The benefits and limitations of reaction cell and sector field inductively coupled plasma mass spectrometry in the detection and quantification of phosphopeptides.

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The phosphorylation of proteins is one of the most important post-translational modifications in nature. Knowledge of the quantity or degree of protein phosphorylation in biological samples is extremely important. A combination of liquid chromatography (LC) and inductively coupled plasma mass spectrometry (ICP-MS) allows the absolute and relative quantification of the phosphorus signal. A comparison between dynamic reaction cell quadrupole ICP-MS (DRC-Q-ICP-MS) and high-resolution sector field ICP-MS (SF-ICP-MS) in detecting signals of phosphorus-containing species using identical capillary LC (reversed-phase technology) and nebulizer settings was performed. A method to diminish the reversed-phase gradient-related signal instability in phosphorus detection with LC/ICP-MS applications was developed. Bis(4-nitrophenyl)phosphate (BNPP) was used as a standard to compare signal-to-noise ratios and limits of detection (LODs) between the two instrumental setups. The LOD reaches a value of 0.8 µg L(-1) when applying the DRC technology in Q-ICP-MS and an LOD of 0.09 µg L(-1) was found with the SF-ICP-MS setup. This BNPP standard was further used to compare the absolute quantification possibilities of phosphopeptides in these two setups. This one-to-one comparison of two interference-reducing ICP-MS instruments demonstrates that absolute quantification of individual LC-separated phosphopeptides is possible. However, based on the LOD values, SF-ICP-MS has a higher sensitivity in detecting phosphorus signals and thus is preferred in phosphopeptide analysis.