Discovery and Identification of Three Homocysteine Metabolites by Chemical Derivatization and Mass Spectrometry Fragmentation.

Discovery and identification of a new endogenous metabolite are typically hindered by requirements of large sample volumes and multistage purifications to guide synthesis of the standard. Presented here is a metabolomics platform that uses chemical tagging and tandem mass spectrometry to determine structure, direct synthesis, and confirm identity. Three new homocysteine metabolites are reported: N-succinyl homocysteine, 2-methyl-1,3-thiazinane-4-carboxylic acid (MTCA), and homolanthinone.

Isobaric 6-plex and tosyl dual tagging for the determination of positional isomers and quantitation of monounsaturated fatty acids using rapid UHPLC-MS/MS.

Isobaric labelling of fatty acids is complicated by chromatographic co-elution of double bond isomers. This produces contaminated spectra which can mask important biological changes. Here two derivatization strategies are combined to improve throughput and produce MS2 reporters which change mass depending on double bond position. A 6-plex isobaric tag is attached to the acid group, followed by the tosylation of the double bond using chloramine-T. These two derivatizations allowed for the chromatographic resolution of nearly all investigated isomers using a 3.5 minute ultrafast method. Further isomer differentiation is achieved upon fragmentation as reporter masses scale with the double bond location. This occurs by a dual-fragmentation route which reveals the isobaric labelling and fragments along the double bond of each analyte. These unique fragments allowed for accurate quantitation of co-isolated double bond isomers where traditional isobaric tags would experience ratio distortion. Saturated and monounsaturated fatty acids were characterized by this rapid 6-plex method and produced an average signal RSD of 9.3% and R2 of 0.99. The method was then used to characterize fatty acid dysregulation upon inhibition of stearoyl CoA desaturase with CAY10566.

Use of N-(4-aminophenyl)piperidine derivatization to improve organic acid detection with supercritical fluid chromatography-mass spectrometry.

The analysis of organic acids in complex mixtures by LC-MS can often prove challenging, especially due to the poor sensitivity of negative ionization mode required for detection of these compounds in their native (i.e., underivatized or untagged) form. These compounds have also been difficult to measure using supercritical fluid chromatography (SFC)-MS, a technique of growing importance for metabolomic analysis, with similar limitations based on negative ionization. In this report, the use of a high proton affinity N-(4-aminophenyl)piperidine derivatization tag is explored for the improvement of organic acid detection by SFC-MS. Four organic acids (lactic, succinic, malic, and citric acids) with varying numbers of carboxylate groups were derivatized with N-(4-aminophenyl)piperidine to achieve detection limits down to 0.5 ppb, with overall improvements in detection limit ranging from 25-to-2100-fold. The effect of the derivatization group on sensitivity, which increased by at least 200-fold for compounds that were detectable in their native form, and mass spectrometric detection are also described. Preliminary investigations into the separation of these derivatized compounds identified multiple stationary phases that could be used for complete separation of all four compounds by SFC. This derivatization technique provides an improved approach for the analysis of organic acids by SFC-MS, especially for those that are undetectable in their native form.

Evaluation and optimization of PolyJet 3D-printed materials for cell culture studies.

Use of 3D printing for microfluidics is a rapidly growing area, with applications involving cell culture in these devices also becoming of interest. 3D printing can be used to create custom-designed devices that have complex features and integrate different material types in one device; however, there are fewer studies studying the ability to culture cells on the various substrates that are available. This work describes the effect of PolyJet 3D-printing technology on cell culture of two cell lines, bovine pulmonary artery endothelial cells (BPAECs) and Madin-Darby Canine Kidney (MDCK) cells, on two different types of printed materials (VeroClear or MED610). It was found that untreated devices, when used for studies of 1 day or more, led to unsuccessful culture. A variety of device treatment methodologies were investigated, with the most success coming from the use of sodium hydroxide/sodium metasilicate solution. Devices treated with this cleaning step resulted in culture of BPAECs and MDCK cells that were more similar to what is obtained in traditional culture flasks (in terms of cell morphology, viability, and cell density). LC-MS/MS analysis (via Orbitrap MS) was used to determine potential leachates from untreated devices. Finally, the use of a fiber scaffold in the devices was utilized to further evaluate the treatment methodology and to also demonstrate the ability to perform 3D culture in such devices. This study will be of use for researchers wanting to utilize these or other cell types in PolyJet-based 3D-printed devices.

Capillary flow-based sample preparation system for metabolomic analysis of mammalian cells in suspension.

Sample preparation methodology is critical to obtaining reliable data for studying endogenous metabolites. Dependable preparation techniques require separation of cells from culture media, quenching of enzymatic activity, and extraction of metabolites from the cells. Presented here is a simple, rapid, semi-automated metabolomic sample preparation technique for 20 µL samples of RAW 264.7 cells suspended in culture media. This method uses online filter-assisted electroporation-based cell lysis and chilled organic solvent extraction to prepare metabolomic samples from cells in suspension in 2 min. Experiments using an isotopically labeled adenosine triphosphate internal standard were carried out to ensure enzymatic quenching by monitoring the ratio of labeled adenosine diphosphate to adenosine triphosphate. Cells were metabolically labeled with 13C-glucose concurrent with sampling aliquots of the cell suspension over the course of 24 h. Incorporation of 13C into organic acid metabolites such as itaconate Cell lysates was analyzed by nano-reverse-phase liquid chromatography-mass spectrometry (nano-RP-LC-MS), showing incorporation of 13C into organic acid metabolites such as itaconate.

Two-dimensional separation techniques using supercritical fluid chromatography.

High-resolution separation systems are essential for the analysis of complex mixtures in a wide variety of application areas. To increase resolution, multidimensional chromatographic techniques have been one key solution. Supercritical fluid chromatography provides a unique opportunity in these multidimensional separations based on its potential for high solvent compatibility, rapid duty cycles, and orthogonality to other separation modes. This review focuses on two-dimensional chromatography methods from the past decade that use supercritical fluid chromatography because of these advantages. Valving schemes and modulation strategies used to interface supercritical fluid chromatography with other liquid chromatography and gas chromatography techniques are described. Particular applications of multidimensional separations using supercritical fluid chromatography for the analysis of oils and chiral separations of pharmaceutical compounds are highlighted. Limitations of and a potential trajectory for supercritical fluid chromatography in this field are also discussed.

Isomeric Differentiation and Acidic Metabolite Identification by Piperidine-Based Tagging, LC-MS/MS, and Understanding of the Dissociation Chemistries.

We demonstrate a method for facile differentiation of acidic, isomeric metabolites by attaching high proton affinity, piperidine-based chemical tags to each carboxylic acid group. These tags attach with high efficiency to the analytes, increase the signal, and result in the formation of multiply-charged cations. We illustrate the present approach with citrate and isocitrate, which are isomeric metabolites each containing three carboxylic acid groups. We observe a 20-fold increase in signal-to-noise for citrate and an 8-fold increase for isocitrate as compared to detection of the untagged analytes in negative mode. Collision-induced dissociation of the triply tagged, triply charged analytes results in distinct tandem mass spectra. The phenylene spacer groups limit proton mobility and enable access to structurally informative C-C bond cleavage reactions. Modeling of the gas-phase structures and dissociation chemistry of these triply charged analyte ions highlights the importance of hydroxyl proton mobilization in this low proton mobility environment. Tandem mass spectrometric analyses of deuterated congeners and MS3 spectra are consistent with the proposed fragment ion structures and mechanisms of formation. Direct evidence that these chemistries are more generally applicable is provided by subsequent analyses of doubly tagged, doubly charged malate ions. Future work will focus on applying these methods to identify new metabolites and development of general rules for structural determination of tagged metabolites with multiple charges.

Determination of online quenching efficiency for an automated cellular microfluidic metabolomic platform using mass spectrometry based ATP degradation analysis.

As microfluidic cell culture progresses, the need for robust and reproducible intracellular analyses grows. In particular, intracellular metabolites are subject to perturbation and degradation during the lysing process. The reliability of intracellular metabolomic analysis in microfluidic devices depends on the preservation of metabolite integrity during sample preparation and storage. Described here is a novel automated microfluidic system exhibiting the necessary rapid cellular lysis and quenching of enzymatic activity. Quenching efficiency was assessed using a novel ratiometric MALDI-MS-based assay of exogenous isotopic adenosine triphosphate (ATP) hydrolysis to isotopic adenosine diphosphate (ADP) as a marker of metabolite degradation. The lysis system of the microfluidic device was enhanced using a Peltier cooler to chill the lysate and quench aberrant enzymatic activity. Parameter optimization (flow rate, collection time, and temperature control) improved the endogenous and exogenous ADP/ATP ratios by 44.9% and 39.8% respectively consistent with traditional quenching techniques. The effects of chilling/quenching on metabolism were evaluated resulting in over 500 significant features compared to non-chilled from untargeted capillary LC-MS metabolomic analyses. These include increased levels of tryptophan, histidine, and pyruvate as well as decreased levels in UDP-N-acetylglucosamine. The results illustrate the need for both rapid lysis and quenching in microfluidic cell culture platforms. Graphical abstract.

Frequency-Modulated Continuous Flow Analysis Electrospray Ionization Mass Spectrometry (FM-CFA-ESI-MS) for Sample Multiplexing.

A method for multiplexed sample analysis by mass spectrometry without the need for chemical tagging is presented. In this new method, each sample is pulsed at unique frequencies, mixed, and delivered to the mass spectrometer while maintaining a constant total flow rate. Reconstructed ion currents are then a time-dependent signal consisting of the sum of the ion currents from the various samples. Spectral deconvolution of each reconstructed ion current reveals the identity of each sample, encoded by its unique frequency, and its concentration encoded by the peak height in the frequency domain. This technique is different from other approaches that have been described, which have used modulation techniques to increase the signal-to-noise ratio of a single sample. As proof of concept of this new method, two samples containing up to 9 analytes were multiplexed. The linear dynamic range of the calibration curve was increased with extended acquisition times of the experiment and longer oscillation periods of the samples. Because of the combination of the samples, salt had little effect on the ability of this method to achieve relative quantitation. Continued development of this method is expected to allow for increased numbers of samples that can be multiplexed.

Multi-functional derivatization of amine, hydroxyl, and carboxylate groups for metabolomic investigations of human tissue by electrospray ionization mass spectrometry.

Metabolomics, the study of small molecules involved in cellular processes, offers the potential to reveal insights into the pathophysiology of disease states. Analysis of metabolites by electrospray mass spectrometry is complicated by their structural diversity. Amine, hydroxyl, and carboxylate groups all affect signal responses differently based on their polarity and proton affinity. This heterogeneity of signal response, sensitivity, and resistance to competing ionization complicates metabolite quantitation. Such limitations can be mitigated by a dual derivatization scheme. In this work, primary amine and hydroxyl groups are tagged with a linear acyl chloride head containing a tertiary amine tail, followed by carboxylate groups coupled to a linear amine tag with a tertiary amine tail. This tagging scheme increases analyte proton affinity and hydrophobicity. In the case of carboxylate groups, the inherent anionic charge is inverted to a cationic charge. This dual tagging is completed within 2.5 hours, diminishes adduct formation, and improves sensitivity by >75-fold. The average limit of detection for 23 metabolites was 38 nM and the R2 was 0.97. This process was used to investigate metabolite changes from human tissue. Examination of diabetic and non-diabetic human tissue showed marked changes in both energy metabolites and amino acids. Further examination of the tissue showed that HbA1C value is inversely correlated with fumarate levels. This technique potentially allows for the analysis of virtually all metabolites in a single analytical run. Thus, it may lead to a more complete picture of metabolic dysfunction in human disease.

Automated sample preparation in a microfluidic culture device for cellular metabolomics.

Sample pretreatment in conventional cellular metabolomics entails rigorous lysis and extraction steps which increase the duration as well as limit the consistency of these experiments. We report a biomimetic cell culture microfluidic device (MFD) which is coupled with an automated system for rapid, reproducible cell lysis using a combination of electrical and chemical mechanisms. In-channel microelectrodes were created using facile fabrication methods, enabling the application of electric fields up to 1000 V cm(-1). Using this platform, average lysing times were 7.12 s and 3.03 s for chips with no electric fields and electric fields above 200 V cm(-1), respectively. Overall, the electroporation MFDs yielded a ∼10-fold improvement in lysing time over standard chemical approaches. Detection of multiple intracellular nucleotides and energy metabolites in MFD lysates was demonstrated using two different MS platforms. This work will allow for the integrated culture, automated lysis, and metabolic analysis of cells in an MFD which doubles as a biomimetic model of the vasculature.

Extraction and Quantitation of Ketones and Aldehydes from Mammalian Cells Using Fluorous Tagging and Capillary LC-MS.

The extraction and quantitation of carbonyl metabolites from cell lysate was accomplished using a carbonyl-reactive fluorous tag and capillary liquid chromatography coupled to mass spectrometry (capLC-MS). Selective fluorous tagging for ketones and aldehydes provided a 30-fold increase in sensitivity using electrospray ionization MS. Separation of fluorous tagged carbonyl resulted in good separation of all components, and tandem MS was able to differentiate structural carbonyl isomers. The average limit of detection for carbonyl standards was 37 nM (range 1.5-250 nM), with linearity of R(2) > 0.99. Reproducibility for metabolites in cell lysate averaged 9% RSD. Human aortic endothelial cells (HAECs) were exposed to varying levels of glucose, and their carbonyl metabolite levels were quantified. Significant metabolite changes were seen in glycolysis and the propanoate pathway from a glucose challenge. Using an untargeted approach, 120 carbonyl metabolites were found to change in hyperglycemic HAECs. From this list of compounds, multiple metabolites from the pentose phosphate and tryptophan metabolic pathways were discovered. This system provides excellent sensitivity and quantitation of carbonyl metabolites without the need for isotope standards or labels.

Global metabolomic and isobaric tagging capillary liquid chromatography-tandem mass spectrometry approaches for uncovering pathway dysfunction in diabetic mouse aorta.

Despite the prevalence of diabetes and the global health risks it poses, the biochemical pathogenesis of diabetic complications remains poorly understood with few effective therapies. This study employs capillary liquid chromatography (capLC) and tandem mass spectrometry (MS/MS) in conjunction with both global metabolomics and isobaric tags specific to amines and carbonyls to probe aortic metabolic content in diabetic mice with hyperglycemia, hyperlipidemia, hypertension, and stenotic vascular damage. Using these combined techniques, metabolites well-characterized in diabetes as well as novel pathways were investigated. A total of 53,986 features were detected, 719 compounds were identified as having significant fold changes (thresholds ≥ 2 or ≤ 0.5), and 48 metabolic pathways were found to be altered with at least 2 metabolite hits in diabetic samples. Pathways related to carbonyl stress, carbohydrate metabolism, and amino acid metabolism showed the greatest number of metabolite changes. Three novel pathways with previously limited or undescribed roles in diabetic complications--vitamin B6, propanoate, and butanoate metabolism--were also shown to be altered in multiple points along the pathway. These discoveries support the theory that diabetic vascular complications arise from the interplay of a myriad of metabolic pathways in conjunction with oxidative and carbonyl stress, which may provide not only new and much needed biomarkers but also insights into novel therapeutic targets.

Double Cyclization Tandem Mass for Identification and Quantification of Phosphatidylcholines Using Isobaric Six-Plex Capillary nLC-MS/MS.

Multiplexing of phosphatidylcholine analysis is hindered by a lack of appropriate derivatization. Presented here is a tagging scheme that uses a quaternary amine tag and targets the hydroxy group of the phosphate, which switches the net charge from neutral to +2. Quantitative yields were achieved from >99% reaction completion derived by dimethoxymethyl morpholinium (DMTMM) activation. Fragmentation of phosphatidylcholines (PCs) and lysophosphatidylcholines (LPCs) releases two trimethylamines and the acyl chains through neutral loss and generates a unique double cyclization constant mass reporter. Selective incorporation of isotopes onto the tag produces a six-plex set of isobaric reagents. For equivalent six-plex-labeled samples, <14% RSD was achieved, followed by a dynamic range of 1:10 without signal compression. Quantification of PCs/LPCs in human hepatic cancer cells was conducted as six-plex using data-dependent analysis tandem MS. We report a six-plex qualitative and quantitative isobaric tagging strategy expanding the limits of analyzing PCs/LPCs.

Formation of dAMP-glycerol and dAMP-Tris derivatives by Thermococcus kodakaraensis DNA primase.

In the presence of dATP, glycerol, and Tris buffer, the DNA primase isolated from Thermococcus kodakaraensis catalyzed the formation of dAMP and two products that were identified as dAMP-glycerol and dAMP-Tris. These products were formed by the T. kodakaraensis p41 catalytic subunit alone and the T. kodakaraensis p41-p46 complex in the absence of a DNA template. They were not formed with preparations containing the catalytically inactive p41 subunit. Similar glycerol and Tris derivatives as well as dNMPs were also formed with dGTP, dCTP, or dTTP. The mechanism contributing to the formation of these products and its implications in the initiation reaction catalyzed by the T. kodakaraensis primase are discussed.

A novel DNA nuclease is stimulated by association with the GINS complex.

Chromosomal DNA replication requires the spatial and temporal coordination of the activities of several complexes that constitute the replisome. A previously uncharacterized protein, encoded by TK1252 in the archaeon Thermococcus kodakaraensis, was shown to stably interact with the archaeal GINS complex in vivo, a central component of the archaeal replisome. Here, we document that this protein (TK1252p) is a processive, single-strand DNA-specific exonuclease that degrades DNA in the 5' → 3' direction. TK1252p binds specifically to the GINS15 subunit of T. kodakaraensis GINS complex and this interaction stimulates the exonuclease activity in vitro. This novel archaeal nuclease, designated GINS-associated nuclease (GAN), also forms a complex in vivo with the euryarchaeal-specific DNA polymerase D. Roles for GAN in replisome assembly and DNA replication are discussed.

Dual Fragmentation Isobaric Tags for Metabolomics.

Isobaric tags typically leverage an a1 type fragmentation to produce constant mass reporter ions. While this motif allows for efficient reporter formation, isobaric tags lack structural diversity, which limits the number and type of isotopes that are synthetically available. Presented here are two examples of dual fragmentation isobaric tagging. The first example mimics the typical isobaric tag structure through trimethylamine neutral loss and cyclization. Subsequent fragmentation releases a constant mass reporter with high efficiency. This provides a route to create a variety of isobaric tags with regard to both the reporter and the balancer mass. The second example is a set of six-plex isobaric, thiol-reactive tags that produce constant mass reporters by a similar sequential fragmentation mechanism. A trimethylamine neutral loss allows for the incorporation of up to 13 total isotopes in the balancer region, while minimizing deuterium retention time shifts. A subsequent C-S bond cleavage produces a constant mass reporter in the low-mass region. The thiols investigated produced an average RSD of 14% and R2 of 0.98 when analyzed as a six-plex injection. Thiol metabolism was disrupted using the glutamyl-cysteine synthetase inhibitor buthionine sulfoximine (BSO). Endothelial cells were incubated with BSO and showed significant decreases in glutathione and cysteinyl-glycine compared to control. Overall, a new method to generate constant mass reporters using a dual fragmentation scheme is presented.

Supercritical Fluid Nanospray Mass Spectrometry: II. Effects on Ionization.

Nanospraying supercritical fluids coupled to a mass spectrometer (nSF-MS) using a 90% supercritical fluid CO2 carrier (sCO2) has shown an enhanced desolvation compared to traditional liquid eluents. Capillaries of 25, 50, and 75 µm internal diameter (i.d.) with pulled emitter tips provided high MS detection sensitivity. Presented here is an evaluation of the effect of proton affinity, hydrophobicity, and nanoemitter tip size on the nSF-MS signal. This was done using a set of primary, secondary, tertiary, and quaternary amines with butyl, hexyl, octyl, and decyl chains as analytes. Each amine class was analyzed individually to evaluate hydrophobicity and proton affinity effects on signal intensity. The system has shown a mass sensitive detection on a linear dynamic range of 0.1-100 µM. Results indicate that hydrophobicity has a larger effect on the signal response than proton affinity. Nanospraying a mixture of all amine classes using the 75 µm emitter has shown a quaternary amine signal not suppressed by competing analytes. Competing ionization was observed for primary, secondary, and tertiary amines. The 75 and 50 µm emitters demonstrated increased signal with increasing hydrophobicity. Surprisingly, the 25 µm i.d. emitter yielded a signal decrease as the alkyl chain length increased, contrary to conventional understanding. Nanospraying the evaporative fluid in a sub-500 nm emitter likely resulted in differences in the ionization mechanism. Results suggest that 90% sCO2 with 9.99% methanol and 0.01% formic acid yielded fast desolvation, high ionization efficiency, and low matrix effect, which could benefit complex biological matrix analysis.

Subsecond absolute quantitation of amine metabolites using isobaric tags for discovery of pathway activation in mammalian cells.

The absolute quantitation of amine metabolites from mammalian cell samples was achieved by combining amine standards, isobaric tags, and capillary liquid chromatography (LC) tandem mass spectrometry (MS/MS). Our approach allowed 32 specific amines to be analyzed within a single chromatographic run, with the generation of the calibration curve and absolute quantitation of each analyte taking less than 900 ms. Using this strategy, we determined the amine response of human aortic endothelial cells (HAECs) from a glucose challenge. The observed changes of the absolute concentration of these metabolites implied eight enzymatic reactions may change efficiency upon glucose treatment. Five of these reactions have been previously reported as being up-regulated in diabetic conditions. The remaining three reactions were analyzed by measuring the expression of these enzymes, with 66% showing increases. Our data indicate that rapid determination of absolute quantitation is useful in determining novel pathway activation. Furthermore, even though we determined the absolute quantity of 32 metabolites here, the number of analytes that can be measured by this method is limited mainly by commercial availability of amine standards.