Characterisation of tryptic peptides of phosphorylated tyrosine hydroxylase by high-pressure liquid chromatography electrospray ionisation mass spectrometry

Tyrosine hydroxylase (TH) is involved in the biosynthesis of catecholamines and is activated by phosphorylation. Phosphorylated TH was analysed using high-pressure liquid chromatography combined with electrospray mass spectrometry (HPLC ESI-MS). Two mass scanning methods were used to detect tryptic cleavage products of TH. In the positive electrospray ionisation mode (ESI+), the peptides that contain the phosphorylation sites of TH were identified. In the alternative method, a phosphopeptide was detected in the negative electrospray ionisation mode (ESI−) using single ion monitoring in combination with a sequential ESI+ switching experiment. A raised baseline interfered with detection of hydrophilic peptides in ESI−, with the signal-to-noise ratio indicating that the method was operating near the limit of detection for a conventional electrospray source. The switching method improved the certainty of identification of phosphopeptides.