title: Checkpoints for Preliminary Identification of Small Molecules found Enriched in Autophagosomes and Activated Mast Cell Secretions Analyzed by Comparative UPLC/MSe.
abstract: 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/zvalues 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 MSespectra that were in agreement within-silicofragmentation. 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.
authors.affiliation: University of Minnesota, Department of Chemistry, 207 Pleasant Street SE, Minneapolis, MN, 55455-0431, United States.
authors.forename: Chad P
authors.initials: CP
authors.lastname: Satori
authors.affiliation: University of Minnesota, Department of Chemistry, 207 Pleasant Street SE, Minneapolis, MN, 55455-0431, United States.
authors.forename: Marzieh
authors.initials: M
authors.lastname: Ramezani
authors.affiliation: University of Minnesota, Division of Biostatistics, 420 Delaware Street SE, Minneapolis MN, 55455, United States.
authors.forename: Joseph S
authors.initials: JS
authors.lastname: Koopmeiners
authors.affiliation: University of Minnesota, Department of Chemistry, 207 Pleasant Street SE, Minneapolis, MN, 55455-0431, United States.
authors.forename: Audrey F
authors.initials: AF
authors.lastname: Meyer
authors.affiliation: Albert Einstein College of Medicine, Institute for Aging Studies, Marion Besin Liver Research Center, Department of Developmental and Molecular Biology, 1300 Morris Park Avenue, Bronx, NY 10461, United States.
authors.forename: Jose A
authors.initials: JA
authors.lastname: Rodriguez-Navarro
authors.affiliation: University of Minnesota, Department of Chemistry, 207 Pleasant Street SE, Minneapolis, MN, 55455-0431, United States.
authors.forename: Michelle M
authors.initials: MM
authors.lastname: Kuhns
authors.affiliation: University of Minnesota, Department of Chemistry, 207 Pleasant Street SE, Minneapolis, MN, 55455-0431, United States.
authors.forename: Thane H
authors.initials: TH
authors.lastname: Taylor
authors.affiliation: University of Minnesota, Department of Chemistry, 207 Pleasant Street SE, Minneapolis, MN, 55455-0431, United States.
authors.forename: Christy L
authors.initials: CL
authors.lastname: Haynes
authors.affiliation: University of Minnesota, Department of Chemistry, 207 Pleasant Street SE, Minneapolis, MN, 55455-0431, United States.
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Dalluge
authors.affiliation: University of Minnesota, Department of Chemistry, 207 Pleasant Street SE, Minneapolis, MN, 55455-0431, United States.
authors.forename: Edgar A
authors.initials: EA
authors.lastname: Arriaga