Ionic liquid matrices with phosphoric acid as matrix additive for the facilitated analysis of phosphopeptides by matrix-assisted laser desorption/ionization mass spectrometry.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a powerful tool for the analysis and characterization of protein phosphorylation on the peptide level. In this study, the applicability of ionic liquid matrices (ILM) formed by combination of the crystalline MALDI matrix 2,5-dihydroxybenzoic acid (DHB) with pyridine or n-butylamine was tested for the analysis of phosphopeptides. Low ionization efficiency in both positive and negative ion mode was observed in acid-free sample preparations. Upon addition of 0.1% trifluoroacetic acid (TFA), ion formation was increased, but analogously to the situation described earlier for pure DHB, best results were obtained upon use of 1% phosphoric acid as matrix additive. The samples prepared in this way were significantly more homogeneous than preparations with pure DHB, thus avoiding the need for time-consuming search for hot spots. Other characteristics like metastable fragmentation of phosphopeptides did not differ from that observed in classical preparations. The limits of detection for synthetic phosphopeptides and singly or multiply phosphorylated peptides from tryptic digests of alpha- and beta-casein were comparable with those obtained when using pure DHB; in some cases even higher signal intensities could be observed in the ILM. The use of ILM in combination with 1% phosphoric acid as matrix additive significantly facilitates analysis of phosphopeptides by MALDI-MS.