The conformation space occupied by different classes of biomolecules measured by ion mobility-mass spectrometry (IM-MS) is described for utility in the characterization of complex biological samples. Although the qualitative separation of different classes of biomolecules on the basis of structure or collision cross section is known, there is relatively little quantitative cross-section information available for species apart from peptides. In this report, collision cross sections are measured for a large suite of biologically salient species, including oligonucleotides (n=96), carbohydrates (n=192), and lipids (n=53), which are compared to reported values for peptides (n= 610). In general, signals for each class are highly correlated, and at a given mass, these correlations result in predicted collision cross sections that increase in the order oligonucleotides

Recent advances in mass spectrometry approaches to the analysis of lipids include the ability to incorporate both lipid class identification with lipid structural information for increased characterization capabilities. The detailed examination of lipids and their biosynthetic and biochemical pathways made possible by novel instrumental and bioinformatics approaches is advancing research in fundamental cellular and medical studies. Recently, high-throughput structural analysis has been demonstrated through the use of rapid gas-phase separation on the basis of the ion mobility (IM) analytical technique combined with mass spectrometry (IM-MS). While IM-MS has been extensively utilized in biochemical research for peptide, protein and small molecule analysis, the role of IM-MS in lipid research is still an active area of development. In this review of lipid-based IM-MS research, we begin with an overview of three contemporary IM techniques which show great promise in being applied towards the analysis of lipids. Fundamental concepts regarding the integration of IM-MS are reviewed with emphasis on the applications of IM-MS towards simplifying and enhancing complex biological sample analysis. Finally, several recent IM-MS lipid studies are highlighted and the future prospects of IM-MS for integrated omics studies and enhanced spatial profiling through imaging IM-MS are briefly described.

The high chemical complexity of the lipidome is one of the major challenges in lipidomics research. Ion-mobility spectrometry (IMS), a gas-phase electrophoretic technique, makes possible the separation of ions in the gas phase according to their charge, shape, and size. IMS can be combined with mass spectrometry (MS), adding three major benefits to traditional lipidomic approaches. First, IMS-MS allows the determination of the collision cross section (CCS), a physicochemical measure related to the conformational structure of lipid ions. The CCS is used to improve the confidence of lipid identification. Second, IMS-MS provides a new set of hybrid fragmentation experiments. These experiments, which combine collision-induced dissociation with ion-mobility separation, improve the specificity of MS/MS-based approaches. Third, IMS-MS improves the peak capacity and signal-to-noise ratio of traditional analytical approaches. In doing so, it allows the separation of complex lipid extracts from interfering isobaric species. Developing in parallel with advances in instrumentation, informatics solutions enable analysts to process and exploit IMS-MS data for qualitative and quantitative applications. Here we review the current approaches for lipidomics research based on IMS-MS, including liquid chromatography-MS and direct-MS analyses of "shotgun" lipidomics and MS imaging.

Ion mobility-mass spectrometry (IM-MS) provides rapid two-dimensional separations based on analyte apparent surface area or collision cross section (CCS, Å 2 ) and mass-to-charge, respectively. Recently, traveling-wave (t-wave) IM-MS was developed which uses electrodynamic rather than electrostatic fields commonly used in drift cell IM-MS instruments. The underlying theory for obtaining CCS data is well developed for drift cell IM-MS, while strategies for obtaining CCS values from t-wave IM-MS data remains an active area of research. In this report, methods were developed and validated to obtain CCS values of phospholipids and peptides directly from thin tissue sections by MALDI t-wave IM-MS using CCS calibrants measured by MALDI drift cell IM-MS. Importantly, the average percent difference between t-wave and drift cell CCS measurements is minimized by calibrating with the same biomolecular class. Calibrating t-wave phospholipid CCS values with drift cell peptide CCS measurements results in an average percent difference of ca. 7% between the same lipids measured using t-wave and drift cell IM-MS, while this improves to <0.5% when drift cell phospholipid CCS values are used for calibrating t-wave data. A suite of CCS values are reported for lipids and peptides that were determined directly from tissue, i.e. without the need for tissue extraction and further purification steps.

Profiling and imaging of cholesterol and its precursors by mass spectrometry (MS) are important in a number of cholesterol biosynthesis disorders, such as in Smith-Lemli-Opitz syndrome (SLOS), where 7-dehydrocholesterol (7-DHC) is accumulated in affected individuals. SLOS is caused by defects in the enzyme that reduces 7-DHC to cholesterol. However, analysis of sterols is challenging because these hydrophobic olefins are difficult to ionize for MS detection. We report here sputtered silver matrix-assisted laser desorption/ionization (MALDI)-ion mobility-MS (IM-MS) analysis of cholesterol and 7-DHC. In comparison with liquid-based AgNO3 and colloidal Ag nanoparticle (AgNP), sputtered silver NP (10–25 nm) provided the lowest limits-of-detection based on the silver coordinated [cholesterol+Ag]+ and [7-DHC+Ag]+ signals while minimizing dehydrogenation products ([M+Ag-2H]+). When analyzing human fibroblasts that were directly grown on poly-L-lysine-coated ITO glass plates with this technique, in situ, the 7-DHC/cholesterol ratios for both control and SLOS human fibroblasts are readily obtained. The m/z of 491 (specific for [7-DHC+107Ag]+) and 495 (specific for [cholesterol+109Ag]+) were subsequently imaged using MALDI-IM-MS. MS images were co-registered with optical images of the cells for metabolic ratio determination. From these comparisons, ratios of 7-DHC/cholesterol for SLOS human fibroblasts are distinctly higher than in control human fibroblasts. Thus, this strategy demonstrates the utility for diagnosing/assaying the severity of cholesterol biosynthesis disorders in vitro.