Hyphenation of capillary-LC with ICP-MS and parallel on-line micro fraction collection for MALDI-TOF-TOF analysis—complementary tools for protein phosphorylation analysis

Abstract

A new instrumental setup allowing the ‘‘quasi’’ on-line hyphenation of capillary liquid chromatography with elemental mass spectrometry e.g. inductively coupled plasma mass spectrometry (ICP-MS) and parallel micro fraction collection for the complementary application of matrix assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF-TOF-MS) has been developed. This approach allows the screening of peptide samples for functionalities such as phosphorylation sites associated with an ICP-MS detectable element e.g. phosphorus and their quantification before a further targeted characterization using MALDI-TOF-TOF-MS. The setup facilitates highly repeatable separations with retention time and peak area RSDs below 0.3% and 5% respectively, which are important for the reliable matching of the phosphorus specific ICP-MS data and the corresponding spot position on the MALDI target. The obtained quantitative data (104 3 pmol for the mono phosphorylated and 101 2 pmol for the tetra phosphorylated peptide), were in good agreement with the theoretical value of 98 pmol, calculated on the basis of the digested protein amount. The described approach has the potential to overcome some of the limitations (non-detectability of certain low abundant peptides, ionization suppression effects, co-eluting sample constituents, limitations in terms of data acquisition rates) related with the analysis of samples such as tryptic protein digests, which have been directly spotted on a MALDI target or which have been analysed via the frequently used on-line hyphenation of capillary or nano-LC to electrospray ionization based MS approaches (ESI-MS).