Protein Extraction

Plasma Membrane Protein Enrichment (IFN Screens)

PM profiling was used to enrich PM proteins in THP-1s and primary leukocytes, prior to analysis by mass spectrometry (MS) (data in chapter 3); this was performed as previously described (Weekes et al, 2010, 2012). All spins were at 4 ºC, and PBS containing calcium and magnesium (D8662, Sigma) was used for the biotinylation and enrichment; all reagents were kept ice cold.

The suspension cells were transferred to a 15 ml falcon tube and centrifuged to collect (400 g, 5 mins), then washed twice in cold PBS. Sialic acid residues were oxidised with sodium meta-periodate and biotinylated with aminooxy-biotin, with the reaction catalysed by Aniline. This was performed by removing the supernatant from the washed cells, wrapping the falcon tubes in foil and adding 3 ml of biotinylation mix to each (see below). The falcon tubes were rocked at for 30 mins at 4 ºC in the dark. The reaction was then quenched with 600 µl 5x glycerol solution (1M glycerol (Sigma) diluted 1:2000 in PBS), mixed by inversion and centrifuged (400g, 5 mins). The supernatant was removed and the cells washed twice in PBS, before being resuspended in 1 ml 1 % triton lysis buffer (see below) and transferred to an Eppendorf. Lysis proceeded on ice for 30 mins, and then the tube was centrifuged at 13,000 g for 5 mins and the supernatant transferred. Two further spins were performed, retaining the supernatant with a minimal amount of debris. The final supernatant was snap frozen in liquid nitrogen prior to pull- down and digestion.

Once multiple samples of biotinylated glycoproteins had been collected, they were enriched with high affinity streptavidin agarose beads (Thermo). The following method is described for 10 samples, and volumes were adjusted according to the number of samples. Streptavidin agarose beads were resuspended and 500 µl and added to a Poly- Prep column (Bio-Rad) on a vacuum manifold. The beads were washed 4 times with 800 µl cold 1 % triton lysis buffer, before resuspending in 1200 µl lysis buffer. 95 µl beads were added to each lysate, and they were incubated on a rotor for 75 mins at 4 ºC. Each sample was resuspended by pipetting several times and then transferred to an individual polyprep column on the vacuum manifold. Several rounds of washing were then

performed. In each case, the required volume was added to each polyprep column using a repeat pipettor, the wash reagent removed under vacuum and then this was repeated the required number of times. Cells were first washed with 4x 1 ml lysis buffer, and then 4x 1 ml 0.5 % SDS in PBS (diluted from 20 % SDS, Invitrogen) before incubation for 20 mins in 500 µl 0.5 % SDS/PBS/DTT (add 550 µl 1M DTT (Sigma) to 5ml 0.5 % SDS/PBS) in order to denature and reduce proteins. The samples were subsequently washed with 8x 1.5 ml urea solution (see below), once with 10 ml urea solution, and then alkylated by incubating for 20 mins in the dark with 500 µl urea solution /iodoacetamide (IAA) (5ml urea solution with 500 µl 500 mM IAA dissolved in urea solution). A further 2x 1.5 ml washes were performed with urea solution, 3x 1.5 ml washes with ultra pure H2O (JT Baker), and finally one 10 ml wash with H2O. The samples were transferred

in 2x 300 µl H2O into a screw cap column (Pierce). The column was centrifuged to

remove the H2O at 2000 g for 1 min, and 40 µl trypsin / HEPES solution (one vial of

Promega trypsin dissolved in 1 ml 200 mM hepes pH 8.5) added for on-bead digestion. After 3 h in a 37 ºC shaker, spin columns were placed in fresh eppendorfs and centrifuged at 2000 g for 1 min. The tryptic peptides were stored at -80 ºC prior to TMT labelling.

Reagents for PMP:

Biotinylation mix - for two samples: 25-40 mg sodium periodate (Thermo) was dissolved in ice-cold PBS pH 6.7 (weight in mg x 0.47 ml, to make 10 mM solution), keeping on ice and agitating periodically. In a foil covered tube 6 ml PBS at pH 6.7, 6.7 µl aminooxy- biotin (Biotium) and 6.1 µl aniline (Acros Organics) were combined, and mixed immediately. 660 µl of the sodium periodate was then added to this.

500 mM IAA – The weight of IAA (Sigma) in mg was multiplied by 10.8 µl and the IAA was diluted in this volume of lysis buffer, urea solution or HEPES as indicated.

1 % Triton Lysis Buffer - For 50 ml lysis buffer, 500 µl 1M Tris-HCL (Sigma), 25 ml 2 % Triton (Thermo, one vial 10 % Trition X-100 added to 40 ml ultrapure H2O), 1.5 ml

5M NaCL (Sigma) and 23 ml ultra pure H2O were combined. 100 µl 500 mM IAA

Urea Solution – 90 g urea was dissolved in 25 ml 1M Tris-HCL (Sigma) and 156.5 ml ultra pure H2O (JT Baker).

Whole Cell Protein Extraction (VACV Screens)

For the proteomic studies of VACV infection (chapter 4), WCL protein extractions were performed by Dr Jonas Albarnaz. Briefly, cells were washed twice with PBS and then scraped in 250 µl guanidine lysis buffer (200mM HEPES pH 8.4 is produced from a 1M HEPES stock (Sigma), and 3 ml of this is added to 9 ml 8 M guanidine-HCL (Pierce), to make 6M guanidine / 50 mM HEPES pH 8.5 lysis buffer). Cells were collected in eppendorfs, centrifuged briefly and sonicated prior to centrifugation at 21,000 g for 10 mins to remove debris. The supernatant was transferred, centrifuged again and the second supernatant snap frozen in liquid nitrogen. These samples were then given to me for further processing.

A portion of the lysate (50 µl) was digested for proteomic analysis, and the rest retained for immunoblot analysis where required. Proteins were reduced and then alkylated by incubating for 20 mins with 2.5 µl 100 mM DTT and then for an additional 20 mins in the dark with 1.5 µl 10x IAA (dissolved in 200 mM HEPES). Excess IAA was quenched by incubating for 15 mins with a further 2.5 µl 100 mM DTT and samples were diluted in 143.5 µl 200 mM HEPES to achieve a final concertation of 1.5 M guanidine. Samples were then digested in 2 µl endoproteinase LysC (Wako) for 3 h at room temperature. They were then further diluted in 398 µl HEPES to a final concentration of 0.5 M guanidine, prior to adding 50 µl trypsin (Pierce), and incubating in a 37 ºC shaker overnight. The next day, the reaction was quenched with 65 µl 50 % formic acid (FA, Sigma, diluted to 50 % in H2O) to achieve a concentration of 5 % FA, and centrifuged at

21,000 g for 10 mins to remove undigested protein.

Peptides were then subjected to C18 solid-phase extraction (Sep-Pak, Waters). A 50 mg SepPak was activated with 2x 1 ml 100 % acetonitrile (AcN, Merck) followed by 1 ml 70 % AcN / 1 % FA. The column was washed with 3x 1 ml 1 % FA. Samples were diluted with 300 µl 1 % FA, loaded onto the SepPak and allowed to pass through slowly. The SepPak was washed again with 3x 1 ml 1 % FA prior to elution of the peptides in 2x 350 µl 70 % AcN / 1 % FA. Samples were dried completely by vacuum centrifugation in a

speedvac and resuspended in 75 µl 200 mM HEPES pH 8.5. The amount of peptides was quantified using a micro BCA protein assay kit (Pierce) following the manufacturer’s instructions.