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

Journal of Proteomics
Li ZhuYinlong Guo

Abstract

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 pH9. 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 Fe(3+) 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 gre...Continue Reading

References

Jan 15, 1999·Journal of the American Society for Mass Spectrometry·J M Asara, J Allison
Jul 29, 1999·Analytical Chemistry·M C Posewitz, P Tempst
Dec 26, 2001·Chemical Reviews·L N Johnson, R J Lewis
May 30, 2007·Journal of the American Society for Mass Spectrometry·Yuebo WangXinghua Xia
Dec 5, 2008·Journal of Proteome Research·Dominic WinterWolf D Lehmann
Aug 1, 1993·Journal of the American Society for Mass Spectrometry·L M Nuwaysir, J T Stults

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