Automated peak width measurements for targeted analysis of ion mobility unresolved species

•A new automated data analysis tool is introduced for untargeted identification of ion mobility (IM) overlapping compounds based on IM peak shapes.•Species with multiple conformations can be rapidly identified in complex mixtures.•Further ultrahigh resolution mass spectrometry and ion fragmentation experiments confirm the validity of using IM peak widths for conformational analyses.

Peak broadening in ion mobility (IM) is a relatively predictable process and abnormally broad peaks can be indicative of the presence of unresolved species. Here, we introduce a new ion mobility peak fitting (IM_FIT) software package for automated and systematic determination of traveling wave ion mobility (TWIM) unresolved species. To identify IM unresolved species, the IM_FIT software generates a trend line by plotting ions' mobility peak widths as a function of their arrival times. Utilizing user-defined thresholds, IM_FIT allows for automated and rapid detection of ions that deviate from the peak width trend line. To demonstrate the advantages of IM_FIT for automated detection of IM unresolved species, IM-mass spectrometry (IM-MS) data from a sample mixture containing polypropylene glycol and multiple peptides were analyzed. A total of 14 out of the 34 observed singly-charged IM peaks above 5% relative abundance (i.e., signal-to-noise ratios above ∼200) were tagged as potentially co-eluting ions by IM_FIT. Subsequently, the 14 IM peaks tagged as potentially unresolved (presumably, peaks corresponding to co-eluting compounds), were further analyzed by automated IM deconvolution (AIMD), liquid chromatography-IM-MS (LC-IM-MS), and/or ultra-high resolution mass spectrometry. Using the aforementioned techniques, more than 85% of the tagged IM peaks (12 out of 14) were confirmed to contain co-eluting ions. As an additional new finding, IM_FIT facilitated the discovery of an unexpected sequence-scrambled y-type fragment ion.

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