Proteomics :On-bead/in-solution digestion and phosphopeptide enrichment

Protein digestion of GST samples was performed following published protocols

Digestion buffer (50 mM NH₄HCO3) was added to

the sample, followed by treatment with 1 mM dithiothreitol (DTT, Thermo

Scientific Cat#R0861) at room temperature for 30 min. Subsequently, 5 mM

iodoacetamide (IAA, Sigma Cat#I1149) was added to the mixture and incubated at

room temperature for 30 min in the dark.

Proteins were digested with 2 µg of lysyl endopeptidase (Wako, Cat# 125-05061) at room temperature (overnight).

Further digestion was performed with 2 µg trypsin (Promega, Cat#VA9000) at room temperature

(overnight). In contrast, for phosphopeptide enrichment, proteins were digested

overnight at room temperature with 1 µg of Asp-N (Promega, Cat# VA1160) for samples in solution or 5 µg of Asp-N for the samples

on-beads.

The clean peptides were concentrated using a high-select Fe-NTA

phosphopeptide enrichment kit (Thermo Scientific Cat#A32992) All resulting

peptides were desalted and dried using an HLB column (Waters, Cat#186002034)

and vacuum.