•Glyphosate has appropriate and stable chelating ability with Fe3 +.•Mono- and multi-phosphopeptides can be eluted sequentially by adjusting pH value.•Glyphosate can suppress nonspecific binding without additional metal salts.•S/N of phosphopeptides can be enhanced 3–5 folds in MALDI MS detection.•The eluate can be analyzed directly without desalting.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 significanceThis 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.

•Use nontoxic and inexpensive natural nanomaterial bentonite to the enrichment of phosphopeptides.•The operation is facile, fast, and highly effective, without nanomaterial preparation, desalting and elution process. Phosphopeptides binded on bentonite can be detected directly.•All the phosphorylation sites in a-casein and ß-casein can be detected unambiguously even at low fmol level.•Open up new horizons for the application of bentonite and natural nanomaterials.A novel strategy for facile, fast and highly effective enrichment of phosphopeptides by bentonite followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is presented. Bentonite is a Al(III)-containing, nontoxic and inexpensive natural nanomaterial with good biocompatibility. By employing bentonite into phosphopeptides enrichment, phosphopeptides binded on bentonite can be detected directly after simple wash, without nanomaterial preparation, desalting and elution process. The whole enrichment procedure can be easily completed within 10 min. Tryptic digest products from several standard proteins and nonfat milk are pretreated using bentonite to demonstrate the efficiency of this method, all the phosphorylation sites in a-casein and ß-casein can be detected unambiguously even at low fmol level. With all the advantages mentioned above, this method is of great potential for future studies of complex phosphoproteomes, and opens up new horizons for bentonite application.