Development and characterisation of a new interface for coupling capillary LC with collision-cell ICP–MS and its application for phosphorylation profiling of tryptic protein digests
AbstractA comparison of different nebulisers for the direct hyphenation of capillary and nano-liquid chromatography (cap-LC, nano-LC) and quadrupole based collision cell inductively coupled plasma mass spectrometry (CC-ICP-MS) for phosphorylation profiling of tryptic protein digests is described. Helium has been used as cell gas and specially tuned instrumental conditions have been employed to achieve background minimisation at the mass of phosphorus due to kinetic energy discrimination of the interfering polyatomic ions.
The proposed setup is based on a modified capillary electrophoresis interface and a home made 4 mL spray chamber. It allows the untilisation of gradient conditions with highly concentrated organic mobile phase as often used in protein phosphorylation analysis, without the necessity to apply membrane desolvation for removal of the organic phase or further background minimisation. No significant signal suppression or other negative effects caused by the organic mobile phase appear as a result of the low flow rates used in capillary or nano-LC and the robust plasma conditions of the CC-ICP-MS instrument.
Tryptic digests of alpha and beta-casein were investigated as model compounds to demonstrate the applicability of the proposed setup for phosphorylation profiling in protein analysis using quadrupole based collision-cell ICP-MS as phosphorus specific detector. Detection limits for phosphorylated peptides down to the sub pico-mole level have been obtained. As a complementary technique, electrospray ionisation mass spectrometry (ESI-MS/MS) with data base searching has been applied to further characterise the phosphorylated peptides detected.