Enhanced detection and desalting free protocol for phosphopeptides eluted from immobilized Fe (III) affinity chromatography in direct MALDI TOF analysis

IMAC strategy is widely used in phosphopeptide enrichment, but most of the current eluents contain large amount of salt, which must be discarded before MS detection. Here, we present techniques to elute phosphopeptides with low ionization efficiency reagents, which could be left in the eluate for direct MS analysis, thus saving desalting and the following steps. Several reagents were studied, including 5-sulfosalicylic acid dihydrate, acetyl acetone and glyphosate. The results show that glyphosate has very outstanding advantages: only monophosphopeptides can be eluted with glyphosate solution, while all phosphopeptides can be eluted with negatively charged glyphosate ions with pH 9. Moreover, the high ionic strength can minimize nonspecific electrostatic interactions in elution step and limit the generation of potential phosphopeptide metal ion adducts such as sodium or Fe3+ counterparts. S/N of phosphopeptides could be enhanced 3-5 folds in MALDI MS detection and phosphopeptide recovery is greatly improved while compared with its counterparts eluted by commonly used elution buffers. By applying this reagent into IMAC elution, the whole experimental process could be more convenient, time-saving and cost-saving, which is of great importance to the enrichment and detection of phosphopeptides in phosphoproteomics research. Biological significance This potent desalting-free and signal enhanced elution method can improve the sensitivity and detection of phosphopeptides in MALDI TOF MS analysis, both time saving and cost saving. With these advantages, it's highly appropriate for the high throughout analysis of phosphoproteomics. (c) 2013 Elsevier B.V. All rights reserved.