Convenient structural analysis of glycosphingolipids using MALDI-QIT-TOF mass spectrometry with increased laser power and cooling gas flow
ABSTRACT Matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (MALDI-QIT-TOF MS) was applied to the structural characterization of neutral glycosphingolipids. Lithium adduct ions of glycosphingolipids were analyzed using MALDI-QIT-TOF MS under strong conditions of increased laser power and cooling gas flow. The relative intensities of fragment ions were increased under the strong conditions, and the resulting spectra revealed the presence of oligosaccharide ions fragmented from the glycosphingolipids. Consequently, the oligosaccharide sequences of the glycosphingolipids were readily obtained. To obtain more detailed structural information, MS/MS (MS2) and MS/MS/MS (MS3) analyses were performed with selection of the lactosylceramide and ceramide ions, respectively. The resulting data were sufficient to determine the structures of both the oligosaccharide and the ceramide moiety of each glycosphingolipid. The fragmentation patterns of MS2 and MS3 for Forssman glycolipid under the strong conditions were comparable to those of MS3 and MS4 obtained under standard conditions, respectively. Thus, MALDI-QIT-TOF MS with increased laser power and cooling gas flow is a convenient method for glycosphingolipid analysis.
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- "Matrix assisted laser desorption ionization has been successfully used to ionize multiple classes of SL such as Cer, HexCer, LacCer, ST, globosides, gangliosides, and SM   . Sample preparation for MALDI analysis usually occurs by mixing a solution of small organic acid (matrix) with a solution containing the analyte of interest. "
ABSTRACT: Sphingolipids are a highly diverse category of molecules that serve not only as components of biological structures but also as regulators of numerous cell functions. Because so many of the structural features of sphingolipids give rise to their biological activity, there is a need for comprehensive or "sphingolipidomic" methods for identification and quantitation of as many individual subspecies as possible. This review defines sphingolipids as a class, briefly discusses classical methods for their analysis, and focuses primarily on liquid chromatography tandem mass spectrometry (LC-MS/MS) and tissue imaging mass spectrometry (TIMS). Recently, a set of evolving and expanding methods have been developed and rigorously validated for the extraction, identification, separation, and quantitation of sphingolipids by LC-MS/MS. Quantitation of these biomolecules is made possible via the use of an internal standard cocktail. The compounds that can be readily analyzed are free long-chain (sphingoid) bases, sphingoid base 1-phosphates, and more complex species such as ceramides, ceramide 1-phosphates, sphingomyelins, mono- and di-hexosylceramides, sulfatides, and novel compounds such as the 1-deoxy- and 1-(deoxymethyl)-sphingoid bases and their N-acyl-derivatives. These methods can be altered slightly to separate and quantitate isomeric species such as glucosyl/galactosylceramide. Because these techniques require the extraction of sphingolipids from their native environment, any information regarding their localization in histological slices is lost. Therefore, this review also describes methods for TIMS. This technique has been shown to be a powerful tool to determine the localization of individual molecular species of sphingolipids directly from tissue slices.Biochimica et Biophysica Acta 07/2011; 1811(11):838-53. DOI:10.1016/j.bbalip.2011.06.027 · 4.66 Impact Factor
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- "d fatty acids in the lipid backbones ( Fig . 4 ) . MALDI usually uses matrix molecules to assist the efficient production of singly charged ions , which in conjunction with the high m / z range of TOF instruments , has aided in the observation of higher molecular weight GSPs . Therefore , studies of more complex GSPs often utilize this technique ( Suzuki et al . 2006 ; Landoni et al . 2008 ; Chan et al . 2009 ) . One disadvantage of MALDI is that matrices produce abundant background chemical noise at lower m / z values , which precludes analysis of free sphingoid bases . However , new advances in matrix choices and high pressure sources may provide an opportunity to study these lower molecular weigh"
ABSTRACT: Sphingolipids (SP) are a complex class of molecules found in essentially all eukaryotes and some prokaryotes and viruses where they influence membrane structure, intracellular signaling, and interactions with the extracellular environment. Because of the combinatorial nature of their biosynthesis, there are thousands of SP subspecies varying in the lipid backbones and complex phospho- and glycoheadgroups. Therefore, comprehensive or "sphingolipidomic" analyses (structure-specific, quantitative analyses of all SP, or at least all members of a critical subset) are needed to know which and how much of these subspecies are present in a system as a step toward understanding their functions. Mass spectrometry and related novel techniques are able to quantify a small fraction, but nonetheless a substantial number, of SP and are beginning to provide information about their localization. This review summarizes the basic metabolism of SP and state-of-art mass spectrometric techniques that are producing insights into SP structure, metabolism, functions, and some of the dysfunctions of relevance to neuromedicine.Neuromolecular medicine 12/2010; 12(4):306-19. DOI:10.1007/s12017-010-8132-8 · 3.89 Impact Factor
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- "2.5. MALDI, mass spectrometry, and tandem mass spectrometry and gangliosides (Suzuki et al., 2006). Samples are prepared for MALDI by mixing a solution containing the analyte with a solution containing a MALDI matrix compound, which is typically a small substituted organic acid that contains a moiety that can absorb the photonic energy of the laser. "
ABSTRACT: Due to the large number of highly bioactive subspecies, elucidation of the roles of sphingolipids in cell structure, signaling, and function is beginning to require that one perform structure-specific and quantitative (i.e., "sphingolipidomic") analysis of all individual subspecies, or at least of those are relevant to the biologic system of interest. As part of the LIPID MAPS Consortium, methods have been developed and validated for the extraction, liquid chromatographic (LC) separation, and identification and quantitation by electrospray ionization (ESI), tandem mass spectrometry (MS/MS) using an internal standard cocktail that encompasses the signaling metabolites (e.g., ceramides, ceramide 1-phosphates, sphingoid bases, and sphingoid base 1-phosphates) as well as more complex species (sphingomyelins, mono- and di-hexosylceramides). The number of species that can be analyzed is growing rapidly with the addition of sulfatides and other complex sphingolipids as more internal standards become available. This review describes these methods as well as summarizes others from the published literature. Sphingolipids are an amazingly complex family of compounds that are found in all eukaryotes as well as some prokaryotes and viruses. The size of the sphingolipidome (i.e., all of the individual molecular species of sphingolipids) is not known, but must be immense considering mammals have over 400 headgroup variants (for a listing, see http://www.sphingomap.org), each of which is comprised of at least a few-and, in some cases, dozens-of lipid backbones. No methods have yet been developed that can encompass so many different compounds in a structurally specific and quantitative manner. Nonetheless, it is possible to analyze useful subsets of the sphingolipidome, such as the backbone sphingolipids involved in signaling (sphingoid bases, sphingoid base 1-phosphates, ceramides, and ceramide 1-phosphates) and metabolites at important branchpoints, such as the partitioning of ceramide into sphingomyelins, glucosylceramides, galactosylceramides, and ceramide 1-phosphate versus turnover to the backbone sphingoid base. This review describes methodology that has been developed as part of the LIPID MAPS Consortium (www.lipidmaps.org) as well as other methods that can be used for sphingolipidomic analysis to the extent that such is currently feasible. The focus of this review is primarily mammalian sphingolipids; hence, if readers are interested in methods to study other organisms, they should consult the excellent review by Stephen Levery in another volume of Methods in Enzymology (Levery, 2005), which covers additional species found in plants, fungi, and other organisms. It should be noted from the start that although many analytical challenges remain in the development of methods to analyze the full "sphingolipidome," the major impediment to progress is the limited availability of reliable internal standards for most of the compounds of interest. Because it is an intrinsic feature of mass spectrometry that ion yields tend to vary considerably among different compounds, sources, methods, and instruments, an analysis that purports to be quantitative will not be conclusive unless enough internal standards have been added to correct for these variables. Ideally, there should be some way of standardizing every compound in the unknown mixture; however, that is difficult, if not impossible, to do because the compounds are not available, and the inclusion of so many internal standards generates a spectrum that may be too complex to interpret. Therefore, a few representative internal standards are usually added, and any known differences in the ion yields of the analytes of interest versus the spiked standard are factored into the calculations. Identification of appropriate internal standards has been a major focus of the LIPID MAPS Consortium, and the methods described in this review are based on the development of a certified (i.e., compositionally and quantitatively defined by the supplier) internal standard cocktail that is now commercially available (Avanti Polar Lipids, Alabaster, AL). For practical and philosophical reasons, an internal standard cocktail was chosen over the process of an investigator adding individual standards for only the analytes of interest. On the practical level, addition of a single cocktail minimizes pipetting errors as well as keeping track of whether each internal standard is still usable (e.g., has it degraded while in solution?). Philosophically, the internal standard cocktail was chosen because an underlying premise of systems analysis asserts that, due to the high relevancy of unexpected interrelationships involving more distant components, one can only understand a biological system when factors outside the primary focus of the experiment have also been examined. Indeed, the first payoffs of "omics" and systems approaches involve the discoveries of interesting compounds in unexpected places when a "sphingolipidomic" analytical method was being used as routine practice instead of a simpler method that would have only measured the compound initially thought to be important (Zheng et al., 2006). Thus, routine addition of a broad internal standard cocktail at the outset of any analysis maximizes the opportunity for such discoveries, both at the time the original measurements are made and when one decides to return to the samples later, which can fortunately be done for many sphingolipids because they remain relatively stable in storage.Methods in Enzymology 02/2007; 432:83-115. DOI:10.1016/S0076-6879(07)32004-1 · 2.19 Impact Factor