Complete Structural Elucidation of Triacylglycerols by Tandem Sector Mass Spectrometry

Since the 1950s, X-ray crystallography has been the mainstay of structural biology, providing detailed atomic-level structures that continue to revolutionize our understanding of protein function. From recent advances in this discipline, a picture has emerged of intimate and specific interactions between lipids and proteins that has driven renewed interest in the structure of lipids themselves and raised intriguing questions as to the specificity and stoichiometry in lipid-protein complexes. Herein we demonstrate some of the limitations of crystallography in resolving critical structural features of ligated lipids and thus determining how these motifs impact protein binding. As a consequence, mass spectrometry must play an important and complementary role in unraveling the complexities of lipid-protein interactions. We evaluate recent advances and highlight ongoing challenges towards the twin goals of (1) complete structure elucidation of low, abundant, and structurally diverse lipids by mass spectrometry alone, and (2) assignment of stoichiometry and specificity of lipid interactions within protein complexes.Key words: Lipid-protein interactions, Structural biology, Structural lipidomics, Protein mass spectrometry Lipid-Protein Interactions P roteins and lipids, both integral for the function of all biological systems, are chemically distinct. A protein's three-dimensional structure and ultimately its function are defined by peptide-bonded amino acids and subsequent posttranslational modifications. In contrast, lipids are smaller molecules than proteins, but have a larger array of building blocks and linkage chemistries. Because of these inherent chemical differences, the optimal tools for de novo structural characterization differ between proteins and lipids.Lipid-protein interactions are critical for maintaining biological function in the membrane bilayer, the cytosol, and extracellular space. In the membrane, the chemical structures of lipid solvent molecules are important for the correct folding, insertion, structure, and function of membrane proteins. The interactions between enzymes and their respective lipid substrates are critical for correct substrate recognition and catalysis (e.g., lipoxygenase [1]). Additionally, lipid-protein interactions are increasingly being realized as important in maintaining cellular structure (e.g., cytoskeletal anchoring [2]), scaffolding and localization of multi-subunit protein complexes (e.g., associated kinase anchoring protein complexes [3]), and as second messengers in signal transduction (e.g., protein kinase C [4]).

Section: Locating Carbon-carbon Double Bondsmentioning

confidence: 99%

Time to Face the Fats: What Can Mass Spectrometry Reveal about the Structure of Lipids and Their Interactions with Proteins?

Brown

Mitchell

Oakley

et al. 2012

“…More recently, electrospray ionization and atmospheric pressure ion-ization have been employed to analyze TAGs at the molecular species level using considerably gentler conditions° [12][13][14][15].°Notable°advances°were°made°by°Han and°Gross° [16]°and°Hsu°and°Turk° [17]°using°lithium adducts of TAGs with collisional activation of the [M ϩ Li] ϩ ions to yield diacyl product ions that revealed specific information about acyl groups esterified to individual TAG molecular species. Also, Byrdwell and Neff employed MS 3 to study the composition of synthetic°TAG°species° [18].°Cheng,°Gross,°and°Pittenauer used high-energy collisions of ammonium and sodium adducts of TAGs to elucidate acyl position on the glycerol backbone and double-bond location of unsaturated°acyl°moieties° [19]°of°pure°TAG°molecular°spe-cies.…”

mentioning

confidence: 99%

Direct qualitative analysis of triacylglycerols by electrospray mass spectrometry using a linear ion trap

McAnoy

Murphy

2005

161

149

Triacylglycerols (TAGs) isolated from a biological sample provide a challenge for mass spectrometric analysis because of the complexity of naturally occurring TAGs, which may contain different fatty acyl substituents resulting in a large number of molecular species having the identical elemental composition. We have investigated the use of mass spectrometry to obtain unambiguous information as to the individual TAG molecular species present in a complex mixture of triacylglycerols using a linear ion trap mass spectrometer. Ammonium adducts of TAGs, [M ϩ NH 4 ] ϩ , were generated by electrospray ionization, which permitted the molecular weight of each TAG molecular species to be determined. The mechanisms involved in the decomposition of the [M ϩ NH 4 ] ϩ and subsequent fragment ions were investigated using deuterium labeling, MS/MS, and MS 3 experiments. Collision induced decomposition of [M ϩ NH 4 ] ϩ ions resulted in the neutral loss of NH 3 and an acyl side-chain (as a carboxylic acid) to generate a diacyl product ion. MS/MS data were used to identify each acyl group present for a given [M ϩ NH 4 ] ϩ ion, and this information could be combined with molecular weight data to identify possible TAG molecular species present in a biological extract. Subsequent MS 3 experiments on the resultant diacyl product ions, which gave rise to acylium (RCO ϩ ) and related ions, enabled unambiguous TAG molecular assignments. These strategies of MS, MS/MS, and MS 3 experiments were applied to identify components within a complex mixture of neutral lipids extracted from RAW 264.7 cells. (J Am Soc Mass Spectrom 2005, 16, 1498 -1509

“…At 12 l/min the increasing of the internal energy is more dramatic and all the precursor ions are over the threshold of fragmentation; therefore, the formation of the FFA dominates. It is important to mention that fragmentation of TALG has been observed by using atmospheric pressure chemical ionization [2] and tandem sector MS [3] but in any of these works the FFA ammoniated adduct was observed. The fragmentation of TALG into FFA as a result of varying the drying gas flow in ESI-MS experiments, to our knowledge, has not been previously reported.…”

Section: Modelling Of the Lcitms Variables And Estimation Of Their Opmentioning

confidence: 99%

“…A s an essential polyunsaturated fatty acid, ␣-linoleic acid (ALA, 18:3n-3) is not synthesized de novo in animals, but must be provided in their diets in the form of triacylglycerol [1]. The development of analytical methods in the analysis and quantification of ALA and more general triacylglycerols (TAG) is of considerable importance [2,3] in view of their involvement in many vital biochemical processes and their association with the alleviation of a wide variety of diseases [4,5]. Quantification of TAG using coupled techniques such as LCMS depends mainly on two aspects, the sample preparation technique and the instrumental condition settings.…”

mentioning

confidence: 99%

Chemometric approaches in calibration experiments of trilinolenoylglycerol by liquid chromatography ion-trap mass spectrometry

Araujo¹,

Nguyen²,

Frøyland³

2005

The article presents a comprehensive account of the application of chemometric approaches to determine the factors that influence the tri-␣-linolenoylglycerol (TALG) ammoniated adduct signal m/z 890.6 in an ion-trap mass spectrometer coupled to a liquid chromatograph and the estimation of different sources of errors involved in TALG calibration experiments. It was found that by using experimental design, the influence of the nebulizer pressure on the analytical signal is less pronounced than the influence of the drying gas flow and the chromatographic flow rate. The results revealed that without using tandem mass or atmospheric pressure chemical ionization, it is possible to fragment the TALG backbone and obtain the free ␣-linolenic acid by varying the drying gas flow and using an electrospray interface. The error decomposition approach revealed that the preparation error was 26 times higher than the instrumental error. [4,5]. Quantification of TAG using coupled techniques such as LCMS depends mainly on two aspects, the sample preparation technique and the instrumental condition settings. Well-established protocols and books [6 -8] have been devoted to the former and nowadays many research laboratories around the world invest time and resources to investigate new, rapid, and simpler techniques. Works regarding the latter aspect are not widespread in the literature, perhaps because variation of the instrumental conditions can cause drastic alterations to the results obtained with a tested and optimized method [9]. Continuity of results within long periods of time (years) is of paramount importance in clinical studies [10], impairing in some degree the interest in the exploration of new or existing methodologies aiming at optimizing the setting of the LCMS instrumental conditions used in the quantitative analysis of TAG. Superb LCMS instruments equipped with automatic optimization devices could be another reason for the limited number of publications on this field. It has been reported that the sole chromatographic system is influenced by more than 50 factors [11], hence the complexity of chromatography coupled to mass spectrometry overwhelms even the most sophisticated software intended for automatic optimization and method development. The classic one-at-the-time methodology for optimization has been widely used in TAG quantification studies. One of the drawbacks of this method is that it is a tedious procedure and the true optimum can be missed if there are important interactions between the factors. The optimization of a LCMS method is prone to difficulties resulting from the inherent high number of instrumental factors involved and their interactions. The determination of the local response surface and exploitation of the local factor dependence is essential. The discovery and application of methods to that effect are extremely important. This study was undertaken, first to estimate the optimal combination of liquid chromatography-ion-trap-mass spectrometry (LCITMS) experimental variables, specifically nebu...