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The identification of phosphorylated peptides by LC-MS is a challenging task. It is in part due to the relative low abundance of phosphopeptides; and their low ionization efficiency in positive ESI-MS ionization mode. The problem is further exacerbated by the phosphopeptides’ ability to form complexes with metals such as Fe (III) or Al (III) accumulated in the LC systems. Thus, the amount of phosphopeptides eluted from the LC system may be less than the amount injected, especially for multiply phosphorylated species. It has been reported that adding chelating agents such as EDTA into the sample before LC injection can disrupt the formation of phosphopeptide-metal complexes and improve the phosphopeptide detection. However, EDTA introduces several high background ions upon mass spectrometry detection.
We utilized mobile phases with triethylammonium ion-pairing agent to eliminate the loss of phosphopeptides in a nano-scale ultra-performance liquid chromatorgraphy. Triethylammonium ion pairs with the phosphate group(s) in peptides and prevents the binding of phosphopeptides to metalic surfaces in LC systems. Direct comparison reveals a better recovery of phosphopeptides from nanoLC and LC systems with triethylammonium-based mobile phases (UV and MS detection). When using the same LC systems with conventional mobile-phase additives (0.1% formic acid), the majority of the multiply phosphorylated peptides were not detected. Triethylamine concentrations were optimized to obtain the desirable separation of phosphopeptides. The addition of EDTA to samples was no longer necessary to recover multiply phosphorylated peptides. ESI positive ion mode detected mostly singly charged phosphopeptide ions; extensive adduct ion formation (triethylamine/phosphopeptide) was observed. Triethylamine tends to form adduct ions exclusively with phosphopeptides, which can be used as a diagnostic tool to identify phosphopeptides. These adduct ions were dissociated by Tee-in formic acid–containing solvent through an auxiliary pump before their entrance into the ESI source prior to the MS/MS (DDA) experiment.