Automated Peptide Sample Preparation for LC-MRM analysis

Sample preparation for LC-MRM including tryptic digestion and peptide cleanup were automated using Assay Map bravo platform from Agilent technologies with VWorks Automation Control 11.1 software⁵¹. A total of 68 samples (Control_TF (17), DED_TF (17), Control_LF (17), DED_LF (17)) were digested reconstituted with a 8 M urea in 50 mM ABC buffer (pH 8.0). Protein was reduced with 10 mM DTT for 30 min at 37 °C and was alkylated by blocking the cysteine residues using 40 mM IAA at RT for 1 h in dark. Urea concentration was reduced to below 1 M using 50 mM ABC buffer. The proteins were digested using sequence graded trypsin at 50:1 ratio for protein: trypsin at 37 °C overnight. Each sample in 96 well plate was acidified by the addition of 10% TFA and the pH was reduced to 2–3 before desalting. The acidified digests was immediately processed through the Peptide Cleanup Protocol using Sep-Pak C18 96-well plate (100 mg Sorbent per well, Waters). The eluted peptides with 80% acetonitrile (ACN) were dried by speed vaccum.