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High resolving power ion mobility (IM) allows for accurate characterization of complex mixtures in high-throughput IM mass spectrometry (IM-MS) experiments. We previously demonstrated that pure component IM-MS data can be extracted from IM unresolved post-IM/collision-induced dissociation (CID) MS data using automated ion mobility deconvolution (AIMD) software [Matthew Brantley, Behrooz Zekavat, Brett Harper, Rachel Mason, and Touradj Solouki, J. Am. Soc. Mass Spectrom., 2014, 25, 1810-1819]. In our previous reports, we utilized a quadrupole ion filter for m/z-isolation of IM unresolved monoisotopic species prior to post-IM/CID MS. Here, we utilize a broadband IM-MS deconvolution strategy to remove the m/z-isolation requirement for successful deconvolution of IM unresolved peaks. Broadband data collection has throughput and multiplexing advantages; hence, elimination of the ion isolation step reduces experimental run times and thus expands the applicability of AIMD to high-throughput bottom-up proteomics. We demonstrate broadband IM-MS deconvolution of two separate and unrelated pairs of IM unresolved isomers (viz., a pair of isomeric hexapeptides and a pair of isomeric trisaccharides) in a simulated complex mixture. Moreover, we show that broadband IM-MS deconvolution improves high-throughput bottom-up characterization of a proteolytic digest of rat brain tissue. To our knowledge, this manuscript is the first to report successful deconvolution of pure component IM and MS data from an IM-assisted data-independent analysis (DIA) or HDMSE dataset.

Existing instrumental resolving power limitations in ion mobility spectrometry (IMS) often restrict adequate characterization of unresolved or co-eluting chemical isomers. Recently, we introduced a novel chemometric deconvolution approach that utilized post-IM collision-induced dissociation (CID) mass spectrometry (MS) data to extract "pure" IM profiles and construct CID mass spectra of individual components from a mixture containing two IM-overlapped components [J. Am. Soc. Mass Spectrom., 2012, 23, 1873-1884]. In this manuscript we extend the capabilities of the IM-MS deconvolution methodology and demonstrate the utility of energy resolved IM deconvolution for successful characterization of ternary and quaternary isomer mixtures with overlapping IM profiles. Furthermore, we show that the success of IM-MS deconvolution is a collision-energy dependent process where different isomers can be identified at various ion fragmentation collision-energies. Details on how to identify a single collision-energy or suitable collision-energy ranges for successful characterization of isomer mixtures are discussed. To confirm the validity of the proposed approach, deconvoluted IM and MS spectra from IM overlapped analyte mixtures are compared to IM and MS data from individually run mixture components. Criteria for "successful" deconvolution of overlapping IM profiles and extraction of their corresponding pure mass spectra are discussed.