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.
A comparison of different nebulisers for 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 was used as cell gas and specially tuned instrumental conditions were used to achieve background minimisation at the mass of phosphorus, because of kinetic energy discrimination of the interfering polyatomic ions. The proposed set-up is based on a modified capillary electrophoresis interface and a home-made 4 mL spray chamber. It enables the use of gradient conditions with a highly concentrated organic mobile phase as often used in protein phosphorylation analysis, without the need 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 occur, because of the low flow rates used in Cap-LC and the robust plasma conditions of the CC-ICP-MS instrument. A tryptic digest of beta-casein was investigated as model compound to demonstrate the applicability of the proposed set-up 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 picomole level were obtained. As a complementary technique, electrospray ionisation tandem mass spectrometry (ESI-MS-MS) with data base searching was used for further characterisation of the phosphorylated peptides detected.
Pröfrock
Daniel
D
GKSS Research Centre Geesthacht, Institute for Coastal Research, Marine Bioanalytical Chemistry, Max Planck Street 1, 21502 Geesthacht, Germany.
Leonhard
Peter
P
Ruck
Wolfgang
W
Prange
Andreas
A
D002853:Chromatography, Liquid / Q000295:instrumentation* / Q000379:methods; D013058:Mass Spectrometry / Q000295:instrumentation* / Q000379:methods; D010449:Peptide Mapping; D010766:Phosphorylation; D011506:Proteins / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D014357:Trypsin / Q000737:chemistry*
D011506:Proteins; D014357:Trypsin