This application note describes the use of an Ion Trap LC/MS/MS intelligent database search to identify phosphorylation sites in peptides resulting from proteolytic digestion of a phosphorylated protein
This document describes an application using automated tandem mass spectrometry coupled to online capillary liquid chromatography to identify post-translational modifications, specifically phosphorylation sites in proteins. The technique utilizes enzymatic digestion, capillary LC separation, and data-dependent scan functions to select parent ions for collision-induced dissociation (CID).
| Column | Zorbax 300SB-C18, 150 x 0.3 mm, 5 µm |
|---|---|
| Flow rate | 5.5 µl/min |
| Mobile phase A | 0.1% formic acid in water |
| Mobile phase B | 0.1% formic acid in acetonitrile |
| Ionization mode | Positive ESI |
| Nebulizer pressure | 15 psig |
| Automatic MS/MS | Number of parents: 2 |
| Isolation width | 3 m/z |
| Fragmentation amplitude | 1.0 V |
The Mascot database search program was used to search the uninterpreted MS/MS spectra.
Beta-casein was digested with trypsin in ammonium bicarbonate buffer.
Search parameters were set to consider possible phosphorylation at all serine, threonine, or tyrosine residues.