Ultramicrospin c18 column

Peptide samples were subjected to ubiquitin remnant immunoaffinity. Ten microliters of PTMScan® Ubiquitin Remnant Motif (K-ε-GG) Antibody Bead Conjugate purification (Cell Signaling Technology) slurry was used per 1 mg peptide sample. Ubiquitin remnant beads were washed twice with IAP buffer, then split into individual 1.7 ml low bind tubes (Eppendorf) for binding with peptides. Peptides were dried with a centrifugal evaporator for 12 h to remove TFA in the elution. The lyophilized peptides were resuspended in 1 ml of IAP buffer (50 mM 4- morpholinepropnesulfonic acid, 10 mM disodium hydrogen phosphate, 50 mM sodium chloride, pH 7.5). Peptides were sonicated and centrifuged for 5 min at 16,100 × g. The soluble peptide supernatant was incubated with the beads at 4°C for 90 min with rotation. Unbound peptides were separated from the beads after centrifugation at 700 × g for 60 s. Beads containing peptides with diGly remnants were washed twice with 500 µl of IAP buffer, then washed twice with 500 µl of water, with a 700 × g 60-s centrifugation to allow the collection of each wash step. Peptides were eluted twice with 60 µl of 0.15% TFA. diGly remnant peptides were desalted with UltraMicroSpin C18 column (The Nest Group). Desalted peptides were dried with a centrifugal adaptor and stored at −20°C until analysis by liquid chromatography and mass spectrometry.

Peptide samples were subjected to ubiquitin remnant immunoaffinity. 10 uL of PTMScan^® Ubiquitin Remnant Motif (K-ε-GG) Antibody Bead Conjugate purification (Cell Signaling) slurry was used per 1 mg peptide sample. Ubiquitin remnant beads were washed twice with IAP buffer, then split into individual 1.7 mL low bind tubes (Eppendorf) for binding with peptides. Peptides were dried with a centrifugal evaporator for 12 hours to remove TFA in the elution. The lyophilized peptides were resuspended in 1 ml of IAP buffer (50 mM 4- morpholinepropnesulfonic acid, 10 mM disodium hydrogen phosphate, 50 mM sodium chloride, pH 7.5). Peptides were sonicated and centrifuged for 5 minutes at 16,100g. The soluble peptide supernatant was incubated with the beads at 4°C for 90 minutes with rotation. Unbound peptides were separated from the beads after centrifugation at 700g for 60 seconds. Beads containing peptides with di-glycine remnants were washed twice with 500 μL of IAP buffer, then washed twice with 500 μL of water, with a 700g 60s centrifugation to allow the collection of each wash step. Peptides were eluted twice with 60 μL of 0.15% TFA. Di-glycine remnant peptides were desalted with UltraMicroSpin C18 column (The Nest Group). Desalted peptides were dried with a centrifugal adaptor and stored at −20°C until analysis by liquid chromatograph and mass spectrometry.