Deep Profiling of Aminophospholipids Reveals a Dysregulated Desaturation Pattern in Breast Cancer Cell Lines.

Phosphatidylethanolamines (PEs), ether-PEs, and phosphatidylserines (PSs) are glycerophospholipids harboring a primary amino group in their headgroups. They are key components of mammalian cell membranes and play pivotal roles in cell signaling and apoptosis. In this study, a liquid chromatography-mass spectrometry (LC-MS) workflow for deep profiling of PEs, ether-PEs, and PSs has been developed by integrating two orthogonal derivatizations: (1) derivatization of the primary amino group by 4-trimethylammoniumbutyryl-N-hydroxysuccinimide (TMAB-NHS) for enhanced LC separation and MS detection and (2) the Paternò-Büchi (PB) reaction for carbon-carbon double bond (C═C) derivatization and localization. Significant improvement of the limit of identification down to the C═C location has been achieved for the standards of PSs (3 nM) and ether-PEs (20 nM). This workflow facilitates an identification of more than 200 molecular species of aminophospholipids in the porcine brain, two times more than those identified without TMAB-NHS derivatization. Importantly, we discovered that the n-10 isomers in C16:1 and C18:1 of aminophospholipids showed elevated contribution among other isomers, which correlated well with an increased transcription of the corresponding desaturase (FADS2) in the human breast cancer cell line (MDA-MB-231) relative to that in the normal cell line (HMEC). The abovementioned data suggest that lipid reprograming via forming different C═C location isomers might be an alternative mechanism in cancer cells.

A liquid chromatography-mass spectrometry workflow for in-depth quantitation of fatty acid double bond location isomers.

Tracing compositional changes of fatty acids (FAs) is frequently used as a means of monitoring metabolic alterations in perturbed biological states. Given that more than half of FAs in the mammalian lipidome are unsaturated, quantitation of FAs at a carbon-carbon double bond (C=C) location level is necessary. The use of 2-acetylpiridine (2-acpy) as the charge-tagging PB reagent led to a limit of identification in the subnanomolar range for mono- and polyunsaturated as well as conjugated FAs. Conjugated free FAs of low abundance such as FA 18:2 (n-7, n-9) and FA 18:2 (n-6, n-8) were quantified at concentrations of 0.61 ± 0.05 and 0.05 ± 0.01 mg per 100 g in yak milk powder, respectively. This workflow also enabled deep profiling of eight saturated and 37 unsaturated total FAs across a span of four orders of magnitude in concentration, including ten groups of C=C location isomers in pooled human plasma. A pilot survey on total FAs in plasma from patients with type 2 diabetes revealed that the relative compositions of FA 16:1 (n-10) and FA 18:1 (n-10) were significantly elevated compared with that of normal controls. In this work, we have developed a workflow for global quantitation of FAs, including C=C location isomers, via charge-tagging Paternò-Büchi (PB) derivatization and liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Evaluation of ultraviolet photodissociation tandem mass spectrometry for the structural assignment of unsaturated fatty acid double bond positional isomers.

Fatty acids are a major source of structural diversity within the lipidome due to variations in their acyl chain lengths, branching, and cyclization, as well as the number, position, and stereochemistry of double bonds within their mono- and poly-unsaturated species. Here, the utility of 193 nm UltraViolet PhotoDissociation tandem mass spectrometry (UVPD-MS/MS) has been evaluated for the detailed structural characterization of a series of unsaturated fatty acid lipid species. UVPD-MS/MS of unsaturated fatty acids is shown to yield pairs of unique diagnostic product ions resulting from cleavages adjacent to their C=C double bonds, enabling unambiguous localization of the site(s) of unsaturation within these lipids. The effect of several experimental variables on the observed fragmentation behaviour and UVPD-MS/MS efficiency, including the position and number of double bonds, the effect of conjugated versus non-conjugated double bonds, the number of laser pulses, and the influence of alkali metal cations (Li, Na, K) as the ionizing adducts, has been evaluated. Importantly, the abundance of the diagnostic ions is shown to enable relative quantitation of mixtures of fatty acid isomers across a range of molar ratios. Finally, the practical application of 193 nm UVPD-MS/MS is demonstrated via characterization of changes in the ratios of fatty acid double bond positional isomers in isogenic colorectal cancer cell lines. This study therefore demonstrates the practicality of UVPD-MS/MS for the structural characterization of fatty acid isomers in lipidome analysis workflows.

Establishing Signature Fragments for Identification and Sequencing of Dityrosine Cross-Linked Peptides Using Ultraviolet Photodissociation Mass Spectrometry.

Dityrosine cross-linking of Aß peptides and α-synuclein is increasingly becoming recognized as a biomarker of neuropathological diseases. However, there remains a need for the development of analytical methods that enable the specific and selective identification of dityrosine cross-linked proteins and peptides in complex biological samples. Here, we report that the gas-phase fragmentation of protonated dityrosine cross-linked peptides under ultraviolet photodissociation (UVPD) tandem mass spectrometry (MS/MS) conditions results in the cleavage across Cα and Cß atoms of the dityrosine residue. This Cα-Cß cleavage in UVPD-MS/MS results in the formation of diagnostic pairs of product ions, providing information on the two individual peptides involved in the cross-linking, resolving the intrinsic "n2 problem" plaguing the identification of this post-translational modification (PTM) by tandem mass spectrometry. Sequencing of a heterodimeric dityrosine cross-linked peptide was demonstrated using hybrid UVPD-MS/MS and CID-MS3 on a diagnostic pair of product ions. In combination with dedicated MS-cleavable MSn software, UVPD-MSn therefore provides an avenue to selectively discover and describe dityrosine cross-linked peptides. Additionally, observation of dityrosine-specific "reporter ions" at m/z 240.1019 and m/z 223.0752 in UVPD-MS/MS will be useful for the validation of the dityrosine cross-linked peptides.

Structural Characterization and Absolute Quantification of Microcystin Peptides Using Collision-Induced and Ultraviolet Photo-Dissociation Tandem Mass Spectrometry.

Microcystin (MC) peptides produced by cyanobacteria pose a hepatotoxic threat to human health upon ingestion from contaminated drinking water. While rapid MC identification and quantification in contaminated body fluids or tissue samples is important for patient treatment and outcomes, conventional immunoassay-based measurement strategies typically lack the specificity required for unambiguous determination of specific MC variants, whose toxicity can significantly vary depending on their structures. Furthermore, the unambiguous identification and accurate quantitation of MC variants using tandem mass spectrometry (MS/MS)-based methods can be limited due to a current lack of appropriate stable isotope-labeled internal standards. To address these limitations, we have systematically examined here the sequence and charge state dependence to the formation and absolute abundance of both "global" and "variant-specific" product ions from representative MC-LR, MC-YR, MC-RR, and MC-LA peptides, using higher-energy collisional dissociation (HCD)-MS/MS, ion-trap collision-induced dissociation (CID)-MS/MS and CID-MS3, and 193 nm ultraviolet photodissociation (UPVD)-MS/MS. HCD-MS/MS was found to provide the greatest detection sensitivity for both global and variant-specific product ions in each of the MC variants, except for MC-YR where a variant-specific product uniquely formed via UPVD-MS/MS was observed with the greatest absolute abundance. A simple methodology for the preparation and characterization of 18O-stable isotope-labeled MC reference materials for use as internal standards was also developed. Finally, we have demonstrated the applicability of the methods developed herein for absolute quantification of MC-LR present in human urine samples, using capillary scale liquid chromatography coupled with ultra-high resolution / accurate mass spectrometry and HCD-MS/MS. Graphical abstract ᅟ.