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.

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.

Isobaric 4-Plex Tagging for Absolute Quantitation of Biological Acids in Diabetic Urine Using Capillary LC-MS/MS.

Isobaric labeling in mass spectrometry enables multiplexed absolute quantitation and high throughput, while minimizing full scan spectral complexity. Here, we use 4-plex isobaric labeling with a fixed positive charge tag to improve quantitation and throughput for polar carboxylic acid metabolites. The isobaric tag uses an isotope-encoded neutral loss to create mass-dependent reporters spaced 2 Da apart and was validated for both single- and double-tagged analytes. Tags were synthesized in-house using deuterated formaldehyde and methyl iodide in a total of four steps, producing cost-effective multiplexing. No chromatographic deuterium shifts were observed for single- or double-tagged analytes, producing consistent reporter ratios across each peak. Perfluoropentanoic acid was added to the sample to drastically increase retention of double-tagged analytes on a C18 column. Excess tag was scavenged and extracted using hexadecyl chloroformate after reaction completion. This allowed for removal of excess tag that typically causes ion suppression and column overloading. A total of 54 organic acids were investigated, producing an average linearity of 0.993, retention time relative standard deviation (RSD) of 0.58%, and intensity RSD of 12.1%. This method was used for absolute quantitation of acid metabolites comparing control and type 1 diabetic urine. Absolute quantitation of organic acids was achieved by using one isobaric lane for standards, thereby allowing for analysis of six urine samples in two injections. Quantified acids showed good agreement with previous work, and six significant changes were found. Overall, this method demonstrated 4-plex absolute quantitation of acids in a complex biological sample.

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.

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.