Matthew J. Sniatynski

Speciation in reverse: morphological and genetic evidence of the collapse of a three‐spined stickleback (Gasterosteus aculeatus) species pair

Historically, six small lakes in southwestern British Columbia each contained a sympatric species pair of three‐spined sticklebacks (Gasterosteus aculeatus). These pairs consisted of a ‘benthic’ and ‘limnetic’ species that had arisen postglacially and, in four of the lakes, independently. Sympatric sticklebacks are considered biological species because they are morphologically, ecologically and genetically distinct and because they are strongly reproductively isolated from one another. The restricted range of the species pairs places them at risk of extinction, and one of the pairs has gone extinct after the introduction of an exotic catfish. In another lake, Enos Lake, southeastern Vancouver Island, an earlier report suggested that its species pair is at risk from elevated levels of hybridization. We conducted a detailed morphological analysis, as well as genetic analysis of variation at five microsatellite loci for samples spanning a time frame of 1977 to 2002 to test the hypothesis that the pair in Enos Lake is collapsing into a hybrid swarm. Our morphological analysis showed a clear breakdown between benthics and limnetics. Bayesian model‐based clustering indicated that two morphological clusters were evident in 1977 and 1988, which were replaced by 1997 by a single highly variable cluster. The most recent 2000 and 2002 samples confirm the breakdown. Microsatellite analysis corroborated the morphological results. Bayesian analyses of population structure in a sample collected in 1994 indicated two genetically distinct populations in Enos Lake, but only a single genetic population was evident in 1997, 2000, and 2002. In addition, genetic analyses of samples collected in 1997, 2000, and 2002 showed strong signals of ‘hybrids’; they were genetically intermediate to parental genotypes. Our results support the idea that the Enos Lake species pair is collapsing into a hybrid swarm. Although the precise mechanism(s) responsible for elevated hybridization in the lake is unknown, the demise of the Enos Lake species pair follows the appearance of an exotic crayfish, Pascifasticus lenisculus, in the early 1990s.

Lipidomics Profiling by High-Resolution LC−MS and High-Energy Collisional Dissociation Fragmentation: Focus on Characterization of Mitochondrial Cardiolipins and Monolysocardiolipins

A liquid chromatography-mass spectrometry (LC-MS) method was used for separation of lipid classes as well as both qualitative and semiquantitative detection of individual lipids in biological samples. Data were acquired using high-resolution full-scan MS and high-energy collisional dissociation (HCD) all ion fragmentation. The method was evaluated for efficient separation and detection in both positive and negative ionization mode using standards spanning six lipid classes. Platform linearity and robustness, related to the mitochondrial lipid cardiolipin (CL), were assessed using extracted ion chromatograms with mass tolerance windows of 5 ppm or less from full scan exact mass measurements. The platform CL limit of detection was determined to be 5 pmol (0.9 μM) on the column, with mass accuracy <1.5 ppm, retention time coefficients of variation (CV) < 0.5%, and area CV < 13%. This mass accuracy was critical to the identification of unknown CL species in mitochondria samples, through the elimination of false positives. In addition to detection and relative quantitation of CL species in mitochondria, CL structures were characterized through the use of alternating HCD scans at different energies to produce diagnostic fragmentations on all ions in the analysis. The developed lipid profiling method was applied to mitochondrial samples from an animal study related to the linkages between diet, mitochondrial function, and disease. The analysis identified 28 unique CL species and two monolysocardiolipin species that are often associated with mitochondrial stress and dysfunction.

Serum lipidomics profiling using LC-MS and high-energy collisional dissociation fragmentation: focus on triglyceride detection and characterization.

There is a growing need both clinically and experimentally to improve the characterization of blood lipids. A liquid chromatography-mass spectrometry (LC-MS) method, developed for the qualitative and semiquantitative detection of lipids in biological samples and previously validated in mitochondrial samples, was now evaluated for the profiling of serum lipids. Data were acquired using high-resolution, full scan MS and high-energy, collisional dissociation (HCD), all ion fragmentation. The method was designed for efficient separation and detection in both positive and negative ionization mode and evaluated using standards spanning seven lipid classes. Platform performance, related to the identification and characterization of serum triglycerides (TGs), was assessed using extracted ion chromatograms with mass tolerance windows of 5 ppm or less from full scan exact mass measurements determined using SIEVE nondifferential LC-MS analysis software. The platform showed retention time coefficients of variation (CV) of <0.3%, mass accuracy values of <2 ppm error, and peak area CV of <13%, with the majority of that error coming from sample preparation and extraction rather than the LC-MS analysis, and linearity was shown to be over 4 orders of magnitude (r(2) = 0.999) for the standard TG (15:0)(3) spiked into serum. Instrument mass accuracy and precision were critical to the identification of unknown TG species, in part because these parameters enabled us to reduce false positives. In addition to detection and relative quantitation of TGs in serum, TG structures were characterized through the use of alternating HCD scans at different energies to produce diagnostic fragmentations on all ions in the analysis. The lipidomics method was applied to serum samples from 192 rats maintained on diets differing in macronutrient composition. The analysis identified 86 TG species with 81 unique masses that varied over 3.5 orders of magnitude and showed diet-dependency, consistent with TGs linking diet and disease risk.

Current approaches for global post-translational modification discovery and mass spectrometric analysis

More and more attention is being focused on the analysis of post-translational modifications (PTMs) on proteins as researchers are continually learning how essential they are for proper cellular function. As there are hundreds of different types of known PTMs, traditional methods of modification analysis are incapable of comprehensively monitoring for post-translational modifications, a task which is a necessity for truly understanding a cells biology. This review highlights recent developments in novel multiplexed methods of PTM analysis including: fluorescent stain and immuno-based methods, hardware-based mass spectrometric methods and computational-based mass spectrometric methods. Many of these techniques show great promise and will likely be a valuable resource for the biological community.

Dietary macronutrients modulate the fatty acyl composition of rat liver mitochondrial cardiolipins

The interaction of dietary fats and carbohydrates on liver mitochondria were examined in male FBNF1 rats fed 20 different low-fat isocaloric diets. Animal growth rates and mitochondrial respiratory parameters were essentially unaffected, but mass spectrometry-based mitochondrial lipidomics profiling revealed increased levels of cardiolipins (CLs), a family of phospholipids essential for mitochondrial structure and function, in rats fed saturated or trans fat-based diets with a high glycemic index. These mitochondria showed elevated monolysocardiolipins (a CL precursor/product of CL degradation), elevated ratio of trans-phosphocholine (PC) (18:1/18:1) to cis-PC (18:1/18:1) (a marker of thiyl radical stress), and decreased ubiquinone Q9; the latter two of which imply a low-grade mitochondrial redox abnormality. Extended analysis demonstrated: i) dietary fats and, to a lesser extent, carbohydrates induce changes in the relative abundance of specific CL species; ii) fatty acid (FA) incorporation into mature CLs undergoes both positive (>400-fold) and negative (2.5-fold) regulation; and iii) dietary lipid abundance and incorporation of FAs into both the CL pool and specific mature tetra-acyl CLs are inversely related, suggesting previously unobserved compensatory regulation. This study reveals previously unobserved complexity/regulation of the central lipid in mitochondrial metabolism.

Simple LC-MS Method for Differentiation of Isobaric Phosphatidylserines and Phosphatidylcholines with Deuterated Mobile Phase Additives.

Lipids from different classes sometimes can exhibit the same exact mass upon electrospray ionization; this presents an analytical challenge in lipidomics. In the negative ionization mode, for example, this can occur with phosphatidylcholines (PCs) and phosphatidylserines (PSs), making them indistinguishable in the absence of fragmentation data. PSs are found at low concentrations in biological samples, making MS/MS spectra difficult to obtain. Moreover, while PCs and PSs are distinguishable in the positive mode, PSs do not ionize as well as PCs, and their ionization is suppressed by the PCs. Here, we show that, in the negative ionization mode, substituting protiated LC-MS additives with their deuterated forms provides a way to distinguish PCs and PSs without chemical derivatization. The method described leverages the differential ionization mechanism of PCs and PSs. PCs are ionized via adduction with salts, whereas PSs ionize via hydrogen abstraction. Substituting the salts used for LC-MS with their deuterated form shifts the mass of PCs by the number of deuterium atoms in the salt, while the mass of PSs remains the same. This comparative shift enables their direct differentiation. We demonstrate that the use of deuterated formate shifts the mass of PCs and provides a direct method to distinguish PCs and PSs, even at biologically relevant low concentrations. The utility of the method was established and validated in the simultaneous analysis of PCs and PSs in lipid extracts from isolated liver mitochondria in two different rat strains. Thirteen low concentration PSs were identified that would otherwise not have been distinguishable from low concentration PCs.

Optimization of ESI-Source Parameters for Lipidomics Reduces Misannotation of In-Source Fragments as Precursor Ions

Lipidomics requires the accurate annotation of lipids in complex samples to enable determination of their biological relevance. We demonstrate that unintentional in-source fragmentation (ISF, common in lipidomics) generates ions that have identical masses to other lipids. Lysophosphatidylcholines (LPC), for example, generate in-source fragments with the same mass as free fatty acids and lysophosphatidylethanolamines (LPE). The misannotation of in-source fragments as true lipids is particularly insidious in complex matrixes since most masses are initially unannotated and comprehensive lipid standards are unavailable. Indeed, we show such LPE/LPC misannotations are incorporated in the data submitted to the National Institute of Standards and Technology (NIST) interlaboratory comparison exercise. Computer simulations exhaustively identified potential misannotations. The selection of in-source fragments of highly abundant lipids as features, instead of the correct recognition of trace lipids, can potentially lead to (i) missing the biologically relevant lipids (i.e., a false negative) and/or (ii) incorrect assignation of a phenotype to an incorrect lipid (i.e., false positive). When ISF is not eliminated in the negative ion mode, ∼40% of the 100 most abundant masses corresponding to unique phospholipids measured in plasma were artifacts from ISF. We show that chromatographic separation and ion intensity considerations assist in distinguishing precursor ions from in-source fragments, suggesting ISF may be especially problematic when complex samples are analyzed via shotgun lipidomics. We also conduct a systematic evaluation of electrospray ionization (ESI) source parameters on an Exactive equipped with a heated electrospray ionization (HESI-II) source with the objective of obtaining uniformly appropriate source conditions for a wide range of lipids, while, at the same time, reducing in-source fragmentation.