Lipid kinase PIK3C3 maintains healthy brown and white adipose tissues to prevent metabolic diseases.

Adequate mass and function of adipose tissues (ATs) play essential roles in preventing metabolic perturbations. The pathological reduction of ATs in lipodystrophy leads to an array of metabolic diseases. Understanding the underlying mechanisms may benefit the development of effective therapies. Several cellular processes, including autophagy and vesicle trafficking, function collectively to maintain AT homeostasis. Here, we investigated the impact of adipocyte-specific deletion of the lipid kinase phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3) on AT homeostasis and systemic metabolism in mice. We report that PIK3C3 functions in all ATs and that its absence disturbs adipocyte autophagy and hinders adipocyte differentiation, survival, and function with differential effects on brown and white ATs. These abnormalities cause loss of white ATs, whitening followed by loss of brown ATs, and impaired "browning" of white ATs. Consequently, mice exhibit compromised thermogenic capacity and develop dyslipidemia, hepatic steatosis, insulin resistance, and type 2 diabetes. While these effects of PIK3C3 largely contrast previous findings with the autophagy-related (ATG) protein ATG7 in adipocytes, mice with a combined deficiency in both factors reveal a dominant role of the PIK3C3-deficient phenotype. We have also found that dietary lipid excess exacerbates AT pathologies caused by PIK3C3 deficiency. Surprisingly, glucose tolerance is spared in adipocyte-specific PIK3C3-deficient mice, a phenotype that is more evident during dietary lipid excess. These findings reveal a crucial yet complex role for PIK3C3 in ATs, with potential therapeutic implications.

High-resolution ion mobility based on traveling wave structures for lossless ion manipulation resolves hidden lipid features.

High-resolution ion mobility (resolving power > 200) coupled with mass spectrometry (MS) is a powerful analytical tool for resolving isobars and isomers in complex samples. High-resolution ion mobility is capable of discerning additional structurally distinct features, which are not observed with conventional resolving power ion mobility (IM, resolving power ~ 50) techniques such as traveling wave IM and drift tube ion mobility (DTIM). DTIM in particular is considered to be the "gold standard" IM technique since collision cross section (CCS) values are directly obtained through a first-principles relationship, whereas traveling wave IM techniques require an additional calibration strategy to determine accurate CCS values. In this study, we aim to evaluate the separation capabilities of a traveling wave ion mobility structures for lossless ion manipulation platform integrated with mass spectrometry analysis (SLIM IM-MS) for both lipid isomer standards and complex lipid samples. A cross-platform investigation of seven subclass-specific lipid extracts examined by both DTIM-MS and SLIM IM-MS showed additional features were observed for all lipid extracts when examined under high resolving power IM conditions, with the number of CCS-aligned features that resolve into additional peaks from DTIM-MS to SLIM IM-MS analysis varying between 5 and 50%, depending on the specific lipid sub-class investigated. Lipid CCS values are obtained from SLIM IM (TW(SLIM)CCS) through a two-step calibration procedure to align these measurements to within 2% average bias to reference values obtained via DTIM (DTCCS). A total of 225 lipid features from seven lipid extracts are subsequently identified in the high resolving power IM analysis by a combination of accurate mass-to-charge, CCS, retention time, and linear mobility-mass correlations to curate a high-resolution IM lipid structural atlas. These results emphasize the high isomeric complexity present in lipidomic samples and underscore the need for multiple analytical stages of separation operated at high resolution.

Multidimensional Separations of Intact Phase II Steroid Metabolites Utilizing LC-Ion Mobility-HRMS.

The detection and unambiguous identification of anabolic-androgenic steroid metabolites are essential in clinical, forensic, and antidoping analyses. Recently, sulfate phase II steroid metabolites have received increased attention in steroid metabolism and drug testing. In large part, this is because phase II steroid metabolites are excreted for an extended time, making them a potential long-term chemical marker of choice for tracking steroid misuse in sports. Comprehensive analytical methods, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), have been used to detect and identify glucuronide and sulfate steroids in human urine with high sensitivity and reliability. However, LC-MS/MS identification strategies can be hindered by the fact that phase II steroid metabolites generate nonselective ion fragments across the different metabolite markers, limiting the confidence in metabolite identifications that rely on exact mass measurement and MS/MS information. Additionally, liquid chromatography-high-resolution mass spectrometry (LC-HRMS) is sometimes insufficient at fully resolving the analyte peaks from the sample matrix (commonly urine) chemical noise, further complicating accurate identification efforts. Therefore, we developed a liquid chromatography-ion mobility-high resolution mass spectrometry (LC-IM-HRMS) method to increase the peak capacity and utilize the IM-derived collision cross section (CCS) values as an additional molecular descriptor for increased selectivity and to improve identifications of intact steroid analyses at low concentrations.

New Frontiers in Lipidomics Analyses using Structurally Selective Ion Mobility-Mass Spectrometry.

The growth of lipidomics and the high isomeric complexity of the lipidome has revealed a need for analytical techniques capable of structurally characterizing lipids with a high degree of specificity. Lipids are morphologically diverse molecules that can exist as any one of a large number of isomeric species, and as such are often indistinguishable by mass spectrometry without a complementary separation method. Recent developments in the field of lipidomics aim to address these challenges by utilizing a combination of multiple analytical techniques which are selective to lipid primary structure. This review summarizes two emerging strategies for lipidomic analysis, namely, ion mobility-mass spectrometry and ion fragmentation via ozonolysis.

Conformational ordering of biomolecules in the gas phase: nitrogen collision cross sections measured on a prototype high resolution drift tube ion mobility-mass spectrometer.

Ion mobility-mass spectrometry measurements which describe the gas-phase scaling of molecular size and mass are of both fundamental and pragmatic utility. Fundamentally, such measurements expand our understanding of intrinsic intramolecular folding forces in the absence of solvent. Practically, reproducible transport properties, such as gas-phase collision cross-section (CCS), are analytically useful metrics for identification and characterization purposes. Here, we report 594 CCS values obtained in nitrogen drift gas on an electrostatic drift tube ion mobility-mass spectrometry (IM-MS) instrument. The instrument platform is a newly developed prototype incorporating a uniform-field drift tube bracketed by electrodynamic ion funnels and coupled to a high resolution quadrupole time-of-flight mass spectrometer. The CCS values reported here are of high experimental precision (±0.5% or better) and represent four chemically distinct classes of molecules (quaternary ammonium salts, lipids, peptides, and carbohydrates), which enables structural comparisons to be made between molecules of different chemical compositions for the rapid "omni-omic" characterization of complex biological samples. Comparisons made between helium and nitrogen-derived CCS measurements demonstrate that nitrogen CCS values are systematically larger than helium values; however, general separation trends between chemical classes are retained regardless of the drift gas. These results underscore that, for the highest CCS accuracy, care must be exercised when utilizing helium-derived CCS values to calibrate measurements obtained in nitrogen, as is the common practice in the field.

Resolving Power and Collision Cross Section Measurement Accuracy of a Prototype High-Resolution Ion Mobility Platform Incorporating Structures for Lossless Ion Manipulation.

A production prototype structures for lossless ion manipulation ion mobility (SLIM IM) platform interfaced to a commercial high-resolution mass spectrometer (MS) is described. The SLIM IM implements the traveling wave ion mobility technique across a ∼13m path length for high-resolution IM (HRIM) separations. The resolving power (CCS/ΔCCS) of the SLIM IM stage was benchmarked across various parameters (traveling wave speeds, amplitudes, and waveforms), and results indicated that resolving powers in excess of 200 can be accessed for a broad range of masses. For several cases, resolving powers greater than 300 were achieved, notably under wave conditions where ions transition from a nonselective "surfing" motion to a mobility-selective ion drift, that corresponded to ion speeds approximately 30-70% of the traveling wave speed. The separation capabilities were evaluated on a series of isomeric and isobaric compounds that cannot be resolved by MS alone, including reversed-sequence peptides (SDGRG and GRGDS), triglyceride double-bond positional isomers (TG 3, 6, 9 and TG 6, 9, 12), trisaccharides (melezitose, raffinose, isomaltotriose, and maltotriose), and ganglioside lipids (GD1b and GD1a). The SLIM IM platform resolved the corresponding isomeric mixtures, which were unresolvable using the standard resolution of a drift-tube instrument (∼50). In general, the SLIM IM-MS platform is capable of resolving peaks separated by as little as ∼0.6% without the need to target a specific separation window or drift time. Low CCS measurement biases <0.5% were obtained under high resolving power conditions. Importantly, all the analytes surveyed are able to access high-resolution conditions (>200), demonstrating that this instrument is well-suited for broadband HRIM separations important in global untargeted applications.

High Confidence Shotgun Lipidomics Using Structurally Selective Ion Mobility-Mass Spectrometry.

Ion mobility (IM) is a gas phase separation strategy that can either supplement or serve as a high-throughput alternative to liquid chromatography (LC) in shotgun lipidomics. Incorporating the IM dimension in untargeted lipidomics workflows can help resolve isomeric lipids, and the collision cross section (CCS) values obtained from the IM measurements can provide an additional molecular descriptor to increase lipid identification confidence. This chapter provides a broad overview of an untargeted ion mobility-mass spectrometry (IM-MS) workflow using a commercial drift tube ion mobility-quadrupole-time-of-flight mass spectrometer (IM-QTOF) for high confidence lipidomics.

Collision Cross Section Conformational Analyses of Bile Acids via Ion Mobility-Mass Spectrometry.

Bile acids serve as one of the most important classes of biological molecules in the gastrointestinal system. Due to their structural similarity, bile acids have historically been difficult to accurately annotate in complex biological matrices using mass spectrometry. They often have identical or nominally similar mass-to-charge ratios and similar fragmentation patterns that make identification by mass spectrometry arduous, normally involving chemical derivatization and separation via liquid chromatography. Here, we demonstrate the use of drift tube ion mobility (DTIM) to derive collision cross section (CCS) values in nitrogen drift gas (DTCCSN2) for use as an additional descriptor to facilitate expedited bile acid identification. We also explore trends in DTIM measurements and detail structural characteristics for differences in DTCCSN2 values between subclasses of bile acid molecules.

Evaluating Separation Selectivity and Collision Cross Section Measurement Reproducibility in Helium, Nitrogen, Argon, and Carbon Dioxide Drift Gases for Drift Tube Ion Mobility-Mass Spectrometry.

Previous ion mobility (IM) studies have demonstrated that varying the drift gas composition can be used to enhance chemical selectivity and resolution, yet there are few drift gas studies aimed at achieving quantitatively reproducible mobility measurements. Here, we critically evaluate the conditions necessary to achieve reproducible collision cross section (CCS) measurements in pure drift gases (helium, nitrogen, argon, and carbon dioxide) using a commercial uniform field drift tube instrument. Optimal experimental parameters are assessed based on the convergence of CCS measurements to reproducible values which are compared with literature values. A suite of calibration standards with diverse masses, biological classes, and charge states are examined to assess chemical selectivity and resolution achievable in each drift gas. Results indicate nitrogen and argon perform similarly and are sufficient for most applications where high resolving power and high peak capacity are desired. Carbon dioxide exhibits more selectivity for resolving structurally heterogeneous compounds, which may be preferable in specific analyte pair separations. Helium demonstrated modest separation capabilities but has utility for comparison to theoretical values and previously published work. In drift gases other than nitrogen, pressure differentials up to 230 mTorr between the drift tube and upstream chamber were optimal for improving correlation to literature values, while in nitrogen, the recommended pressure differential of 150 mTorr was found appropriate. We present recommended experimental parameters as well as gas-specific CCS measurements for structurally homogeneous sets of analytes which are suitable for use by other laboratories as standards for purposes of instrument calibration and overall assessment of IM separation performance.

Ion mobility conformational lipid atlas for high confidence lipidomics.

Lipids are highly structurally diverse molecules involved in a wide variety of biological processes. Here, we use high precision ion mobility-mass spectrometry to compile a structural database of 456 mass-resolved collision cross sections (CCS) of sphingolipid and glycerophospholipid species. Our CCS database comprises sphingomyelin, cerebroside, ceramide, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidic acid classes. Primary differences observed are between lipid categories, with sphingolipids exhibiting 2-6% larger CCSs than glycerophospholipids of similar mass, likely a result of the sphingosine backbone's restriction of the sn1 tail length, limiting gas-phase packing efficiency. Acyl tail length and degree of unsaturation are found to be the primary structural descriptors determining CCS magnitude, with degree of unsaturation being four times as influential per mass unit. The empirical CCS values and previously unmapped quantitative structural trends detailed in this work are expected to facilitate prediction of CCS in broadscale lipidomics research.

Collision cross section compendium to annotate and predict multi-omic compound identities.

Ion mobility mass spectrometry (IM-MS) expands the analyte coverage of existing multi-omic workflows by providing an additional separation dimension as well as a parameter for characterization and identification of molecules - the collision cross section (CCS). This work presents a large, Unified CCS compendium of >3800 experimentally acquired CCS values obtained from traceable molecular standards and measured with drift tube ion mobility-mass spectrometers. An interactive visualization of this compendium along with data analytic tools have been made openly accessible. Represented in the compendium are 14 structurally-based chemical super classes, consisting of a total of 80 classes and 157 subclasses. Using this large data set, regression fitting and predictive statistics have been performed to describe mass-CCS correlations specific to each chemical ontology. These structural trends provide a rapid and effective filtering method in the traditional untargeted workflow for identification of unknown biochemical species. The utility of the approach is illustrated by an application to metabolites in human serum, quantified trends of which were used to assess the probability of an unknown compound belonging to a given class. CCS-based filtering narrowed the chemical search space by 60% while increasing the confidence in the remaining isomeric identifications from a single class, thus demonstrating the value of integrating predictive analyses into untargeted experiments to assist in identification workflows. The predictive abilities of this compendium will improve in specificity and expand to more chemical classes as additional data from the IM-MS community is contributed. Instructions for data submission to the compendium and criteria for inclusion are provided.