Building a Library with >1000 Ion Mobility Collision Cross Sections for Ultrafast Small Molecule Analyses (ASMS 2017)

Metabolomics and exposomics studies have been growing rapidly during the past decade and providing insights into cellular metabolism and how a system responds to stimuli. Ion mobility spectrometry-mass spectrometry (IMS-MS) has become an appealing tool for small molecule studies as it enables the analysis and separation of the many isomeric species. However, to facilitate the identification of endogenous metabolites and xenobiotics from complex samples, a library with accurate collision cross sections (CCS) is needed. In this work, we generated a CCS library for >1000 small molecule standards using triplicate analyses in positive and negative polarity. This library was then coupled with ultrafast small molecule analyses using a sub-minute online SPE-IMS-MS method for characterization of different metabolic and xenobiotic conditions.IMS-MS is capable of separating molecules that have the same mass-to-charge (m/z) ratios, but different sizes, shapes or conformations. In this work an Agilent drift tube-IMS-QTOF MS platform was used to characterize over one thousand endogenous metabolites and xenobiotics. The CCS values were measured using seven stepped fields in both positive and negative polarity. For small molecule studies, the samples were analyzed by coupling Rapidfire, an online SPE system to the IMS-MS platform which enables sub-minute ultrafast analyses. Different SPE cartridges such as C18, graphitic carbon, HILIC and phenyl cartridges were explored to effectively extract different classes of molecules. In addition, different ionization sources including ESI, APCI and APPI were evaluated for efficient small molecule studies. A large scale ion mobility CCS library including over 800 endogenous metabolites and over 200 xenobiotics was generated using drift tube IMS-MS. Different classes of metabolites displayed different trend lines for their CCS values. For instance, fatty acids show larger CCS values than carbohydrates or amino acids. We were also able to map the CCS values for key metabolites and intermediates in important metabolite pathways such as glycolysis/gluconeogenesis, pentose phosphate pathway and citrate cycle, etc. These results are essential for future studies aimed at identification of metabolites within different structure classes and pathways. To evaluate the use of this CCS library for metabolite identification, we analyzed how small molecules change in patients with and without exercises, as well as those with and without ethanol use, and compared the results from the SPE-IMS-MS method with those from LC-MS method. <div>Xenobiotics that are important for the evaluation of environmental exposure such as polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) exposures were also studied. Different ionization sources including ESI, APCI and APPI, were evaluated for efficient characterization and detection of the PAHs and PCBs and their metabolites. Preliminary data showed that we were able to detect PAHs in [M∙]+ and [M + H]+ forms and PCBs in the [M-Cl+O]- form using APCI/APPI methods. We also showed that the pre-separation, clean-up and enrichment steps by Rapidfire SPE greatly improved the sensitivity, enabling ultrafast and simultaneous analyses of PAHs and PCBs and their metabolites in complex biological and environmental samples.</div

Petroleomic Characterization using an Ion Mobility-Orbitrap Platform (ASMS 2016)

<p>Ion mobility spectrometry (IMS) is a fast gas-phase separation technique that separates ions in part based upon their shape. Different chemical classes form shape-related ‘trend lines’ in the 2D drift time-m/z chromatogram. These trend lines can be utilized as a quick diagnostic for chemical classes, and hence information that augments that from mass spectrometry. Herein we present a new IMS-Orbitrap platform that couples the benefits of drift-tube IMS with a high mass resolution Orbitrap MS. The new platform was utilized to analyze and profile an array of petroleum products, and highlights the utility of the IMS -Orbitrap platform for analysis of highly chemically complex substances.</p

Collision Cross Section Calibration with Structures for Lossless Ion Manipulations (ASMS 2017)

Ion mobility mass spectrometry (IM-MS) is a powerful separation and structural characterization technique, providing the ability to measure collision cross sections (CCS), revealing information about the three dimensional structure of gaseous ions. In many cases, CCS can be used to identify ions in a mixture, and highly accurate and precise CCS measurements greatly expand IM-MS capabilities. Recently, long path structures for lossless ion manipulations (SLIM) traveling wave (TW) IM modules have allowed extremely high resolution IM separations. However, since SLIM do not utilize uniform low-field drift cells, CCS cannot be directly measured from experiments. To that end, we have developed a CCS calibration framework to provide high resolution CCS assignment.Travelling wave potentials and a combination of lateral DC-only electrodes (guards) and extended RF electrodes aligned with the ion path provided for TWIM separations in several Torr nitrogen in conjunction with efficient ion confinement. Ions from nanoelectrospray ionization of mixtures of multiple classes of compounds (e.g. peptide, glycan, lipid) were injected to the SLIM module. A SLIM ion switch controlled whether ions made multiple passes through the serpentine path of the module, or were sent to the TOF MS for analysis. Multiple mixtures of calibrants of different classes overlapping in CCS space with the compounds studied were prepared and infused as both external and internal calibrants. TWIM-MS features were extracted and calibrated using in-house developed software tools.Recently, multi-pass SLIM separations have been reported, showing very high IM resolutions and peak capacities for a variety of compounds, including peptides, lipids, and carbohydrates. A SLIM ion switch was positioned at the end of a long (>10 meter) serpentine ion path to allow ions to either exit to a TOF MS for mass analysis, or to be shuttled to the beginning of the ion path for addition separation. Resolutions much higher than that from conventional commercially available instruments (both TW and uniform field) have been achieved (e.g., separation powers of over 1000 for singly charged ions for 200 m multi-pass separations). Due to the abundance of information from bottom-up proteomics of many protein standards (e.g. tryptic peptide accurate monoisotopic MW), the first efforts for applying CCS calibration have utilized whole protein digests. Early results have shown baseline separations of peptides in a protein digest (serum albumin) that are inseparable by conventional IM instruments. Initially, a poly-alanine mixture was used to begin evaluating CCS calibrations for peptides and was used as external and internal calibration standards. The protein digest was then run on an Agilent 6560 IM-MS to compare the calibrated CCS values against values measured directly by a uniform low field instrument. The presentation will detail the efficacy of CCS calibration in SLIM TWIM measurements as well as effects resulting from the choice of calibrant, internal vs. external calibration, and other biological compound classes