Abstract
Objectives Urine as a biofluid is commonly used in clinical diagnostics, including during pregnancy. Urine is a rich source of polypeptides and protein degradation products. Electron transfer dissociation (ETD) techniques have recently been applied to peptides, and improved fragmentation of long polypeptides has been demonstrated; ETD further facilitates analysis of labile post-translational modifications. We therefore applied this advanced technique for peptide dissociation to analyse and identify urinary polypeptides for the first time. Methods We examined the urinary peptidome from normal pregnant women during pregnancy, to demonstrate that peptides are readily observed. We utilised conventional MS/MS techniques and then used ETD (LC–MS/MS performed on an orbitrap equipped with ETD source, coupled to a nanoAcquity HPLC) to increase the identification rate of the peptides within these samples, as the polypeptide species observed are large and highly charged. Results An increase in the number of peptides whose identities could be ascribed using routine database searching methods was enabled via use of ETD. These peptides, as anticipated, were large and highly charged (typically giving charge states of 4–8+ over a mass-to-charge range of 400–2000 m/z). Conclusions Urinary proteomic profiling has not been able to discriminate between normal and pre-eclampsia pregnancies at gestations early enough to enable preventative strategies. Enhanced polypeptide identification achieved through ETD may facilitate clinically useful predictive tests. Disclosures S. Hart: None. L. Kenny: Commercial Interest: Metabolomic Diagnostics. J. Myers: None. P. Baker: Commercial Interest: Metabolomic Diagnostics.
Published Version
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