaminants containing the key words: ubiquitin, keratin, globin, DEAD, myosin and ribosomal were removed, in accordance with the non-specific binding partners identified by Trinkly-Mulcahy et al27 A batch NCBI conserved domain database (CDD) search of all protein hits, using default parameters, returned a further data set linking proteins to domain hits, with descriptions of each domain. These domains were then manually assigned to five categories, one domain was allowed to appear in multiple categories: nuclear, proteasomal, trafficking, signalling and “known ubiquitin binding”. A table was produced for each category to show the proteins assigned to that group and the entire dataset was uploaded to figshare.com to make it available for meta-analysis.