Checkpoints for Preliminary Identification of Small Molecules found Enriched in Autophagosomes and Activated Mast Cell Secretions Analyzed by Comparative UPLC/MSe.

We report the use of ultra high performance liquid chromatography (UPLC) coupled with acquisition of low- and high-collision energy mass spectra (MSe) to explore small molecule compositions that are unique to either enriched-autophagosomes or secretions of chemically activated murine mast cells. Starting with thousands of features, each defined by a chromatographic retention time, m/z values and ion intensities, manual examination of the extracted ion chromatograms (XIC) of chemometrically selected features was essential to eliminate false positives, occurring at rates of 33, 14 and 37% in samples of three biological systems. Forty-six percent of features that passed the XIC-based checkpoint, had IDs in compound databases used here. From these, 19% of IDs had experimental high-collision energy MSe spectra that were in agreement with in-silico fragmentation. The importance of this second checkpoint was highligthed through validation with selected commercially available standards. This work illustrates that checkpoints in data processing are essential to ascertain reliability of unbiased metabolomic studies, thereby reducing the risk of generating 'false identifications' which are is a major concern as 'omics' data continue to proliferate and be used as platforms to lauch novel biological hypotheses.

Electrostatically defying cation-cation clusters: can likes attract in a low-polarity environment?

Like-charge ion pairing is commonly observed in protein structures and plays a significant role in biochemical processes. Density functional calculations combined with the conductor-like polarizable continuum model were employed to study the formation possibilities of doubly charged noncovalently linked complexes of a series of model compounds and amino acids in the gas phase and in solution. Hydrogen bond interactions were found to offset the Coulombic repulsion such that cation-cation clusters are minima on the potential energy surfaces and neither counterions nor solvent molecules are needed to hold them together. In the gas phase the dissociation energies are exothermic, and the separation barriers span from 1.7 to 15.6 kcal mol(-1). Liquid-phase computations indicate that the separation enthalpies of the cation-cation complexes become endothermic in water and nonpolar solvents with dielectric constants of ≥7 (i.e., the value for THF). These results reveal that electrostatically defying noncovalent complexes of like-charged ions can overcome their Coulombic repulsion even in low-polarity environments.