Sequencing grade modified porcine trypsin

Antibodies to Mcl-1 (sc-12756 and sc-20679) and Protein A/G PLUS-Agarose Beads (sc-2003) were from Santa Cruz Biotechnology (Dallas, TX); antibody to phospho-Bcl-xL (ab30655) and full length His-tagged human Mcl-1 protein (ab131682) were from Abcam (Cambridge, MA); antibodies to Bcl-xL (2762S), (phospho-histone H3 (9701S) and GAPDH (2118S) were from Cell Signaling Technology (Danvers, MA); purified active Cdk1/cyclin A2 was obtained from SignalChem (Richmond, BC, Canada); [γ-³²P]ATP (BLU002A250UC) was from PerkinElmer (Waltham, MA); lambda protein phosphatase (P0753S) was from New England BioLabs (Ipswich, MA); OmniCleave^™ endonuclease was from Epicentre (Madison, WI); tributylphosphine (T7567-10VL) and cycloheximide (C104450) were from Sigma-Aldrich (St. Louis, MO); Bio-Lyte 5/7 Ampholytes (163-1112) were from Bio-Rad (Hercules, CA); and porcine sequencing grade modified trypsin was from Promega (Madison, WI).

Proteins were digested in solution with porcine sequencing grade modified trypsin (catalogue number V5111, Promega) in 5 mM Tris-HCl pH 8.0 supplemented with 1 mM CaCl_2, for 16 h at 37°C using a substrate: enzyme ratio of 50∶1.

Proteins were eluted from beads by heating at 95 °C for 5 min in 100 μL 1x Laemmli buffer (Boston Bioproducts), followed by separation in a 4–12% Bis-Tris Deep Well gel and visualization by Coomassie staining. Each gel lane was then collected and cut into 12 equal-volume gel slices, which were subject to in-gel trypsin digestion as described before^{80 (link)}. In brief, gel segments were destained (in 50 mM ammonium bicarbonate buffer with 50% methanol) first, followed by reduction (in 10 mM TCEP), alkylation (in 50 mM iodoacetamide), dehydration (in acetonitrile) and finally digestion at 37 °C for 12–16 h in 50 mM ammonium bicarbonate buffer containing 100 ng porcine sequencing grade modified trypsin (Promega). Tryptic peptide products were then acidified (in 0.1% formic acid) and separated by an in-line 150 × 0.075 mm column loaded with reverse phase XSelect CSH C18 2.5 um resin (Waters) using a nanoAcquity UPLC system (Waters). Eluted peptides were ionized by electrospray (2.15 kV) and analyzed using an Orbitrap Fusion Tribrid mass spectrometer (Thermo), with MS data and MS/MS data acquired by the FTMS (profile mode; with resolution of 240,000 and range from 375 to 1500 m/z) and ion trap (centroid mode; with normal mass range and precursor mass-dependent normalized collision energy between 28.0 and 31.0) analyzer, respectively.

Mass spectrometry was performed as previously described [27 (link)] with minor modifications. Proteins from whole extracts of IPAM, BPAM and SAM from three individuals were precipitated using cold acetone (repeated until DTT scent was not detectable), then subjected to trypsin digest at 37°C overnight (1 µg of trypsin per 50 µg of extracted protein; modified porcine trypsin, sequencing grade from Promega). Tryptic peptides were analysed by reverse-phase HPLC-ESI-MS/MS using an Eksigent NanoLC 400 2D Ultra Plus HPLC system connected to a TripleTOF 6000 quadrupole time-of-flight mass spectrometer (AB Sciex, Concord, ON, Canada). After injection, peptide mixtures were transferred to a AB Sciex column (3C18-CL 75 µm × 15 cm) and eluted at a flow rate of 300 nl min⁻¹. MS data was acquired using the TripleTOF 6000 mass spectrometer fitted with a Nanospray III source (AB Sciex) using a pulled quartz tip as the emitter (New Objectives, MA, USA).

Chromatography grade acetonitrile from Merck KGaA was used. Modified porcine trypsin (sequencing grade) was obtained from Promega (Madison, WI). The iTRAQ 4-plex reagent kits were purchased from Applied Biosystems (Framingham, MA). Desalting spin columns were purchased from Pierce (Thermo Scientific, San Jose, CA). The column packing materials for the analytical column were purchased from MACHEREY-NAGEL (Düren, Germany). The packing materials for the trap column were purchased from Michrom Bioresources (Auburn, CA). All other chemicals and reagents were of analytical grade and purchased from Sigma-Aldrich (St. Louis, MO), unless otherwise stated.