96 well low protein binding deep well plate

Using a Dionex Ultimate 3000 Bio-RS UHPLC system (Thermo Scientific), lysates were injected (100 μl per injection) onto a BioBasic SEC1000 column (300 × 7.8 mm, 5 μm particles, 1000Å pores) equilibrated with 0.2% SDS, 100 mm NaCl and 10 mm NaPO₄ pH 6.0 at 30 °C. A buffer at pH 6.0 is used to prolong column lifetime. The flow rate was 0.2 ml min⁻¹ and for each sample two injections were performed. For each injection 48 × 125 μl fractions were collected separately using a 96-well thin-walled PCR plate (Eppendorf) and heated to 95 °C for 30 min in a PCR machine, using a heated lid, to break all crosslinks. After heat reversal of crosslinks, samples were transferred to a 96-well low protein binding deep-well plate (Eppendorf) and Tris-HCl (1 m pH 8.0) was added to each fraction to a final concentration of 0.1 m to adjust the pH to 8.0. Proteins in each fraction were digested to peptides using either LysC alone (injection 1), or LysC and trypsin (injection 2), which were diluted in 0.1 m Tris-HCl and added at a ratio of 1:50 by weight, based upon an EZQ protein assay of the fractions, then incubated for 18 h at 37 °C.

SDS in peptide samples was removed using 96-well detergent removal plates (Thermo Scientific) and centrifugation according to manufacturer's instructions. Briefly, the resin in each well was washed three times with 300 μl of room temperature PBS, with centrifugation at 1000 × g to remove the solution after each wash. Peptide samples were applied to the resin in each well and incubated for 2 min at room temperature before collecting the filtrate (containing clean peptides) by centrifugation for 2 min at 1,000 g at room temperature into a 96-well low protein binding deepwell plate (Eppendorf). Peptides were then desalted after trifluoroacetic acid (TFA) was added to 1% (v/v) final concentration and peptides were purified using a Sep-Pak tC18 96-well u-elution plate (Waters). Peptides were eluted in 200 μl of 50% (v/v) acetonitrile 0.1% TFA and evaporated to dryness in a rotary evaporator prior to re-suspension in 5% (v/v) formic acid. Peptide concentrations were determined using the CBQCA assay (Thermo Scientific) and peptide standards derived from a BSA digest, after 25-fold dilution of peptide samples in 0.1 m borate buffer pH 9.3.