Rapid LC-MS assay for targeted metabolite quantification by serial injection into isocratic gradients.

Liquid chromatography mass spectrometry (LC-MS) has emerged as a mainstream strategy for metabolomics analyses. One advantage of LC-MS is that it can serve both as a biomarker discovery tool and as a platform for clinical diagnostics. Consequently, it offers an exciting opportunity to potentially transition research studies into real-world clinical tools. One important distinction between research versus diagnostics-based applications of LC-MS is throughput. Clinical LC-MS must enable quantitative analyses of target molecules in hundreds or thousands of samples each day. Currently, the throughput of these clinical applications is limited by the chromatographic gradient lengths, which-when analyzing complex metabolomics samples-are difficult to conduct in under ~ 3 min per sample without introducing serious quantitative analysis problems. To address this shortcoming, we developed sequential quantification using isotope dilution (SQUID), an analytical strategy that combines serial sample injections into a continuous isocratic mobile phase to maximize throughput. SQUID uses internal isotope-labelled standards to correct for changes in LC-MS response factors over time. We show that SQUID can detect microbial polyamines in human urine specimens (lower limit of quantification; LLOQ = 106 nM) with less than 0.019 normalized root mean square error. Moreover, we show that samples can be analyzed in as little as 57 s. We propose SQUID as a new, high-throughput LC-MS tool for quantifying small sets of target biomarkers across large cohorts.

Role of metabolism in uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) is responsible for more than 75% of urinary tract infections (UTIs) and has been studied extensively to better understand the molecular underpinnings of infection and pathogenesis. Although the macromolecular adaptations UPEC employs - including the expression of virulence factors, adhesion molecules, and iron-acquisition systems - are well described, the role that metabolism plays in enabling infection is still unclear. However, a growing body of literature shows that metabolic function can have a profound impact on which strains can colonize the urinary tract. The goal of this review is to critically appraise this emerging body of literature to better understand the role that nutritional selection plays in enabling urinary tract colonization and the progression of UTIs.