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We report the use of ultra high performance liquid chromatography (UPLC) coupled with acquisition of low- and high-collision energy mass spectra (MS e ) 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 MS e 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.

analytical methods

Checkpoints for preliminary identification of small molecules found enriched in autophagosomes and activated mast cell secretions analyzed by comparative UPLC/MS<sup>e</sup>

Checkpoints for preliminary identification of small molecules found enriched in autophagosomes and activated mast cell secretions analyzed by comparative UPLC/MSe

Checkpoints for preliminary identification of small molecules found enriched in autophagosomes and activated mast cell secretions analyzed by comparative UPLC/MS^e