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There is emerging evidence that lipids play an important role in many neurodegenerative processes, for example in Alzheimer's disease (AD). Although different lipid alterations in the AD brain have been reported, there have only been very few investigations of lipid changes in the cerebrospinal fluid (CSF). Recent developments in mass spectrometry (MS) have enabled fast and sensitive detection of lipid species in different biological matrixes. In this study we developed an on-line HPLC-MS method for phospholipid profiling in the CSF based on nano-HPLC separation using an Amide column and detection with electrospray (ESI) quadrupole-time of flight (QTOF) MS. We achieved good separation, reproducibility, and sensitivity in monitoring of the major phospholipid classes, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI), and sphingomyelin (SM) in CSF. To emphasize the applicability of the method, a pilot study was performed on a group of CSF samples (N = 16) from individuals with probable AD and non-demented controls. We observed a statistically significant increase of SM levels (24.3 ± 2.4%) in CSF from probable AD individuals vs. controls. Our findings indicate that SM levels in the CSF could potentially provide a new lead in AD biomarker research, and show the potential of the method for disease-associated CSF phospholipid screening.

In less than 5 years, an impressive number of methods based on nano-LC and HPLC-chip coupled online to MS were developed and implemented to comprehensively address structural heterogeneity of glycoconjugates and glycans in biological matrices. C18, graphitized carbon and amide-based stationary phases were adapted to nanoflow level and on chip format, leading to improved sensitivity of structural analysis and superior level of information on highly complex glycan and glycoconjugate mixtures. This review offers a summary of the recent progress in the application of nano-LC and HPLC-chip-MS in glycoanalytics of glycopeptides, glycoprotein glycans, glycosaminoglycans, oligosaccharides and glycosphingolipids.

The introduction of micro- and nanochip front end technologies for electrospray mass spectrometry addressed a major challenge in carbohydrate analysis: high sensitivity structural determination and heterogeneity assessment in high dynamic range mixtures of biological origin. Chip-enhanced electrospray ionization was demonstrated to provide reproducible performance irrespective of the type of carbohydrate, while the amenability of chip systems for coupling with different mass spectrometers greatly advance the chip/MS technique as a versatile key tool in glycomic studies. A more accurate representation of the glycan repertoire to include novel biologically-relevant information was achieved in different biological sources, asserting this technique as a valuable tool in glycan biomarker discovery and monitoring. Additionally, the integration of various analytical functions onto chip devices and direct hyphenation to MS proved its potential for glycan analysis during the recent years, whereby a new analytical tool is on the verge of maturation: lab-on-chip MS glycomics. The achievements until early beginning of 2007 on the implementation of chip- and functional integrated chip/MS in systems glycobiology studies are reviewed here., ((c) 2009 Wiley Periodicals, Inc.)

A superior approach involving nano-high-performance liquid chromatography (nano-HPLC) in on-line conjunction to electrospray ionization quadrupole time-of-flight mass spectrometry (ESI QTOF MS) and tandem MS for screening and structural characterization of complex mixtures of neutral glycosphingolipids (GSLs) is here described. Neutral GSLs purified from human erythrocytes were efficiently separated according to the differences in carbohydrate chain length by an optimized nano-HPLC protocol and flow-through detected by ESI QTOF MS at the low femtomole level. Additionally, GSL species were accurately distinguished from the accompanying lipids in the mixture, thus permitting the determination of detailed structural characteristics by data-dependent analysis for identification of GSL constitution within single experiments. An alternative nano-HPLC/ESI QTOF MS approach was designed for dissection of unsaturation/saturation degree of the ceramide moieties defining the hydrophobic portion of GSLs and subsequent localization by nano-HPLC/ESI QTOF MS/MS of the -CH=CH- within the ceramide regions. The method is fast, highly sensitive, and high-throughput amenable and is highlighted as a new and valuable analytical dimension in glycolipidomics.

Currently two types of chip systems are used in conjunction with MS: out-of-plane devices, where hundreds of nozzles, nanospray emitters are integrated onto a single silicon substrate from which electrospray is established perpendicular to the substrate, and planar microchips, embedding a microchannel at the end of which electrospray is generated in-plane, on the edge of the microchip. In the last two years, carbohydrate research greatly benefited from the introduction and implementation of the chip-based MS. In two laboratories the advantages of the chip electrospray in terms of ionization efficiency, sensitivity, reproducibility, quality of data in combination with high mass accuracy, and resolution of detection were systematically explored for several carbohydrate classes: O- and N-glycopeptides, oligosaccharides, gangliosides and glycoprotein-derived O- and N-glycans, and glycopeptides. The current state-of-the-art in interfacing the chip electrospray devices to high-performance MS for carbohydrate analysis, and the particular requirements for method optimization in both positive and negative ion modes are reviewed here. The recent applications of these miniaturized devices and their general potential for glycomic-based surveys are highlighted.

We report on the development of a novel methodology to extend the limits of capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) general applicability. A sheathless on-line CE-ESI-MS setup was optimized on standard monosaccharide mixture to operate in reverse polarity and negative ion mode for MS detection without pressure to assist the sample migration, coating of the capillaries, and/or sample derivatization. This approach was further applied for screening of a complex glycopeptide mixture obtained from the urine of a patient diagnosed with N-acetylhexosaminidase deficiency, known as Schindler's disease. The potential of this methodology in terms of high sensitivity, separation efficiency, resolution, and reproducibility is demonstrated. In combination with the high quality of MS data, a new, significantly improved insight into the sample heterogeneity is possible.

A protocol for negative ion nanoelectrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (-)nanoESI-FTICR MS, investigation of complex biological mixtures consisting of sialylated or sulfated glycosphingolipids (GSL) expressing high heterogeneity in the ceramide portion is described. Different instrumental and solvent conditions were explored and optimized to promote efficient ionization, reduce the in-source fragmentation and consequently enhance the detection of intact molecular species from complex mixtures. Using the novel optimized (-)nanoESI-FTICR MS protocol, a reliable and detailed compositional fingerprint of the polysialylated ganglioside mixture isolated from human brain was obtained. Sustained off-resonance irradiation collision-induced dissociation mass spectrometry (SORI-CID MS2) was introduced for the first time for structural elucidation of polysialylated gangliosides. Under well-defined conditions, an informative fragmentation pattern of the trisialylated ganglioside GT1 was obtained. The compositional mapping of a complex mixture of sulfated glucuronic acid containing neolacto-series GSLs extracted from bovine Cauda equina provided hard evidence upon previously described components and new structures not identified before by any other analytical method. Negative ion nanoESI-FTICR MS at 9.4 T is shown here to represent a valuable method in glycolipidomics, allowing a high resolution and mass accuracy detection of major and minor GSL glycoforms and identification of known and novel biologically relevant structures.

A thin chip polymer-based microsprayer has been coupled to a hybrid quadrupole time-of-flight mass spectrometer (QTOF MS) and introduced in carbohydrate research. The feasibility of the approach is demonstrated for mapping, sequencing and structural elucidation of glycoconjugates originating from human body fluids and tissues such as a glycopeptide mixture from normal human urine and an isolated and purified GT1 ganglioside fraction from normal adult human brain. The optimization procedure required by each glycoconjugate category is described and the advantages of the system in terms of flexibility and adaptability to QTOF MS, stability of the ESI MS signal, carbohydrate ionization and sequencing, sensitivity, speed of analysis and sample consumption are discussed.

The introduction of chip-based electrospray (ESI) ion sources into biological mass spectrometry (MS) addressed the fundamental issue of how to analyze minute amounts of complex biological systems. The automation of sample delivery into the MS combined with the chip-based ESI allows for high quality bioanalysis in a high-throughput fashion. These advantages have already been demonstrated in proteomics, direct screening of drugs and drug discovery. As part of our continuing effort to implement automated chip-based mass spectrometry into the field of complex carbohydrate analysis, we hereby report the development of a chipESI MS and MS/MS methodology for the screening of gangliosides. A strategy to characterize a complex ganglioside mixture from human cerebellar tissue, by automated ESIchip-quadrupole time-of-flight (QTOF) MS and MS/MS is presented here. The feasibility of this method, and the general experimental requirements for automated chipESI MS analysis of these carbohydrate species is described.