Automated Quantitative Analysis of Complex Lipidomes by Liquid Chromatography/Mass Spectrometry

Hermansson, Martin; Uphoff, Andreas; Käkelä, Reijo; Somerharju, Pentti

doi:10.1021/ac048489s

Cited by 197 publications

(130 citation statements)

References 52 publications

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“…Mass Spectrometry and Data Analysis-After the addition of NH 4 OH (4%), the sample was infused at 6 l/min to a Micromass Quattro Micro triple-quadrupole mass spectrometer operated as described previously (37). The D 9 -labeled and unlabeled PC species were detected by scanning for precursors of m/z ϩ193 and m/z ϩ184, respectively.…”

Section: Methodsmentioning

confidence: 99%

Substrate Efflux Propensity Plays a Key Role in the Specificity of Secretory A-type Phospholipases

Haimi¹,

Hermansson²,

Batchu³

et al. 2010

Journal of Biological Chemistry

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To better understand the principles underlying the substrate specificity of A-type phospholipases (PLAs), a high throughput mass spectrometric assay was employed to study the effect of acyl chain length and unsaturation of phospholipids on their rate of hydrolysis by three different secretory PLAs in micelles and vesicle bilayers. With micelles, each enzyme responded differently to substrate acyl chain unsaturation and double bond position, probably reflecting differences in the accommodative properties of their substrate binding sites. Experiments with saturated acyl positional isomers indicated that the length of the sn2 chain was more critical than that of the sn1 chain, suggesting tighter association of the former with the enzyme. Only the first 9 -10 carbons of the sn2 acyl chain seem to interact intimately with the active site. Strikingly, no discrimination between positional isomers was observed with vesicles, and the rate of hydrolysis decreased far more with increasing chain length than with micelles, suggesting that translocation of the phospholipid substrate to the active site is rate-limiting with bilayers. Supporting this conclusion, acyl chain structure affected hydrolysis and spontaneous intervesicle transfer, which correlates with lipid efflux propensity, analogously. We conclude that substrate efflux propensity plays a more important role in the specificity of secretory PLA 2 s than commonly thought and could also be a key attribute in phospholipid homeostasis in which (unknown) PLA 2 s are key players.Type A phospholipases (PLAs) 3 are ubiquitous enzymes involved in many biological phenomena, such as signal transduction (1) and phospholipid homeostasis (2), including acyl chain remodeling (3) and degradation (4). However, the identities of the specific proteins involved in these phenomena have often not been established. PLAs are also clinically relevant because they have been implicated in many common diseases or disorders, including atherosclerosis, allergy, Alzheimer disease, and cancer (5).Several PLAs display significant specificity toward the phospholipid acyl chain(s) in vitro (6, 7). However, despite numerous studies, the factors underlying such specificity have remained largely elusive. In principle, two factors contribute to selective hydrolysis of phospholipids by PLA: 1) accommodation of the acyl chains in the protein lipid binding site and 2) the ease of efflux of the phospholipid substrate from the bilayer (8, 9). The understanding of acyl chain accommodation has been greatly hampered by the lack of crystal structures of PLAs complexed with a physiological substrate. Crystal structures have been obtained for some PLAs with a bound noncleavable shortchain substrate analogue (10 -13), but because these analogues have truncated acyl chains, they only provide very limited information on the factors underlying acyl chain specificity. Furthermore, the information could also be biased, since studies with other lipid-binding proteins have shown that the mode of accommodation of an alkyl...

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Section: Methodsmentioning

confidence: 99%

Substrate Efflux Propensity Plays a Key Role in the Specificity of Secretory A-type Phospholipases

Haimi¹,

Hermansson²,

Batchu³

et al. 2010

Journal of Biological Chemistry

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“…The lipids were then extracted according to Folch et al (32), except that the solvent contained 0.1 M HCl. For quantification of the lipid species by mass spectrometry, a mixture of internal standards was added at the one-phase stage of extraction (33). After evaporation of the solvents, the lipids were redissolved in chloroform/methanol (1:2, v/v) and stored at Ϫ20°C.…”

Section: Methodsmentioning

confidence: 99%

“…Mass Spectrometry and Data Analysis-After addition of aqueous NH 3 (4%), the sample was infused (6 l/min) into a Micromass Quattro Micro triple-quadrupole mass spectrometer operated as previously (33). The D 4 -labeled and unlabeled PE species were selectively detected using constant neutral-loss (NL) scanning of 145 and 141, respectively, in the positive ion mode (34).…”

Section: Methodsmentioning

confidence: 99%

Electrospray Ionization Mass Spectrometry and Exogenous Heavy Isotope-labeled Lipid Species Provide Detailed Information on Aminophospholipid Acyl Chain Remodeling

Kainu

Hermansson

Somerharju

2008

Journal of Biological Chemistry

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Mammalian cells maintain the phospholipid compositions of their different membranes remarkably constant. Beside de novo synthesis, degradation, and intracellular trafficking, acyl chain remodeling plays an important role in phospholipid homeostasis. However, many key details of this process remain unresolved, largely because of limitations of existing methodologies. Here we describe a novel approach that allows one to study metabolism of individual phospholipid species in unprecedented detail. Forty different phosphatidylethanolamine (PE) or -serine (PS) species with a deuterium-labeled head group were synthesized and introduced to BHK21 or HeLa cells using cyclodextrin-mediated transfer. Their metabolism was then monitored in detail by electrospray ionization mass spectrometry. Atypical PE and PS species were rapidly remodeled at both sn1 and sn2 position, yielding a molecular species profile similar to that the endogenous PE and PS. In contrast, remodeling of exogenous species identical or similar to major endogenous ones was more limited and much slower. Major differences in remodeling pathways and kinetics were observed between species within a class, as well as between corresponding PE and PS species. These data along with those obtained with pharmacological inhibitors strongly suggest that multiple lipid class-specific A-type phospholipases and acyl transferases are involved in aminophospholipid remodeling. In conclusion, the approach described here provides highly detailed information on remodeling of exogenously added (amino)glycerophospholipids and should thus be very helpful when elucidating the proteins and processes maintaining molecular species homeostasis.

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“…Most of the experiments were performed using phospholipid mixtures composed of PtdGro, PtdCho, and PtdEtn at a molar ratio of 1:10:15, which was approximately the molar ratio of these three classes of phospholipid in biological samples [24]. In addition, lipid analysis after intrasource separation and selective ionization was conducted in the low concentration region since many studies have demonstrated that the linear dynamic range for quantitative analysis of lipids occurs in the region of Ͻ50 pmol of a lipid class/ L in chloroform/ methanol (1:1, vol/vol) [13,[25][26][27].…”

Section: Intrasource Separation and Selective Ionization Of Phospholimentioning

confidence: 99%

Factors influencing the electrospray intrasource separation and selective ionization of glycerophospholipids

Han

Yang

et al. 2006

J. Am. Soc. Mass Spectrom.

119

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The external electric field induces a separation of cations from negative electrolyte ions in the infusate while differential ionization of molecular species that possess differential electrical propensities can be induced in either the positive-or negative-ion mode during the electrospray ionization process. These physical and electrical processes that occur in the electrospray ion source have been used to selectively ionize lipid classes possessing different electrical propensities that are now known as "intrasource separation and selective ionization". However, the chemical principles underlying charge-dependent alterations in ionization efficiencies responsible for the selective ionization of lipid classes are not known with certainty. Herein, we examined the multiple factors that contribute to intrasource separation and selective ionization of lipid classes under optimal instrumental conditions. We demonstrated that many different lipid classes could be selectively ionized in the ion source and that intrasource resolution of distinct molecular constituents was independent of lipid concentration, flow rate, and residual ions under most experimental conditions. Moreover, the presence of alkaline conditions facilitates the selective ionization of many lipid classes through a mechanism independent of the design of the ESI ion source. Collectively, this study provides an empirical foundation for understanding the chemical mechanisms underlying intrasource separation and selective ionization of lipid classes that can potentially be used for global analysis of cellular lipidomes without the need for chromatographic separation. (J Am Soc Mass Spectrom 2006, 17, 264 -274)

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Automated Quantitative Analysis of Complex Lipidomes by Liquid Chromatography/Mass Spectrometry

Cited by 197 publications

References 52 publications

Substrate Efflux Propensity Plays a Key Role in the Specificity of Secretory A-type Phospholipases

Substrate Efflux Propensity Plays a Key Role in the Specificity of Secretory A-type Phospholipases

Electrospray Ionization Mass Spectrometry and Exogenous Heavy Isotope-labeled Lipid Species Provide Detailed Information on Aminophospholipid Acyl Chain Remodeling

Factors influencing the electrospray intrasource separation and selective ionization of glycerophospholipids

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