Access to the Phospho-proteome via the Mitigation of Peptide-Metal Interactions

Liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS) has become the default platform for proteomics due to its specificity, sensitivity and sample compatibility. However, interactions between transition metals in LC systems and analytes containing phosphate groups result in poor chromatographic performance or even analyte loss. The use of systems where the metal surfaces had been treated with a hybrid inorganic/organic surface material to form an effective surface barrier mitigated these undesired interactions. When employed for the analysis of tryptic digests of Alpha and Beta Casein, along with synthetic "PhosphoMix" standards, the use of such a system showed significant improvements in chromatographic peak shape and analyte response together with superior spectral quality and sequence coverage. These improvements resulted in the detection of phosphorylated peptides ADEPSSESDLEIDK and ELSNSPLRENSFGSPLEFR from the PhosphoMix which went undetected with a standard, untreated LC system. The hybrid surface system thus offered significant advantages for the analysis of phosphopeptides compared to conventional LC/MS.