Selective detection and identification of phosphorylated proteins by simultaneous ligand-exchange fluorescence detection and mass spectrometry

Abstract

A ligand-exchange method for the detection and identification of phosphorylated peptides in complex mixtures is presented that is based on the characterization of phosphorylated species by solution-phase interactions with Fe(III) ions and subsequent fluorescence readout. After the separation of the peptides and digest products on a reversed-phase LC column, the flow is split between the two detection systems. One part is directed towards an electrospray mass spectrometer for direct detection and identification of all the peptides present in the sample. The other part of the flow is directed towards a ligand-exchange detection system. This system relies on the specific release of a fluorescent reporter ligand from a Fe(III)-complex in the presence of phosphorylated peptides. To recognize false positive signals due to high-affinity non-phosphorylated high-acidic peptides and other compounds which are known to be a problem in for instance immobilized metal affinity chromatography (IMAC), a second run is performed after incubation of the sample with alkaline phosphatase. A positive signal in this second run indicates a high-affinity non-phosphorylated compound. The method is illustrated using digest from a phosphorylated α-casein. Automated switching between MS and MS–MS was performed to obtain additional information about the compounds present in the sample. The linearity of the method was tested in the range of 0.5–80 μM of phosphorylated peptides. A limit of detection (LOD) of 0.5 μM was obtained for a mono-phosphorylated peptide. The interday ( n = 4) and intraday precision ( n = 3) expressed as relative standard deviation was better than 10%.