Peptides were redissolved in 20 µl 0.1% TFA and subjected to nano-liquid chromatography (nano-LC) tandem mass spectrometry (MS/MS). Peptides were delivered with a
FAMOS autosampler at 30 µl/min to a
Pepmap C18 trap column (5 mm × 300 µm i.d.; Dionex) and separated on an Alltima analytical capillary C18 column (150 mm × 100 µm i.d.) at 400 nl/min using the LCPacking Ultimate system. The peptides were separated using a linearly increasing concentration of acetonitrile from 5–35% in 80 min, from 35–45% in 7 min, from 45–90% in 2 min, and then held at 90% for an additional 11 min. The eluent was mixed with matrix (7 mg α-cyano-hydroxycinnaminic acid in 1 ml 70% acetonitrile, 0.1% trifluoroacetic acid, 10 mM ammonium monobasic phosphate) delivered at a flow rate of 1.5 µl/min and deposited offline to the Applied Biosystems metal target every 15 s for a total of 384 spots, using an automatic robot (
Probot; Dionex). A
5800 proteomics analyzer (Applied Biosystems) was used for peptide analysis. CID was performed at 2 kV (the collision gas was nitrogen). MS/MS spectra were collected from 2,500 laser shots. The peptides with signal to noise ratio above 50 at the MS mode were selected for the MS/MS experiment; a maximum of 25 MS/MS were allowed per spot. The precursor mass window was 200 relative resolution (FWHM).
Schreiber J., Végh M.J., Dawitz J., Kroon T., Loos M., Labonté D., Li K.W., Van Nierop P., Van Diepen M.T., De Zeeuw C.I., Kneussel M., Meredith R.M., Smit A.B, & Van Kesteren R.E. (2015). Ubiquitin ligase TRIM3 controls hippocampal plasticity and learning by regulating synaptic γ-actin levels. The Journal of Cell Biology, 211(3), 569-586.
