Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> MALDI-TOF <s> Fast atom bombardment-produced [M + Na]+ ions of tristearoylglycerol and [M - H]- ions of stearic or nervonic acid undergo charge-remote fragmentations (CRFs) to produce one series of product ions reflecting CnH2n + 2, losses, whereas electrospray ionization-produced ions fragment to give two series of product ions reflecting CnH2n + 2 and CnH2n + 1 losses. These results and those from previous studies show that the mechanisms and energetics of CRFs are complex and unsettled. We demonstrate that several pathways are simultaneously involved in CRFs, and the preference for certain pathways (by CnH2n + 1 and CnH2n + 2 losses) is determined by the internal energy of the compound itself and the ionization and activation energies that are applied to it. <s> BIB001 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> MALDI-TOF <s> Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is an established tool for the analysis of proteins, whereas it gained by far less interest in the field of lipid analysis. This method works well with phospholipids as well as organic cell extracts and provides high sensitivity and reproducibility. The aim of the present paper is to extend our previous studies to the analysis of lysophospholipids and phospholipid mixtures. To study the suitability of MALDI-TOF mass spectrometry for the analysis of lysophospholipids, different phospholipids like phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, and phosphatidylinositol as well as their mixtures were digested with phospholipase A(2). Positive and negative ion mass spectra of all phospholipids before and after digestion were recorded. In all these cases, the molecular ions of the expected digestion products could be detected and only a very small extent of further fragmentation was observed. On the other hand, spectra of phospholipid mixtures containing phosphatidylcholine were strongly dominated by phosphatidylcholine and lysophosphatidylcholine signals, which prevented the detection of further phospholipids even if those lipids were present in comparable amounts. This is of paramount interest for the analysis of tissue and cell extracts. <s> BIB002 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> MALDI-TOF <s> A novel method is presented for direct coupling of high-performance thin-layer chromatography (HPTLC) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the analysis of biomolecules. A first key feature is the use of a liquid matrix (glycerol), which provides a homogeneous wetting of the silica gel and a simple and fast MALDI preparation protocol. A second is the use of an Er:YAG infrared laser, which ablates layers of ∼10-μm thickness of analyte-loaded silica gel and provides a soft desorption/ionization of even very labile analyte molecules. The orthogonal time-of-flight mass spectrometer employed in this study, finally provides a high accuracy of the mass determination, which is independent of any irregularity of the silica gel surface. The analytical potential of the method is demonstrated by the compositional mapping of a native GM3 (II3-α-Neu5Ac−LacCer) ganglioside mixture from cultured Chinese hamster ovary cells. The analysis is characterized by a high relative sensi... <s> BIB003 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> MALDI-TOF <s> A fully automated interface to couple high-performance thin-layer chromatography (HPTLC) with mass spectrometry (MS) is described. This universal hands-free interface connects intact normal-phase plates to any liquid chromatography/mass spectrometry (LC/MS) system without any adjustments or modifications to the mass spectrometer. The interface extracts the complete substance band with its depth profile and thus allows detections in the pg/band range. The high performance of the automated interface was evaluated through caffeine quantification in real samples, viz., energy drinks and pharmaceutical tablets, without internal standard. Following chromatographic separation on silica gel 60 F(254) HPTLC plates, caffeine bands were eluted from the plate by means of the automated interface to the electrospray ionization (ESI) source of a triple-quadrupole mass spectrometer. Since in full scan mode only the protonated molecule [M+H](+) was observed, caffeine quantification was performed using the selected-ion monitoring (SIM) mode at m/z 195. The validation showed highly reliable results for the linear range (R(2) = 0.9973), repeatability (RSD = 5.6%, n = 6) and intermediate precision (RSD = 1.5%, n = 3). Regarding accuracy the results obtained by HPTLC/MS were not statistically different (F-test, t-test) from those obtained by validated HPTLC/UV methods. Hence, this interface proved to be one of the most reliable and universal interfaces for HPTLC/MS. <s> BIB004 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> MALDI-TOF <s> The application of a recently developed direct coupling of high-performance thin-layer chromatography (HPTLC) and infrared matrix-assisted laser desorption/ionization orthogonal extracting time-of-flight mass spectrometry (Dreisewerd, K.; Müthing, J.; Rohlfing, A.; Meisen, I.; Vukelic, Z.; Peter-Katalinic, J.; Hillenkamp, F.; Berkenkamp, S. Anal. Chem. 2005, 77, 4098-4107) to the analysis of phospholipid mixtures is demonstrated. Mixtures of six phospholipid types were exemplarily analyzed. The sensitivity was found to be in the range between about 10 and 150 pmol of material spotted for HPTLC, depending on phospholipid acidity, Rf value, and ion polarity. The lateral resolution of the analysis is on the order of the laser focus diameter of about 220 x 300 microm2, allowing differentiation between phospholipid species of different acyl chain composition within one single HPTLC band, which were undistiguishable by a mere visual assessment. Analyte diffusion due to the addition of glycerol to the HPTLC plate was found to be-if at all notable-of only minor importance. <s> BIB005 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> MALDI-TOF <s> The thin-layer chromatography (TLC) immunoenzyme overlay assay is a widely used tool for antibody-mediated identification of glycosphingolipids (GSLs) in mixtures. However, because the majority of GSLs is left unexamined in a chromatogram of a single assay, we developed a novel method that permits detection of various GSLs by sequential multiple immunostaining combined with individual coloring of GSLs in the same chromatogram. Specific staining was achieved by means of primary anti-GSL antibodies, directed against lactosylceramide, globotriaosylceramide, and globotetraosylceramide, in conjunction with alkaline phosphatase (AP)- or horseradish peroxidase (HRP)-conjugated secondary antibodies together with the appropriate chromogenic substrates. Triple coloring with 5-bromo-4-chloro-3-indolyl phosphate (BCIP)-AP, Fast Red-AP, and 3,3'-diaminobenzidine (DAB)-HRP resulted in blue, red, and black precipitates, respectively, following three sequential immunostaining rounds. Structures of antibody-detected GSLs were determined by direct coupling of TLC with infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry. This combinatorial technique was used to demonstrate structural GSL profiling of crude lipid extracts from human hepatocellular cancer. This powerful technology allows efficient structural characterization of GSLs in small tissue samples and marks a further step forward in the emerging field of glycosphingolipidomics. <s> BIB006 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> MALDI-TOF <s> Triacylglycerols were analyzed as cationized species (Li(+), Na(+), K(+)) by high-energy CID at 20 keV collisions utilizing MALDI-TOF/RTOF mass spectrometry. Precursor ions, based on [M + Li](+)-adduct ions exhibited incomplete fragmentation in the high and low m/z region whereas [M + K](+)-adducts did not show useful fragmentation. Only sodiated precursor ions yielded product ion spectra with structurally diagnostic product ions across the whole m/z range. The high m/z region of the CID spectra is dominated by abundant charge-remote fragmentation of the fatty acid substituents. In favorable cases also positions of double bonds or of hydroxy groups of the fatty acid alkyl chains could be determined. A-type product ions represent the end products of these charge-remote fragmentations. B- and C-type product ions yield the fatty acid composition of individual triacylglycerol species based on loss of either one neutral fatty acid or one sodium carboxylate residue, respectively. Product ions allowing fatty acid substituent positional determination were present in the low m/z range enabling identification of either the sn-1/sn-3 substituents (E-, F-, and G-type ions) or the sn-2 substituent (J-type ion). These findings were demonstrated with synthetic triacylglycerols and plant oils such as cocoa butter, olive oil, and castor bean oil. Typical features of 20 keV CID spectra of sodiated triacylglycerols obtained by MALDI-TOF/RTOF MS were an even distribution of product ions over the entire m/z range and a mass accuracy of +/-0.1 to 0.2 u. One limitation of the application of this technique is mainly the insufficient precursor ion gating after MS1 (gating window at 4 u) of species separated by 2 u. <s> BIB007 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> MALDI-TOF <s> Glycosphingolipids (GSLs) play key roles in the manifestation of infectious diseases as attachment sites for pathogens. The thin-layer chromatography (TLC) overlay assay represents one of the most powerful approaches for the detection of GSL receptors of microorganisms. Here we report on the direct structural characterization of microbial GSL receptors by employment of the TLC overlay assay combined with infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry (IR-MALDI-o-TOF-MS). The procedure includes TLC separation of GSL mixtures, overlay of the chromatogram with GSL-specific bacteria, detection of bound microbes with primary antibodies against bacterial surface proteins and appropriate alkaline phosphatase labeled secondary antibodies, and in situ MS analysis of bacteria-specific GSL receptors. The combined method works on microgram scale of GSL mixtures and is advantageous in that it omits laborious and time-consuming GSL extraction from the silica gel layer. This technique was successfully applied to the compositional analysis of globo-series neutral GSLs recognized by P-fimbriated Escherichia coli bacteria, which were used as model microorganisms for infection of the human urinary tract. Thus, direct TLC/IR-MALDI-o-TOF-MS adds a novel facet to this fast and sensitive method offering a wide range of applications for the investigation of carbohydrate-specific pathogens involved in human infectious diseases. <s> BIB008 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> MALDI-TOF <s> Abstract The fragmentation behavior of various types of precursor ions ( [M−H] − , [M+H] + , [M+Na] + , [M+2Na−H] + and [M+3Na−2H] + ) of the six major classes of glycerophospholipids (glycerophosphatidic acid, glycerophosphatidylglycerol, glycerophosphatidylinositol, glycerophosphatidylserine, glycerophosphatidylethanolamine and glycerophosphatidylcholine) was studied in detail by high-energy collision-induced dissociation (CID)-TOF/RTOF mass spectrometry. Whereas negative-ion CID-spectra of the [M−H] − ion of these lipids primarily exhibited the molecular mass of the fatty acid substituents, protonated and monosodiated precursor ions showed abundant loss of the polar head group identifying the class of glycerophospholipid. In contrast, doubly and triply sodiated singly charged glycerophospholipid precursor ions not only yielded charge-remote fragmentation of the fatty acid substituents, but also several abundant low-mass product ions of diagnostic value for the identification of the phospholipid class as well as specific phosphate-related sodium adduct ions for determining the number of sodium cations attached to the precursor ion. These findings finally lead to the establishment of common fragmentation rules as well as a universal product ion nomenclature for all types of glycerophospholipid precursor ions investigated (applicable for low- and high-energy CID). This nomenclature is based on the A -, B -, C -, D -, E -, F -. and G -product ion nomenclature previously published for sodiated triacylglycerol precursor ions [C. Cheng, M.L. Gross, E. Pittenauer, Anal. Chem. 70 (1998) 4417–4426] with adaptations for the type of precursor ion and class of glycerophospholipid investigated. <s> BIB009
Although not as widely used as ESI instruments, MALDI-TOF is a good complementary choice for lipids in the mass range above m/z 500. The soft ionization properties of MALDI result in intact molecular adduct ions. Paired with the speed of MALDI-TOF analysis this fact renders the technology very suitable for fast screening of lipids. MALDI-TOF instruments equipped with a reflectron nowadays regularly achieve 10,000 resolution and 30 ppm mass accuracy, which is sufficient for assigning intact molecular ions of lipid species. Choice of the right MALDI matrix is an important step for good sensitivity. 2,5-Dihydroxy benzoic acid, α-cyano-4-hydroxy-cinnamic acid, 9-amino-acridine and 2-mercaptobenzothiazole are often used matrix compounds. On the downside of this technology, the mass range below m/z 500 is usually not amenable due to matrix interferences. MALDI-TOF has been used for analysis of various lipid classes  , but, similar to ESI, MALDI also has certain quantitative limits for crude mixtures due to ion suppression effects BIB002 . This effect can become quite severe, particularly as MALDI does not allow any chromatographic separation to be coupled directly to the instrument. Recently, TLC/MALDI was proposed by several groups as an interesting alternative BIB004 BIB003 . Instead of a MALDI target, a developed TLC plate with separated lipid spots is used as target. This approach is very promising, because TLC is still widely used in lipid analysis and coupling with MALDI-TOF vastly increases identification certainty of TLC spots. In such a setup, visualization of TLC spots should be reversible as well as non destructive and is either achieved by dyes non-covalently bound to lipids BIB003  or by immunodetection with antibodies BIB006 BIB008 . Conventional UV lasers are used with most MALDI instruments and only penetrate the applied MALDI matrix but not the silica gel itself. Therefore UV lasers are prone to distortion of results, because only lipids at the surface of the silica particles can be detected. In contrast, IR lasers do penetrate silica gel particles and are thus certainly the better choice for TLC/MALDI targets. Although this technology is still in its infancy, there are already promising results on glycosphingolipids BIB006 BIB008 and glycerophospholipids BIB005 . Further technological progress could develop TLC/MALDI-TOF into a fast and easy to use survey method for lipidomic analysis. While most instruments nowadays are equipped with low energy CID devices, MALDI-TOF/TOF offers an alternative for high energy CID. The advantage of high energy CID at 20 keV is induction of abundant charge remote fragmentation, which in turn allows structural analysis of lipids at the level of fatty acyl sn position and double bond location BIB001 . With the decline of sector instruments in lipid mass spectrometry, MALDI-TOF/TOF has the potential to fill this gap. Pittenauer et al. show the use of MALDI-TOF/TOF for such applications in an excellent manner BIB009 BIB007 . Alkali cationized TG show a wealth of structure specific fragments indicative for location of fatty acids and their double bonds ( Figure 4) . One challenge still to be solved is the quite wide isolation window of about 4 Da which makes precursor selection especially in biological lipid extracts problematic. Although this application is clearly not a high throughput method and lacks automated software solutions, it is still highly useful for structural determination of selected lipid species. 