This note describes the characteristics and importance of protein phosphorylation, as well as the method of qualitative analysis of protein phosphorylation using HPLC-Chip electron transfer dissociation ion trap mass spectrometry technology. By comparing the differences between collision-induced dissociation (CID) and electron transfer dissociation (ETD) mass spectrometry techniques, it demonstrates the ability to analyze samples using both CID and ETD in the same LC/MS run. Examples of the identification of site-specific phosphorylation using an Agilent HPLC-Chip system are provided.
This document describes a method for characterizing protein phosphorylation using Electron Transfer Dissociation (ETD) on an Agilent 6340 Ion Trap mass spectrometer. It compares ETD with Collision-Induced Dissociation (CID), highlighting that ETD preserves phosphate groups while CID often causes their loss, making ETD superior for site-specific identification.
The note details the experimental setup involving an Agilent HPLC-Chip system interfaced with the 6340 MS. It presents results from analyzing synthetic phosphopeptides and mixtures, showing that ETD provides nearly complete sequence coverage and clear identification of phosphorylation sites, whereas CID yields limited fragmentation data.
| Loading flow rate | 4 pL/min |
|---|---|
| Flow rate | 600 nL/min |
| Injection volume | 100 nL |
| Drying gas flow | 4 L/min |
| Drying gas temperature | 325°C |
| Capillary voltage | -1800 V |
| ETD Temperature | 55°C |
| Emission current | 4.0 mA |
ETD is gentler than CID; the phosphate group remains attached to the amino acid during fragmentation, allowing specific location determination, whereas phosphate groups easily dissociate under CID.
The reaction time for the ETD process is set to 80 ms.