Reprint of: Chip-based nanoelectrospray mass spectrometry of brain gangliosides

Biochimica et Biophysica Acta 1811 (2011) 897–917

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Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbalip

Reprint of: Chip-based nanoelectrospray mass spectrometry of brain gangliosides ☆ Corina Flangea a, b, 1, Alina Serb b, c, 1, Eugen Sisu c, d, Alina D. Zamfir a, b,⁎ a

Department of Chemical and Biological Sciences, “Aurel Vlaicu” University of Arad, 310130, Arad, Romania Mass Spectrometry Laboratory, National Institute for Research and Development in Electrochemistry and Condensed Matter, 300224, Timisoara, Romania c Department of Biochemistry, “Victor Babes” University of Medicine and Pharmacy, 300054, Timisoara, Romania d Chemistry Institute of Romanian Academy, 300223 Timisoara, Romania b

a r t i c l e

i n f o

Available online 18 September 2011 Keywords: Brain gangliosides Mass spectrometry Chip-based electrospray Microfluidics Brain diseases

a b s t r a c t In the past few years, a considerable effort was invested in interfacing mass spectrometry (MS) to microfluidicsbased systems for electrospray ionization (ESI). Since its first introduction in biological mass spectrometry, chipbased ESI demonstrated a high potential to discover novel structures of biomarker value. Therefore, recently, microfluidics for electrospray in conjunction with advanced MS instruments able to perform multistage fragmentation were introduced also in glycolipid research. This review is focused on the strategies, which allowed a successful application of chip technology for ganglioside mapping and sequencing by ESI MS and tandem MS (MS/MS). The first part of the review is dedicated to the progress of MS methods in brain ganglioside research, which culminated with the introduction of two types of microfluidic devices: the NanoMate robot and a polymer microchip for electrospray. In the second part a systematic description of most relevant results obtained by using MS in combination with the two chip systems is presented. Chip-based ESI accomplishments for determination of ganglioside expression and structure in normal brain regions and brain pathologies such as neurodegenerative diseases and primary brain tumors are described together with some considerations upon the perspectives of microfluidics-MS to be routinely introduced in biomedical investigation. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Abbreviations: AD, Alzheimer disease; CAD, collision activated dissociation; Cer, ceramide; CI, chemical ionization; CID, collision induced dissociation; CNS, central nervous system; DE MALDI TOF MS, delayed extraction matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; ECD, electron capture dissociation; ESI, electrospray ionization; FAB MS, fast atom bombardment mass spectrometry; FTICR, Fourier transform ion cyclotron resonance; Fuc, fucose; Gal, galactose; Glc, glucose; GalNAc, N-acetylgalactosamine; GC, gas chromatography; GD1a, Neu5Acα3Galβ3GalNAcβ4(Neu5Acα3) Galβ4GlcCer; GD1b, Galβ3GalNAcβ4(Neu5Acα 8Neu5Acα3)Galβ4GlcCer; GD2, GalNAcβ4(Neu5Acα8Neu5Acα3)Galβ4GlcCer; GD3, Neu5Acα 8Neu5Acα 3Galβ4GlcCer; GlcNAc, N-acetylglucosamine; GM1a, Galβ3GalNAcβ4(Neu5Acα3)Galβ4GlcCer; GM1b, Neu5Acα 3Galβ3GalNAcβ4Galβ4GlcCer; GM2, GalNAcß4(Neu5Acα 3)Galβ4GlcCer; GM3, Neu5Acα3Galβ4GlcCer; GSL, glycosphingolipid; GT1a, Neu5Acα8Neu5Acα 3Galβ3GalNAcβ4(Neu5Acα3)Galβ4GlcCer; GT1b, Neu5Acα 3Galβ3GalNAcβ4(Neu5Acα8Neu5Acα3)Galβ4GlcCer; GT1c, Galβ3GalNAcβ4(Neu5Acα8Neu5Acα8Neu5Acα3) Galβ4GlcCer; HCT, high capacity ion trap; HPLC, high performance liquid chromatography; HPTLC, high performance thin layer chromatography; IRMPD, infrared laser multiphoton; LC, liquid chromatography; LCB, long chain base; MS/MS, tandem mass spectrometry; MSn, multistage mass spectrometry; Neu, neuraminic acid; Neu5Ac, N-acetyl neuraminic acid; Neu5Gc, N-glycolylneuraminic acid; QTOF MS, quadrupole time-offlight mass spectrometer/spectrometry; SORI-CID, sustained off-resonance irradiation collision-induced dissociation; TLC, thin layer chromatography; TSD, Tay–Sachs disease ☆ A publishers' error resulted in this article appearing in the wrong issue. The article is reprinted here for the reader's convenience and for the continuity of the special issue. For citation purposes, please use the original publication details:, Biochimica et Biophysica Acta 1811 (2011) 513–535. DOI of original article: 10.1016/j.bbalip.2011.06.008. ⁎ Corresponding author at: Plautius Andronescu Str. 1, RO-300224, Timisoara, Romania. Tel.: +40 256 494413; fax: +40 256 204698. E-mail address: alina.zamfi[email protected] (A.D. Zamfir). 1 These authors have contributed equally. 1388-1981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2011.09.006

Sphingolipids are among the most complex, structurally diverse, and enigmatic lipids found in nature. The high degree of their structural variety is a direct result of the thousands of possible combinations of head groups, fatty acids, and sphingoid base backbones, which include variations in chain length, substitution, unsaturation, or branching in these moieties [1]. Glycosphingolipids (GSLs) are classified several ways, the most common being according to the general types of carbohydrates of which they are composed: 1) neutral GSL contain one or more uncharged sugars such as Glc, hence, glucosylceramide is GlcCer, Gal, GlcNAc, GalNAc, Fuc; and 2) acidic glycosphingolipids contain ionized functional groups (phosphate or sulfate) attached to neutral sugars, or ionizable sugar residues such as sialic acid. Gangliosides are GSLs containing one or more sialic acid residues. In humans, the most abundant sialic acid present in gangliosides is Neu5Ac. A variant of O-acetyl derivative of Neu5Ac is Neu5Gc known to occur in species-, tissue- and physiological condition-dependent way [2]. Gangliosides occur ubiquitously in vertebrate cell plasma membranes and are particularly abundant in the nervous system. Gangliosides are inserted in the outer layer of the plasma membrane via their hydrophobic and heterogeneous ceramide moiety while their oligosaccharide moiety faces the external medium; this makes them free to interact with soluble extra cellular molecules and with the hydrophilic portion of other membrane components [3,4]. Gangliosides in

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the cell membrane are not randomly or homogeneously distributed, but rather organized in domains with particular physical–chemical and functional properties, different from those of the surrounding membrane environment [4]. Ganglioside expression is particularly high in mammalian central nervous system (CNS) where it is developmentally regulated and closely related to cell differentiation. They trigger a variety of biological events and participate in cell-to-cell recognition/communication and cell signaling [5]. Systematic investigations [6,7] showed that CNS contains ten times higher ganglioside concentration than the extraneural tissue. Moreover, ganglioside composition was demonstrated to change specifically during brain development, maturation, aging [8], and neurodegeneration [9,10]. Certain structures were found to be expressed differentially in primary brain tumors [11,12]; therefore, gangliosides are considered as diagnostic markers of brain ailments and are in the current focus of research as potential therapeutic agents [13,14]. Changes in the amounts and types of cellular sphingolipids arise from both de novo biosynthesis as well as the turnover of more complex sphingolipids. Therefore, to determine which sphingolipids are being perturbed from their basal state, it is critical to precisely identify, elucidate the structures, and quantify all species in these pathways [1]. Due to the large number of gangliosides, elucidation of their roles in cell structure, signaling, and function require structure-specific and quantitative analysis of all individual subspecies with high accuracy, reproducibility and sensitivity. From the beginning, mass spectrometry (MS) has emerged as a powerful tool for the structural analysis of complex GSLs such as gangliosides [15]. MS was for the first time employed in ganglioside analysis in 1978 when an unknown human brain monosialosyl ganglioside could be characterized using gas chromatography coupled to MS (GC MS) [15]. The carbohydrate moiety was identified as trimethylsillyl ethers, whereas the long-chain base (LCB) and the fatty acids as their methyl derivatives. With the development few years later of chemical ionization (CI) larger species such as di-sialo-oligosaccharides extracted from human brain could be examined by MS [16]. However, for CI MS analysis gangliosides needed to be permethylated [16]. Further improvement of CI MS methods allowed characterization of more complex structures such GM4, GM5, GM2, and GM1 from normal brain adult tissue extracts [17]. Introduction of the novel ionization technique based on fast atom bombardment (FAB) and development of efficient protocols for sample derivatization have considerably broadened the applicability of mass spectrometry in the field of gangliosides. Thus, based on carbohydrate mapping by FAB MS, oligosaccharide moieties of the GSLs in complex mixtures of normal and pathological human brain tissues could be reliably identified for the first time. The lactonic form of GD1b was identified by FAB MS experiments from complex mixture extracts of 51–70 year old normal adult brain specimens [18]. Using the same technique, GSLs belonging to Globo, Ganglio and Neolacto series were for the first time evidenced in brain fetuses in the 22nd to 23rd weeks of gestation [19]. Further improvement of FAB MS reproducibility and ionization efficiency as well development of liquid chromatography (LC), collisionactivated dissociation (CAD) and sophisticated derivatization procedures in combination with FAB MS provided new insights into the structural diversity of brain gangliosides in health and disease [20–22]. Hence, FAB CAD MS/MS analysis of GM2 ceramides isolated from human neuroblastoma tumor vs. normal human brain GM2, highlighted the higher degree of hydroxylation of the fatty acids in neuroblastoma ceramides [20]. Lysogangliosides LGM1, LGM2 and LGM3 – each carrying a single sphingoid base – from brain of a patient with infantile Sandhoff disease could be also structurally characterized using the resourceful procedure based on LC combined with FAB MS [21].

Silicon chip bearing a 20 x 20 array of nanospray nozzles

High Voltage 850-1500 V

Sample

MS Orifice conductive pipette tip

Fig. 1. Sample infusion into MS by nanoESI using the silicon chip technology integrated in the NanoMate robot (adapted from Zhang et al. [39]).

With an original protocol combining FAB MS, permethylation and methanolysis, in 1992 the group of Hakomori has discovered two unusual GSLs from human cerebellum as new plasmal (fatty aldehyde) conjugates of psychosine with cyclic acetal linkage at the galactosyl residue [23]. Using a similar strategy, the same group proceeded with the isolation and characterization of two unknown plasmalocerebrosides from human brain [24]. Permethylation, trimetylsililation and methanolysis, followed by reduction and full acetylation, were employed as derivatization procedures to enhance MS ionization and detection of gangliosides. To characterize the sugar moiety of unknown GSLs extracted from normal human brain, such complex derivatization steps were applied by several groups [25,26]. In conjunction with GC MS, this strategy successfully discovered two new GSL species: Gal β(1–4)[Fucα(1–3)]GlcNAc β(1–3)Gal β(1–4) Glc-Cer [25] and sulfate-3-GlcA β (1–3)Gal β (1–4) GlcNAc β (1–3)Gal β (1–4) Glc β (1–1)-Cer [26]. At the beginning of 90's potentials of MS for sensitive structural analysis of gangliosides increased significantly after the introduction of matrix-assisted laser desorption/ionization (MALDI) and electrospray (ESI) methods from one side and the possibility to sequence complex ionic species by highly efficient dissociation techniques on the other. Lysogangliosides prepared by microwave-mediated saponification were identified by delayed extraction (DE) MALDI time-offlight (TOF) MS analysis. When GM3, GM2, and GM1 isolated from adult human brain gangliosides were subjected to saponification, GM3 was found to give rise to only lyso-GM3 containing de-iV-

c 7 50 µm

a

3

6

2 4 5 6

b

8

1

Fig. 2. Schematic of polymer microchip. a) top view of the chip structure; b) side view of the chip with the sample reservoir; c) zoom insert view of the chip showing the microchannel spray exit; 1, polyimide thin layer substrate; 2, ESI-spray exit; 3, microchannel fabricated by plasma ablation of the polyimide substrate; 4, sealing layer of polyethylene/ polyethylene terephthalate laminate; 5, electrode for high voltage application; 6, electrode contacts; 7, sharp tip angle prepared by plasma etching of the substrate; 8, sample reservoir (from Rossier et al. [45]).

C. Flangea et al. / Biochimica et Biophysica Acta 1811 (2011) 897–917

899

Fig. 3. Positioning of the polymer microchip in the front of the mass spectrometer for electrospray generation (from Gobry et al. [47]).

acetylneuraminic acid (de-JV-acetyl-lyso-GM3), whereas GM2 produced both lyso-GM2 and de-iV-acetyl compounds, and GM1 also gave both lyso-GM1 and the de-iV-acetyl compound. DE MALDI TOF MS analysis of the prepared lysogangliosides showed that their LCB consisted of d18:1 and d20:1 sphingosines in various ratios reflecting

those of the different mammalian brain gangliosides [27]. LCB composition of gangliosides could be determined without any chemical modification by DE MALDI TOF MS analysis. The analytical results for the LCB compositions of various samples of GM1 from the brain tissue of patients with different diseases at different ages confirmed that the

Fig. 4. a) Microchip ESI QTOF MS/MS of the triply charged ion at m/z 717.50 corresponding to GT1 (d18:1/20:0) purified ganglioside fraction extracted from normal adult human cerebrum. ESI voltage, 3 kV; Sampling cone potential, 100 V. b) the structure and the corresponding fragmentation scheme of the GT1b species (from Zamfir et al. [53]). Nomenclature of the fragment ions is according to published recommendations [61,62].

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proportion of d20:1 and d20 of the total sphingosine bases increased quickly until adolescent or adult age and then remained constant slightly exceeding 50% [28]. Electrospray was introduced in brain ganglioside research by the group of Peter-Katalinić [29,30]. The authors accomplished the structural elucidation of human fetal brain ganglioside species GM1, GD1

and GT1 by nanoESI quadrupole time-of-flight (QTOF) MS, using combined data from MS and tandem MS (MS/MS) of isolated native ganglioside fractions. To distinguish between isobaric carbohydrate structures, such as monosialogangliosides GM1a and GM1b, disialogangliosides GD1a, GD1b and GD1c or trisialogangliosides GT1b, GT1c and GT1d, the samples were analyzed after permethylation in

Fig. 5. Fully automated (−) nano ESI chip QTOF MS1 of the ganglioside mixture extracted from human adult cerebellum. Sample concentration, 2–3 pmol/μL in methanol; acquisition time, 3 min; sampling cone potential, 45–135 V; nanoESI voltage, 1.67 kV; n.a. = not assigned: a) m/z (700–980); b) m/z (980–2050) (from Zamfir et al. [54]).

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the positive nanoESI QTOF MS/MS mode. By combining the information obtained by screening in the negative/positive ion mode and sequencing using low energy collision-induced dissociation (CID), identification and characterization of structural isomers in mixtures was possible [29]. Gangliosides from histopathologically-defined human cerebrumresembling remnant and cerebellum from 37 and 30 gestational week-old anenecephalus were identified using nanoESI QTOF MS and CID MS/MS by the same group. In the cerebral remnant GD3, GM2 and GT1b were elevated, while GD1a was decreased in the anencephalic cerebral remnant, but enriched in anencephalic regions. GQ1b was found reduced in both anencephalic regions. In both cerebral and cerebellar anencephalic tissue, GM1b, GD1α, NLm1 and NLd1 were expressed at higher rate in relation to normal tissue [30]. In the past decade Fourier transform ion cyclotron resonance (FTICR) MS has become an indispensable tool for the analysis of complex mixtures, such as those encountered in glycomics. The unique features of the FTICR MS in comparison to all other MS methods are the ultra-high resolution exceeding 10 6 and the mass-determination accuracy very often below 1 ppm. Additionally, FTICR MS provides the advantage of several ion fragmentation techniques based on precursor dissociation such as CID via sustained off-resonance irradiation (SORI-CID), infrared laser multiphoton (IRMPD), or electron capture dissociation (ECD) as well as the possibility to perform multiple stage MS (MS n). For the analysis of ganglioside mixtures, nanoESI FTICR MS in the negative ion mode was shown to be most substantial for screening, sequencing and discovery of novel species with possible biomarker value. For instance, upon FTICR MS analysis of GSLs in a brain affected by Tay–Sachs disease (TSD) a novel structure was detected and identified as a taurine-conjugated GM2 (tauro-GM2) in which the carboxyl group of N-acetylneuraminic acid was amidated by taurine. The presence of tauro-GM2 in TS brains, but not in normal brains, indicated the possible association of this unusual GM2 derivative with the pathogenesis of TSD [31]. Moreover, in another study employing a superior (−) nano ESI FTICR MS protocol, a detailed mapping of gangliosides isolated from human brain was obtained [32]. SORI CID MS 2 introduced here for the first time in structural elucidation of brain gangliosides, provided the full set of product ions diagnostic for the structure of the brain-associated GT1 species [32]. Despite the rapid progress of MS instrumentation and the achievements presented above the data concerning the fine molecular structure of brain GSL, in particular of human brain gangliosides remained rather poor. However, introduction and optimization of chip-based ESI MS for ganglioside analysis opened new perspectives in this field [33]. Initially, application of these systems to gangliosides was limited by their high structural diversity and heterogeneity. Moreover, because of the lower ionization efficiency as compared to peptides and proteins, gangliosides required reconsideration of conditions to promote chip ionization and detection by MS. These conditions were determined by the type of the present labile moieties (Neu5Ac, Fuc, and O-Ac), the ionizability of the functional groups, and the special molecule constitution consisting of a hydrophilic sugar core and a hydrophobic ceramide part. Such characteristics made this class of biomolecules less amenable to chip-based and high-throughput methods. However, in the subsequent parts of this review it will be shown that adequate strategies could lead to successful implementation of this technology also in ganglioside analysis with superior results in their compositional and structural analysis. 2. Microfluidics-MS systems for ganglioside analysis At the beginning of 90's refinement of the electrospray as the ion source for MS yielded the low-flow (micro- and nano ESI) configurations, which provided sensitivities at sub-picomolar level [34]. Later on, the trends in ESI MS started to be oriented toward high-

901

Table 1 Composition of single components in the ganglioside mixture from gray matter of normal human adult cerebellum as detected by a fully automated (−) nanoESI chip QTOF MS. d = dihydroxy sphingoid base. t = trihydroxy sphingoid base; * low intensity ions (from Zamfir et al. [54]). Type of molecular ion

m/z (monoisotopic) Detected

Calculated

[M + 2Na − 4H]2− [M − H]− [M − H]− [M − 2H]2− [M + Na − 2H]− [M − 2H]2− [M − H]− [M − 2H]2− [M − H]− [M − 2H]2− [M − H]− [M − H]− [M − H]− [M − 2H]2− [M − 2H]2− [M − 2H]2− [M + Na − 3H]2− [M − H]− [M + Na − 2H]− [M − 2H]2− [M − 2H]2− [M − 2H]2− [M + Na − 3H]2− [M − H]− [M − 2H]2− [M − 2H]2− [M − 2H]2− [M − 2H]2− [M − 2H]2− [M − 2H]2− [M − 2H]2−

611.40 1179.57 1382.60 734.96 1492.78 748.99 1518.51 771.98 1544.61 786.00 1572.61 1690.55 1716.56 836.46 850.47 917.44 928.45 1835.62 1857.56 926.44 924.44 931.46 942.44 1885.60 940.49 938.44 945.47 954.46 952.47 958.46* 966.44

611.35 1179.74 1382.82 734.91 1492.81 748.93 1518.85 771.93 1544.85 785.92 1572.85 1690.93 1716.94 836.45 850.47 917.48 928.47 1835.96 1857.95 926.48 924.49 931.49 942.48 1885.98 940.50 938.50 945.51 954.51 952.52 958.52 966.53

[M − 2H]2− [M − 2H]2− [M − 2H]2− [M − 2H]2− [M − 2H]2− [M − 2H]2− [M − 2H]2− [M − 2H]2− [M − 3H]3− [M − 2H]2− [M + Na − 3H]2− [M + 2Na − 4H]2− [M − 3H]3− [M + Na − 3H]2− [M − 3H]3− [M − 2H]2− [M + Na − 3H]2− [M + 2Na − 4H]2− [M − 3H]3− [M + Na − 3H]2− [M + Na − 3H]2− [M − 3H]3− [M + Na − 3H]2− [M + Na − 3H]2− [M + Na − 3H]2− [M − 3H]3− [M − 3H]3− [M − 2H]2− [M − 2H]2− [M − 2H]2− [M − 3H]3− [M + Na − 4H]3− [M + 2Na − 4H]2− [M + 3Na − 5H]2− [M − 3H]3− [M + Na − 4H]3− [M + 2Na − 4H]2− [M − 3H]3− [M + Na − 4H]3−

988.40 990.40 999.41* 1002.41 1004.42 1013.44* 1018.99 1032.93* 708.39 1062.96 1073.92 1084.93 714.41 1082.92 717.75 1076.97 1087.95 1098.92 723.75 1096.93 1094.95* 727.11 1101.92 1108.92* 1114.96 722.39 731.74 1128.95 1144.89 1159.89 805.40 812.73 1230.43 1241.43 814.74 822.07 1244.42 819.38* 826.73*

988.49 990.51 999.51 1002.51 1004.52 1013.53 1019.02 1033.03 708.35 1063.03 1074.02 1085.01 714.35 1083.02 717.69 1077.04 1088.03 1099.02 723.70 1097.04 1095.04 727.04 1102.05 1109.06 1115.06 722.35 731.70 1129.05 1145.06 1159.08 805.38 812.71 1230.56 1241.55 814.72 822.05 1244.57 819.38 826.71

Assigned structure

GM3 (d18:1/18:0) GM2 (d18:1/18:0) GD3 (d18:1/18:0) GD3 (d18:1/20:0) GM1, nLM1 and/or LM1 (d18:0/16:0) GM1, nLM1 and/or LM1 (d18:1/18:0) GM1, nLM1 and/or LM1 (d18:1/20:0) Fuc-GM1 (d18:1/18:0) Fuc-GM1 (d18:1/20:1) GD2 (d18:1/18:0) GD2 (d18:1/20:0) GD1, nLD1 and/or LD1 (d18:1/18:0)

GD1, nLD1 and/or LD1 (t18:0/18:0) GD1, nLD1 and/or LD1 (d18:1/19:0) GD1, nLD1 and/or LD1 (d18:1/20:0)

GD1, nLD1 and/or LD1 (t18:0/20:0) GD1, nLD1 and/or LD1 (d18:1/21:0) GD1, nLD1 and/or LD1 (d18:1/22:0) GD1, nLD1 and/or LD1 (t18:0/22:0) GD1, nLD1 and/or LD1 (d18:1/23:0) GD1, nLD1 and/or LD1 (d18:1/24:1) GD1, nLD1 and/or LD1 (d18:1/25:0) or (d20:1/23:0) Fuc-GD1 (d18:1/18:2) Fuc-GD1 (d18:1/18:0) Fuc-GD1 (t18:0/18:0) Fuc-GD1 (d18:1/20:2) Fuc-GD1 (d18:1/20:0) Fuc-GD1 (t18:0/20:0) GalNAc-GD1 (d18:1/18:0) GalNAc-GD1 (d18:1/20:0) GT1 (d18:1/18:0)

GT1 (t18:0/18:0) GT1 (d18:1/20:0)

GT1 (t18:0/20:0) GT1 (d18:1/21:0) GT1 (d18:1/22:0) GT1 (d18:1/23:0) GT1 (d18:1/24:1) O-Ac-GT1 (d18:1/18:0) O-Ac-GT1 (d18:1/20:0) Fuc-GT1 (d18:1/17:0) Fuc-GT1 (t18:0/18:0) Fuc-GT1 (t18:0/20:0) GQ1 (d18:1/18:0)

GQ1 (d18:1/20:0)

O-Ac-GQ1 (d18:1/18:0)

902

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reduction of the ion source size facilitating manipulation and efficient ion transfer by precise positioning with respect to the MS sampling orifice. In ganglioside analysis, devices from two microfluidics-MS categories were introduced: i) a robot belonging to the category of complex systems which nowadays combine robotized sample handling and delivery, fraction collections from liquid chromatography (LC) with a chip for nanoESI microfabricated using silicon technology; ii) a planar microchip belonging to the class of mono-or polyfunctional micro-/ nanosystems with functions integrated into a polymer substrate. The first category of chip-based ESI systems is represented by the out-of plane devices (NanoMate robots produced by Advion BioSciences) where 100 or 400 nozzle-like nanospray emitters are etched onto a single silicon substrate from which electrospray is established perpendicular to the substrate [39]. These devices are particularly well suited to high-throughput sample delivery to ESI MS [33,38–40] and have the potential to completely replace flow-injection analysis assays (Fig. 1). In this configuration, a few microliter aliquots of the working sample solution are loaded into a 96-well plate. The robot is programmed to aspirate a certain volume of sample and afterwards deliver the sample to the inlet side of the 100 or 400 microchip. Following infusion process and MS analysis, the pipette tip is ejected and a new tip and nozzle are used for each sample, thus preventing any cross-contamination or carry-over. The technical quality of the nanosprayers obtained by silicon microtechnology is so high, that in

throughput measurements based on the nanotechnology achievements in automatization and miniaturization of devices. Such novel systems for robotized sample delivery into MS substituted to some extent the classical ESI which requires either manual loading or pumping of the sample liquid through the electrospray capillary [35]. It was observed that by using these devices, significant increase in the analysis throughput and efficiency, allowing minimization of sample volumes and handling can be achieved. Miniaturized, integrated platforms functioning on the principle of “lab-on-a-chip” or micro-total analysis systems were also introduced in mass spectrometry on the basis of microfluidics as front-end technologies for ESI [36,37]. In MS, the option for microfluidics, which refers to all analytical tools where fluids can be driven in microstructured channels and/or narrow capillaries, was driven by a high number of technical, analytical and economical advantages [33,38]. Among these, the most important were: i) simplification of the laborious chemical and biochemical strategies required for MS research; ii) high throughput nanoanalysis/identification of biomolecules; iv) elimination of the time-consuming optimization procedures; v) increase in sensitivity by reduction of the sample and reagent consumption, minimization of sample handling and potential sample loss; vi) elevated reproducibility of the experiments; vii) superior ionization efficiency; viii) versatility; ix) possibility to perform several stages of sample preparation/separation in a single integrated element followed by direct MS structural analysis; x) elimination of possible cross-contamination and carry-overs; xi)

a

e

b

c

d

Fig. 6. Structure and fragmentation scheme corresponding to b ganglioside series: a) GT1; b) GD1; c) GM1; d) CID MS5 of [M − H]− ion at m/z 1282.06 which correspond to asialo Gal-GalNAc-Gal-Glc-Ceramide structure. Inset: ion structure and fragmentation scheme; e) CID MS6 of [M − H]− ion at m/z 592.66 corresponding to Y0− detected in MS5. Inset: general fragmentation scheme of (d20:1/18:0) ceramide variant supported by CID MS6 (from Serb et al. [60]). Nomenclature of the fragment ions is according to published recommendations [61–63].

C. Flangea et al. / Biochimica et Biophysica Acta 1811 (2011) 897–917 Table 2 Comparative assignment of the major ions detected in NEO36, FL36 and OL36 samples as detected by fully automated (−) nanoESI chip HCT MS. d = dihydroxy sphingoid base; t = trihydroxy sphingoid base; + = the species was detected; − = the species was not detected (from Serb et al. [64]). m/z (monoisotopic)

Molecular ion

Proposed structure

NEO 36

FL 36

OL 36

715.72 735.08 749.60 771.76

[M+ Na− 3H]2− [M − 2H]2− [M − 2H]2− [M − 2H]2−

− − − +

+ + + −

− + − −

788.60 805.84 813.23 820.60 823.17 836.48 857.60 885.76 903.29

[M− 2H]2− − H2O [M + 2Na − 4H]2− [M − 3H]3− [M − 2H]2− [M − 3H]3− [M − 2H]2− [M − 2H]2− [M − 2H]2− [M − 2H]2−

− − + − + − − − −

+ + − + − + + + −

− − − − − + − − +

917.31

[M − 2H]2−

+

+

+

924.60

2−

GM2 (d18:1/20:0) GD3 (d18:0/18:0) GD3 (d18:0/20:0) GM1, nLM1 and/or LM1 (d18:1/18:0) GM1 (d18:1/22:2) GM1 (d18:1/20:2) GQ1 (d18:1/20:2) Fuc-GD3 (d18:1/21:0) GQ1 (d18:1/22:1) GM2 (d18:1/18:0) GD2 (d18:1/21:0) GD2 (d18:1/25:0) GD1, nLD1 and/or LD1 (d18:1/16:0) GD1, nLD1 and/or LD1 (d18:1/18:0) GD1, nLD1 and/or LD1 (d18:1/19:0) GD1, nLD1 and/or LD1 (d18:1/18:0) GD1, nLD1 and/or LD1 (d18:1/20:0) GD1, nLD1 and/or LD1 (t18:0/20:0) GD1, nLD1 and/or LD1 (d18:1/22:0) O-Ac-GD2 (d18:1/31:2) O-Ac-GD1 or O-Ac-nLD1 (d18:1/20:0); GD1, nLD1 and/or LD1 (d18:1/23:0) GD1, nLD1 and/or LD1 (d18:1/24:1) GD1 (d18:1/24:1) GD1 (d18:1/22:4) GM4 (d18:1/14:2) GT2 (d18:1/19:0) GD1 (d18:0/28:0) GD1 (d18:1/30:1) GT2 (d18:1/22:2) GM4 (d18:1/19:1) GM4 (d18:1/20:4) or GM4 (d18:1/18:1) GM4 (d18:1/20:2) O-Ac-GD1 (d18:1/33:2) GT1 (d18:1/16:0) GM4 (d18:1/21:1) GT1 (d18:1/18:0) Fuc-GT2 (d18:1/22:0) GT1 (d18:1/20:0) O-Ac-GT1 (d18:1/18:1) or GT1 (d18:1/21:1); GT1 (t18:0/18:0) O-Ac-GT1 (d18:1/20:1); GT1 (d18:1/23:1) GT1 (t18:0/20:0) GT1 (d18:1/20:1) GT1 (d18:1/24:0) O-Ac-GT1 (d18:1/20:4) GM3 (d18:1/13:2) GM3 (t18:1/14:0) or (d18:1/h14:0) or (d18:0/15:0); GM4 (t18:1/24:0) GM3 (d18:1/16:1) GM3 (d18:1/31:0) GT1 (t18:0/18:0) GM3 (d18:0/17:0) or (t18:1/16:0) or (d18:1/h16:0); Fuc-GM4 (d18:1/20:2) GM3 (d18:1/18:0)

−

+

−

+

−

−

−

+

+

+

−

−

−

−

+

+ −

− +

− −

+

−

−

+ − + + + + + + − − − + + + − + − +

− − − − − − + − + + + − + − + − + −

− + − − − − − − − − − − − − + − + −

−

+

−

+ −

− +

− +

− −

+ −

− +

[M − 2H]

928.32

[M + Na − 3H]

931.34

[M − 2H]2−

939.60

[M − 2H]2−

945.35

[M − 2H]

2−

946.67 953.00

[M − 2H]2− [M − 2H]2−

958.86

[M − 2H]2−

969.71 974.44 980.21 988.78 999.71 1011.73 1019.80 1029.80 1037.71 1041.73 1041.40 1049.20 1057.56 1062.83 1074.77 1077.59 1083.08

1096.92

2−

2−

[M + Na − 3H] [M + 3Na − 5H]2[M + Na − 2H]− [M − 2H]2− [M + Na − 3H]2− [M + Na − 3H]2− [M − 2H]2− [M − H]− [M − H]− [M + Na − 2H]− [M − H]− [M − 2H]2− [M − 2H]2− [M − H]– [M − 2H]2− [M − 2H]2− [M − 2H]2− [M − 2H]2− [M + Na − 3H]2− [M − 2H]2− [M + Na − 3H]2−

1127.45 1138.89

[M + 2Na − 4H]2− [M − 2H]2− [M + Na − 3H]2− [M + Na − 2H]− [M − H]−

1149.74 1151.61 1165.33 1167.81

[M + Na − 2H]− [M − H]− [M − H]− [M + Na − 3H]2− [M − H]−

1170.67 1179.91

[M − H]− [M − H]−

1097.79 1104.68

+ − + −

− + − +

− + − −

− +

− +

+ +

(continued on next page)

903

Table 2 (continued) m/z (monoisotopic)

Molecular ion

Proposed structure

NEO 36

FL 36

OL 36

1195.95 1204.70 1208.60 1213.80 1217.01 1219.43 1224.36 1231.90 1237.79 1241.83 1248.96 1263.90 1266.15 1270.69 1277.62 1301.91 1304.69 1307.39 1327.51 1329.40 1331.29 1337.05 1338.60 1341.44 1352.96 1354.97 1356.80 1360.98 1370.59 1383.24 1385.11 1404.62 1437.11 1445.15 1466.95 1471.03 1517.33

[M − H]− [M − H]− [M − H]− [M + Na − 2H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − 2H]2− [M + 3Na − 5H]2− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M + Na − 2H]− [M − H]− [M + Na − 2H]− [M − H]− [M − H]− [M − H]− [M − H]− [M + Na − 2H]− [M − H]− [M − H]− [M − H]− [M − H]− − H2O [M − H]− [M − H]− [M − H]− [M + Na − 2H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]−

+ − − + − + − + − + + + − + − + − − + + + + + − + − − + + − + − − − − − − +

− − − − + − + − + − − + + + − − + − − − − − − − − + + − − − + + − + + + + −

− + + − − − − − + − − − − − + − − + − − − − − + − − − − − + + − + − − − + −

1519.10

[M − H]−

−

+

−

1544.83

[M − H]−

+

+

+

1566.79

[M + Na − 2H]−

GM3 (d18:0/19:0) Fuc-GM3 (d18:0/22:0) Fuc-GM3 (d18:1/11:1) GM3 (d18:1/19:1) Fuc-GM3 (d18:1/23:0) GM3 (d18:1/21:1) Fuc-GM3 (d18:1/12:0) GM3 (d18:1/22:2) GM3 (d18:0/22:0) O-Ac-GQ1 (d18:1/20:2); GQ1 (d18:1/18:0) Fuc-GA2 (d18:1/20:0) GM3 (d18:1/24:0) GM3 (d18:0/24:0) GM2 (d18:0/10:0) GA2 (d18:1/22:0) O-Ac-GM3 (d18:1/24:2) GM2 (d18:1/11:1) GA1 (d18:1/22:1) GM3 (d18:1/27:0) Fuc-GM4 (d18:1/31:0) Fuc-GM4 (d18:0/31:0) GA1 (d18:1/24:1) GM2 (d18:1/15:1) GM2 (d18:1/28:0) GM2 (d18:1/16:1) GM2 (d18:1/16:0) GM2 (d18:0/16:0) GM2 (d18:1/18:2) GM2 (d18:0/17:0) GM2 (d18:1/18:0) GM2 (d18:0/18:0) GM2 (d18:1/18:0) GM2 (d18:1/22:1) GD3 (d18:0/16:0) GD3 (d18:1/18:2) GD3 (d18:1/18:0) GM1, nLM1 and/or LM1 (d18:1/16:0) GM1, nLM1 and/or LM1 (d18:0/16:0) GM1, nLM1 and/or LM1 (d18:1/18:0) GM1, nLM1 and/or LM1 (d18:1/18:0) GM1, nLM1 and/or LM1 (d18:1/20:0) GM2 (d18:0/31:0) GD3 (d18:1/27:1) GD3 (d18:1/29:2) GD3 (d18:1/28:0) GM1 (d18:0/33:0) GM1, nLM1 and/or LM1 (d18:1/24:1) GD3 (d18:1/31:2) GM1 (d18:1/29:2) GM1 (d18:0/27:0) O-Ac-GD3 (d18:0/31:0) O-Ac-GM1 (d18:1/27:2) O-Ac-GM1 (d18:1/28:1) GD2 (d18:0/22:0) GD2 (d18:1/25:2) GT3 (d18:1/16:1) GT3 (d18:1/23:1) GD1, nLD1 and/or LD1 (d18:1/18:0) GT3 (d18:0/22:2) GD1, nLD1 and/or LD1 (d18:1/18:0) GD1, nLD1 and/or LD1 (d18:1/18:0) Fuc-GD2 (d18:1/24:0) GD1 (d18:1/23:1) Fuc-GT3 (d18:1/20:1) Fuc-GD2 (d18:0/27:0)

+

−

−

+

−

+

+ + + + + +

− − − − − −

− − − − − −

+ − + + + + + + + + −

− − − − − − − − − − −

− + − − − − − − − − +

+ +

− −

− +

+

−

−

+ + + +

− − − −

− − − −

1572.02

[M − H]

−

−

1589.20 1595.20 1603.29 1611.40 1617.40 1626.95

[M + Na − 2H] [M − H]− [M − H]− − H2O [M − H]− [M + Na − 2H]− [M − H]−

1649.31 1662.22 1671.36 1697.40 1709.35 1725.20 1732.32 1750.40 1753.36 1830.40 1835.94

[M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M-H]− − H2O [M + Na − 2H]− [M − H]− [M − H]−

1837.64 1858.23

[M + Na − 2H]− [M + Na − 2H]−

1879.94

[M + 2Na − 3H]−

1887.40 1903.40 1916.60 1931.40

[M − H]− [M − H]− [M − H]− [M − H]−

(continued on next page)

904

C. Flangea et al. / Biochimica et Biophysica Acta 1811 (2011) 897–917

Table 2 (continued) m/z (monoisotopic)

Molecular ion

Proposed structure

NEO 36

FL 36

OL 36

1940.52 1956.40 1960.80

[M + Na − 2H]− [M + Na − 2H]− [M − H]−

+ + −

− − −

− − +

1962.53 1984.45 2022.24 2044.40 2062.60 2107.20 2132.01 2167.33 2173.80 2207.58 2244.76 2279.60 2285.80 2307.11 2322.60 2369.80 2382.01 2391.60 2407.82

[M − H]− [M + Na − 2H]− [M + 2Na − 3H]− [M − H]− [M + 2Na − 3H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M + 2Na − 3H]− [M − H]− [M − H]− − H2O [M + Na − 2H]− [M − H]− [M + 2Na − 3H]−

GT3 (d18:1/29:0) GD1 (d18:1/25:0) GT2 (d18:1/18:2) or Fuc-GD1 (d18:1/18:2) GD1 (d18:1/27:0) GD1 (d18:1/27:0) GD1 (d18:1/28:0) GD1 (d18:1/33:1) GD1 (d18:1/31:0) GT2 (d18:0/28:0) Fuc-GT2 (d18:1/21:2) GT1 (d18:1/21:1) GT2 (d18:1/33:1) GT1 (d18:1/24:1) Fuc-GT1 (d18:1/29:2) GT1 (d18:1/29:1) Fuc-GT1 (d18:1/20:0) GT1 (d18:1/28:2) Fuc-GT1 (d18:1/23:2) GQ1 (d18:1/16:1) GQ1 (d18:1/14:1) GQ1 (d18:0/16:0) GT1 (d18:0/14:0)

+ + + + + + + + + + + + + + + + + + +

− − − − − − − − − − − − − − − − − − −

− − − − − − − − − − − − − − − − − − −

some instances, by using this technology, the reported in-run, run-torun and day-to-day experiment reproducibility was almost 100% [33,41]. Due to highly efficient ionization properties, silicon-based chips for nanoESI preferentially form multiply charged ions, and the in-source

fragmentation of labile groups attached to the biomolecular core is minimized. When operating in infusion mode, as the internal diameter of the nozzle is 2.5 μm, flow rates down to 30–50 nL/min were reported [42] which represents a gain in sensitivity by at least 5 times as compared to classical nanospray capillaries. Additionally, the nozzles on the ESI chip were found to provide a long-lasting steady spray, and elimination of sample-to-sample carryover. In LC MS mode with fraction collection, this technology allows coupling of any conventional high performance LC (HPLC) system for ESI MS analysis [43]. The effluent is split post column with a portion directed to a conventional LC MS interface while the remainder of the split effluent is directed to collect at time segments into a multi-well plate. Following LC MS, the system permits selection of fractions for detailed analysis by MS/MS. The second category of microfluidics which was applied to ganglioside analysis by ESI MS consists of disposable planar microchips, made from a thin polymer material, embedding a microchannel at the end of which electrospray is generated in-plane, on the edge of the microchip [44–46]. These microsprayers on polymer substrate were a practical alternative to the silicon or glass chips due to the simpler methods for accurate plastic replications and the lower cost of production. In principle, planar microchips are fabricated by patterning a photoresist on a copper-coated polyimide foil through a printed slide acting as a mask. The photoresist is further developed, and chemically etched to remove the copper where microchannels are to be imprinted. The microchannels are 120 μm wide, 45 μm deep with gold-coated microelectrodes placed at the bottom of the microchannel [47]. A 35 μm polyethylene–polyethylene terephthalate is laminated to close the channels. On end of each channel is manually cut by the aid of a scissor

Fig. 7. Fully automated (−) nanoESI chip HCT MS1 of the native ganglioside mixture extracted from: a) NEO36. Inset: zoomed areas: m/z (1000-1350); m/z (1572–1858) and m/z (1887–2500); b) FL36; c) OL36. Solvent: MeOH; sample concentration, 3 pmol/μL; acquisition time, 2 min; Chip ESI, − 0.85 kV; capillary exit, − 30 V (from Serb et al. [64]). Nomenclature of the fragment ions is according to published recommendations [61,62].

C. Flangea et al. / Biochimica et Biophysica Acta 1811 (2011) 897–917

905

Fig. 7 (continued).

in a tip shape, so that the outlet of the microchannel is located on the edge of the chip (Fig. 2). A reservoir is glued over the inlet of the microchannel to serve as a sample supply and the assembly chip-reservoir is positioned in the front of the mass spectrometer as illustrated in Fig. 3. ESI is generated by applying a high voltage (1–3 kV) between the electrode placed in the inlet reservoir and the mass spectrometer. In comparison with the classical capillaries used for nanoESI, polymer microchips provided superior stability of the spray with time, improved signal-to-noise ratios at various flow rates and high flexibility and adaptability to different ion source configurations. As compared to silicon chips functioning in combination with automatic infusion, these thin microsprayers were found less amenable to high-throughput analyses, however, of higher tolerance to salts and certainly less expensive.

3. Chip-based electrospray for the analysis of ganglioside expression and structure in normal brain regions In the last years different MS configurations were adapted to polymer-based microchip ESI interfacing and optimized mostly for proteomic surveys [45,47–52]. In the field of ganglioside research sample delivery into MS using the robust thin chip polymer-based microsprayer system was for the first time reported only in 2005 [53]. The chip was used in combination with QTOF MS/MS and applied for mapping, sequencing and structural elucidation of a purified GT1 ganglioside fraction extracted from normal adult human cerebrum (45 years of age) [53]. After separation by high performance thin layer chromatography (HPTLC), GT1 ganglioside fraction, showing migration properties of GT1b, was profiled by (−) microchip ESI

906

C. Flangea et al. / Biochimica et Biophysica Acta 1811 (2011) 897–917

QTOF MS. This mapping resulted in a set of triply and doubly charged molecular ions containing a number of lipid variants with only a low level of in-source fragmentation. For further structural analysis, the triply charged precursor ion at m/z 717.63 was fragmented in a MS/ MS step by CID at low energies within 40–70 eV (Elab). The complete sequence of the GT1b was deduced from the ions characterizing the non-reducing end such as Y- and Z-type and ring cleavage X type ions, and also by a set of ions formed from the reducing end, particularly C- and B-type ions, observed to be similarly favored under the employed conditions. Additionally, the (d18:1/20:0) type of ceramide was documented by the presence of both Y0 and Z0 ions at m/z 592.50 and 574.55, respectively (Fig. 4a, b). Healthy CNS contains the highest amount of gangliosides: neuronal membranes hold at least several times higher concentrations of gangliosides than the extraneural cell types, highlighting their special role at the CNS level [54–56]. Mapping of the gangliosides expressed in different regions of normal human brain using TLC, immunochemical and immunohistochemical methods [57–59] offered a low amount of information because of the detection limitations of these methods and their low throughput. Therefore, in last years many efforts to implement automated chip-based ESI MS methodologies into CNS ganglioside analysis have been invested. Thus, using the

NanoMate system, automated ESI chip-QTOF MS and MS/MS was optimized in the negative ion mode, for the characterization of a complex ganglioside mixture from human cerebellar gray matter (20 years old) [54]. NanoESI chip MS screening of cerebellum sample enabled the identification of 46 glycoforms, with a high degree of heterogeneity in the ceramide motifs and biologically relevant modifications. To enhance the ionization/detection efficiency, working conditions have been optimized continuously by increasing the ESI chip and sampling cone potentials (with minimal in-source decay) and adjusting the concentration of the sample to be analyzed (Fig. 5a, b). This way, a more realistic representation of the ganglioside heterogeneity, when compared against TLC and capillary-based ESI was obtained. The mixture was found dominated by GD1 glycoforms: nineteen different doubly charged molecular variants of GD1 have been detected. Also identified in the mixture were GM1, GM2, GM3, GD2, GD3, GT1, and GQ1, expressing different ceramide portions. Biologically important O-acetylated and/or fucosylated GM1, GD1, GT1, and GQ1 exhibiting a high degree of heterogeneity in their ceramide motifs were also detected (Fig. 5, Table 1). For the first time, fully automated chipESI MS infusion was combined also with automated precursor ion selection and fragmentation in datadependant acquisition, allowing to obtain a sufficient set of

Fig. 8. Fully automated (−) nanoESI chip HCT multistage MS of the doubly charged ion at m/z 917.31 corresponding to GD1 (d18:1/18:0) ganglioside species detected in FL36 mixture. − a) MS2; b) fragmentation scheme of the [M− 2H]2− ion at m/z 917.31 corresponding to GD1b isomer; c) MS3 using as a precursor the monocharged Y3β ion detected at m/z 1544.61 in MS2; d) fragmentation scheme of the [M− H]− ion at m/z 1544.61. Each spectrum is a sum of scans acquired over 1.5 min at ion excitation energies within 0.6–1.0 V (from Serb et al. [64]). Nomenclature of the fragment ions is according to published recommendations [61,62].

C. Flangea et al. / Biochimica et Biophysica Acta 1811 (2011) 897–917

907

Fig. 8 (continued).

“fingerprint” ions for the structural elucidation of a GT1b isomer having the configuration (d18:1/18:0) within 1 min of acquiring MS/MS data and only approximately 0.5 pmol of sample consumption. Recently, NanoMate robot was couple to high capacity ion trap (HCT) MS and optimized in an approach based on MS n for the analysis of polysialylated brain gangliosides [60]. The feasibility of this approach was tested for top–down sequencing of a commercially available trisialylated ganglioside fraction from bovine brain (GT1b), which was profiled by MS and sequenced in tandem MS up to MS 6 in the same experiment using CID. The doubly charged ion at m/z 1077.20 detected in MS corresponded to a ubiquitous GT1b structure as confirmed in a top–down fragmentation analysis up to the MS 6 stage. Within MS 2–MS 5 dissociation events, a complete characterization of the oligosaccharide core including discrimination of sialylation sites was achieved by stepwise sequencing of tri- (Fig. 6a), di(Fig. 6b)., mono- (Fig. 6c) and finally asialo (Fig. 6d) fragment ions. Further, the lipid moiety was structurally characterized by CID MS 6 (Fig. 6e). Although GT1 (d18:1/20:0) and (d18:0/20:1) forms are most frequently expressed [60], the signals at m/z 265.80, 283.40 and 308.80 corresponding to P-, V + 16- and respectively T-type of fragment ions supported a less common (d20:1/18:0) ceramide variant having a dihydroxylated sphingoid base with 20 carbon atoms. Combination of automated chip-ESI and MS n allowed for an efficient top–down sequencing experiment, performed in a high-throughput mode in less than 3 min with a sensitivity situated in the subpicomole range. The high-throughput chip-based nanoESI HCT MS n methodology was recently used, for the first time, for comparative screening and sequencing of ganglioside components in three different regions of

human fetal brain in the 36th gestational week: frontal neocortex (NEO36), white matter of the frontal lobe (FL36) and white matter of the occipital lobe (OL36) [64]. Optimized nanoESI chip HCT MS conditions allowed for the simultaneous formation and detection of triply, doubly and singly charged ions, enhanced the ionization of long chain polysialylated GT and GQ structures, provided a fair ionization/detection of minor components and prevented the in-source fragmentation of labile carbohydrate or non-carbohydrate type of modifications. Even under the time-restrictive high-throughput conditions a remarkably rich molecular ion pattern, proving the presence of a large number of glycoforms and an unpredicted diversity of the ceramide chain for certain species especially in neocortex was observed. For the three regions of the human fetal brain, altogether 137 ganglioside and asialo-ganglioside species differing in the composition of either the oligosaccharide core or ceramide chain were detected and identified (Table 2). Direct comparison of NEO36 and FL36 ganglioside extracts indicated that the expression of species in two different tissue layers of the same topographic localization fluctuates significantly (Fig. 7a, b, Table 2): The largest number of ganglioside species (89) was identified in NEO36, which correlates with the functional complexity of neocortex as the newest brain region. Unlike NEO36, MS screening of OL36 disclosed a ganglioside expression and structural diversity comparable to that exhibited by FL36 (Fig. 7c, Table 2). In both mixtures species exhibiting short oligosaccharide chains with reduced sialic acid content dominate numerically. On the other hand, compared with NEO36, the number of O-fucosylated and O-acetylated ganglioside components was found to be severely reduced. This specific expression is in agreement with previous reports, which connect the presence of such modifications to regions

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C. Flangea et al. / Biochimica et Biophysica Acta 1811 (2011) 897–917

occurring in advanced phases of brain development [62,65]. In view of these findings, seemingly, the differences in GG expression in fetal human brain are dependent on the phylogenetic development rather than topographic factors. Fragmentation using the chip-based nanoESI/HCT MS n approach was applied for discrimination of neocortex-associated GD1b (d18:1/18:0) isomer at m/z 917.31. Because MS 2–MS 3 sequencing (Fig. 8a, c) could render fragment ions that are exclusive diagnostic for Neu5Ac attachment at outer galactose due to the symmetry of Gal-GalNAc-Gal chain (Fig. 8b, d), the presence of GD1a isomer could not be excluded. The coupling between the chip-based nanoESI system and the HCT mass spectrometer was also applied to the screening and sequencing

of gangliosides expressed in fetal cerebellum (15th and 40th gestational week—Cc15 and Cc40) (Fig. 9a, b) [66]. A number of 56 ganglioside and asialo ganglioside species differing in either the composition of the glycan core and/or that of the ceramide were identified in the Cc15 and 54 in the Cc40 mixture (Tables 3 and 4). Though the mixtures exhibited a comparable number of structures, apparently similarly expressed, some contrasts in particular with respect to the expression of shorter and larger, polysialylated species were found: Cc15 mixture was found dominated by GM3 group of species with various ceramide composition, while in the Cc40 sample, the GM4 group of monosialylated gangliosides was found predominant. Moreover, GQ species were found only in Cc15, and this finding supported previous hypotheses

Fig. 9. Fully automated (−) nanoESI chip HCT MS1 of ganglioside mixtures extracted from: a) human fetal cerebellum in the 15th gestational week, Cc15; b) human fetal cerebellum in the 40th gestational week, Cc40. Solvent: MeOH; sample concentration, 2 pmol/μL; acquisition time, 1 min; nanoESI chip, − 0.20 kV; capillary exit, − 50 V (from Mosoarca et al. [66]).

C. Flangea et al. / Biochimica et Biophysica Acta 1811 (2011) 897–917 Table 3 Assignment of the major ions detected in Cc15 ganglioside mixture by fully automated (−) nanoESI chip HCT MS. d = dihydroxy sphingoid base (from Mosoarca et al. [66]).

909

Table 4 Assignment of the major ions detected in Cc40 ganglioside mixture by fully automated (−) nanoESI chip HCT MS. d = dihydroxy sphingoid base (from Mosoarca et al. [66]).

m/z monoisotopic

Molecular ion

Proposed structure

m/z monoisotopic

Molecular ion

Proposed structure

932.00 986.88 1003.60 1018.91 1038.88 1052.89 1065.90 1086.89 1104.77 1118.91 1131.82 1152.89 1186.80 1197.80 1218.90 1232.00 1252.88 1297.80 1318.88 1363.80 1381.86 1397.80 1419.80 1431.80 1452.89 1463.80 1467.80 1497.80 1507.80 1519.80 1531.89 1552.88 1584.35 1597.80 1618.85 1631.80 1652.80 1684.82 1702.80 1718.85 1750.80 1784.79 1816.90 1818.95 1850.97 1884.90 1918.98 1948.00 1950.80 1985.80 2016.80 2018.80 2052.60 2150.80 2182.60 2214.40 2383.66

[M − 2H]2− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]−

GD1(d18:1/20:0) or GD1(d/18:0/20:1) GM4(d18:1/16:2) or GM4(d18:0/16:3) GM4(d18:1/17:1) or GM4(d18:0/17:2) GM4(d18:1/18:0) or GM4(d18:0/18:1) GM4(d18:1/20:4) GM4(d18:1/21:4) GA2(d18:1/16:0) or GA2(d18:0/16:1) GM4(d18:1/23:1) or GM4(d18:0/23:2) GM4(d18:0/24:0) GA2(d18:1/20:1) or GA2(d18:0/20:2) GA2(d18:1/21:2) or GA2(d18:0/21:3) GM3(d18:1/16:0) or GM3(d18:0/16:1) GM3(d18:1/19:4) GM3(d18:0/19:0) GM3(d18:1/21:2) or GM3(d18:0/21:3) GM3(d18:1/22:2) or GM3(d18:0/22:3) GM3(d18:0/23:0) GA1(d18:1/21:0) or GA1(d18:0/21:1) GA1(d18:1/23:3) or GA1(d18:0/23:4) GM2(d18:1/17:3) or GM2(d18:0/17:4) GM2(d18:1/23:1) or GM2(d18:0/23:2) GM2(d18:1/18:0) or GM2(d18:0/18:1) GM2(d18:1/20:3) or GM2(d18:0/20:4) GM2(d18:1/21:4) GM2(d18:1/22:1) or GM2(d18:0/22:2) GD3(d18:1/18:4) GD3(d18:1/18:2) or GD3(d/18:0/18:3) GD3(d18:1/20:1) or GD3(d18:0/20:2) GD3(d18:1/21:3) or GD3(d18:0/21:4) GD3(d18:1/22:4) GM1(d18:1/17:0) or GM1(d18:0/17:1) GM1(d18:1/19:4) GM1(d18:1/21:2) or GM1(d18:0/21:3) GM1(d18:1/22:2) or GM1(d18:0/22:3) GM1(d18:0/23:0) GM1(d18:0/24:0) GD2(d18:1/17:4) GD2(d18:1/19:2) or GD2(d18:0/19:3) GD2(d18:1/20:0) or GD2(d18:0/20:1) GD2(d18:0/21:0) GD2(d18:1/24:4) GT3(d18:1/20:3) or GT3(d18:0/20:4) GD1(d18:1/17:3) or GD1(d18:0/17:4) GD1(d18:1/17:2) or GD1(d18:0/17:3) GD1(d18:1/19:0) or GD1(d18:0/19:1) GD1(d18:1/22:4) GD1(d18:1/24:1) or GD1(d18:0/24:2) GT2(d18:1/17:2) or GT2(d18:0/17:3) GT2(d18:1/17:1) or GT2(d18:0/17:2) GT2(d18:1/20:4) GT2(d18:1/22:3) or GT2(d18:0/22:4) GT2(d18:1/22:2) or GT2(d18:0/22:3) GT2(d18:0/24:0) GT1(d18:1/20:3) or GT1(d18:0/20:4) GT1(d18:1/22:1) or GT1(d18:0/22:2) GT1(d18:0/24:0) GQ1(d18:1/16:4)

986.84 1003.60 1019.80 1039.60 1052.84 1065.80 1086.80 1094.91 1105.00 1118.80 1131.80 1152.88 1165.80 1186.88 1197.80 1218.89 1232.00 1252.88 1265.80 1286.80 1297.89 1318.86 1331.80 1352.87 1384.80 1397.83 1411.00 1431.80 1452.89 1464.00 1484.85 1497.80 1518.87 1552.91 1584.83 1618.83 1652.88 1684.88 1721.00 1750.80 1784.80 1811.80 1818.80 1854.80 1884.80 1964.00 1994.05 2008.20 2044.06 2052.60 2110.09 2124.20 2152.02 2214.18

[M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M − H]−

GM4(d18:1/16:2) or GM4(d18:0/16:3) GM4(d18:1/17:1) or GM4(d18:0/17:2) GM4(d18:1/18:0) or GM4(d18:0/18:1) GM4(d18:1/20:4) GM4(d18:1/21:4) GA2(d18:1/16:0) or GA2(d18:0/16:1) GM4(d18:1/23:1) or GM4(d18:0/23:2) GM4(d18:1/24:4) GM4(d18:0/24:0) GA2(d18:1/20:1) or GA2(d18:0/20:2) GA2(d18:1/21:2) or GA2(d18:0/21:3) GM3(d18:1/16:0) or GM3(d18:0/16:1) GM3(d18:1/17:1) or GM3(d18:0/17:2) GM3(d18:1/19:4) GM3(d18:0/19:0) GM3(d18:1/21:2) or GM3(d18:0/21:3) GM3(d18:1/22:2) or GM3(d18:0/22:3) GM3(d18:0/23:0) GM3(d18:1/24:0) or GM3(d18:0/24:1) GA1(d18:0/20.0) GA1(d18:1/21:0) or GA1(d18:0/21:1) GA1(d18:1/23:3) or GA1(d18:0/23:4) GA1(d18:1/24:4) GM2(d18:1/16:2) or GM2(d18:0/16:3) GM2(d18:1/23:0) or GM2(d18:0/23:1) GM2(d18:1/18:0) or GM2(d18:0/18:1) GM2(d18:1/19:0) GM2(d18:1/21:4) GM2(d18:1/22:1) or GM2(d18:0/22:2) GD3(d18:1/18:4) GD3(d18:1/19:1) or GD3(d18:0/19:2) GD3(d18:1/20:1) or GD3(d18:0/20:2) GD3(d18:1/22:4) GM1(d18:1/19:4) GM1(d18:1/21:2) or GM1(d18:0/21:3) GM1(d18:0/23:0) GD2(d18:1/17:4) GD2(d18:1/19:2) or GD2(d18:0/19:3) GD2(d18:1/22:4) GD2(d18:1/24:4) GT3(d18:1/20:3) or GT3(d18:0/20:4) GT3(d18:1/22:4) GD1(d18:1/17:2) or GD1(d18:0/17:3) GD1(d18:0/19:0) GD1(d18:1/22:4) GT2(d18:1/18:2) or GT2(d18:0/18:3) GT2(d18:1/20:0) or GT2(d18:0/20:1) GT2(d18:1/21:0) or GT2(d18:0/21:1) GT2(d18:1/24:3) or GT2(d18:0/24:4) GT2(d18:1/24:0) or GT2(d18:0/24:1) GT1(d18:1/17:3) or GT1(d18:0/17:4) GT1(d18:1/18:2) or GT1(d18:0/18:3) GT1(d18:1/20:2) or GT1(d18:0/20:3) GT1(d18:0/24:0)

[6,7,59,67] according to which a higher sialylation degree of human brain gangliosides is associated with incipient developmental stages of the brain. Biologically important O-Ac- and/or Fuc-modified gangliosides, previously reported as present in the adult human cerebellum [15], were not identified in fetal human cerebellum. Ganglioside chains modified by such attachments were associated with the tissue during its later developmental phase [42]. Therefore, it was hypothesized that these modifications of cerebellum gangliosides start to reach a relevant level only later, during extrauterine brain development and maturation. By combining in high throughput mode fully automated chip ESI MS infusion with CID MS2 at variable RF signal amplitudes, a complete structural characterization of two cerebellum-associated

ganglioside species: GD1 (d18:1/20:0), detected in Cc15 and GM2 (d18:1/19:0) detected in Cc40, was accomplished in only 1 min of signal acquisition for each MS2 experiment and with a sample consumption, situated in the femtomole range. 4. Chip-based electrospray for the analysis of ganglioside expression and structure in pathological brain 4.1. Neurodevelopmental and neurodegenerative disorders Gangliosides participate in induction or development of various neurodegenerative and neurodevelopmental diseases. Some autoimmune-induced neuropathies are probably directly caused by antiganglioside auto-antibodies produced due to a high immunogenicity

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cerebellum were significantly lower than in the corresponding regions of the age-matched brain used as control. In the cerebral remnant, GD3, GM2 and GT1b, GM1b nLM1 and nLD1 were found highly expressed. Oppositely, GD1a was found better expressed in the anencephalic cerebellum, while GQ1b was reduced in both anencephalic regions. In agreement with previously acquired information by immunochemical methods [77], by nanoESI MS, members of the neolactoseries gangliosides were also discovered in anencephalic brain tissues. Additional data corroborating a significant alteration of ganglioside expression in anencephalic vs. age-matched normal brain tissue were recently collected by a novel methodology based on coupling of NanoMate robot to multistage MS on the HCT MS instrument tuned in the negative ion mode [42]. A native ganglioside mixture purified from glial islands of fetal anencephalic brain tissue was investigated in comparison with the ganglioside extract from a normal fetal frontal lobe. Under identical instrumental and solution conditions, 25 distinct species in the mixture from anencephalic tissue (Fig. 10) vs. 44 of which 4 asialylated in the normal tissue were for the first time identified (Table 5). These results indicated that a high number of ganglioside species associated to anencephaly could be ionized and discriminated only by employing chip-based electrospray. Interestingly, GD3 (d18:1/18:0), GD2 (d18:1/18:0), GM1 (d18:1/18:0) and their neolacto or lacto-series isomers were detected as ions of similar low abundances in both mixtures, while GT1 (d18:1/18:0) and GD1 (d18:1/18:0) were found highly expressed in ancencephalic brain tissue. Moreover, several structures such as GT1, GQ1 and GQ2 emerged clearly as associated to anencephaly. This prominent incidence of polysialylated structures in anencephaly was considered an effect, possibly to be used as a diagnostic of brain development stagnation [6,7,59], which occurs in this disease. In view of the results obtained by MS/MS, the earlier report [30] has postulated that GT1b is one of the disease markers; however, because of the limited information obtained by fragmentation analysis in a single CID stage, validation of sialylation sites could not be accomplished. To close this gap, a nanoESI chip CID MS n protocol [42] for fine investigation of the anencephaly-specific GT1 (d18:1/18:0) species was elaborated (Fig. 11a, b). The beneficial combination of chip infusion, high capacity of ion storage and multistage sequencing rendered ions consistent with Neu5Ac2 localization at inner Gal which for the first time corroborated GT1b presence in the cerebral remnant of anencephalic brain.

of gangliosides [68,69]. In some lipidoses [70,71] a group of inherited metabolic disorders, the accumulation of gangliosides occurs in cell bodies due to a blockage of their catabolic and/or maybe even anabolic pathways. Gangliosides, especially GM1, were shown to have neuritogenic and neuronotrophic activity and to facilitate repair of neuronal tissue after mechanical, biochemical or toxic injuries. Continuous intracerebroventricular infusion of GM1 was demonstrated [72] to have a significant beneficial effect in patients with an early onset Alzheimer disease (AD) type I. Moreover, the peripheral treatment [73] of AD mutant mice, by intraperitoneal administration, with GM1, having a high affinity for Aβ, resulted in the reduction of Aβ level in the brain, suggesting that GM1 might serve as the therapeutic agent reducing/ preventing brain amyloidosis by sequestering the plasma Aβ. On the other hand, interactions of Aβ (1–40) with ganglioside-containing membranes, particularly with membrane rafts enriched in GM1 and GM2, were hypothesized [74] to be involved in the pathogenesis of AD. Using conventional high performance thin-layer chromatographic separation/detection, immunochemical and immunohistochemical detection methods specific changes in ganglioside expression and quantity in investigated human brain regions in AD disease were discovered [59]. In anencephaly [75,76], a congenital malformation of the fetal brain occurring when the cephalic end of the neural tube fails to close, the first assessment of ganglioside composition was reported by Cacić [77] on the basis of the evidence obtained by immunostaining on thin-layer chromatograms. In anencephaly where the process of cell differentiation and maturation is severely disturbed, a significant change in ganglioside pattern characterized by a marked reduction in of GM1a, GM1b and GD1a content and a better expression of neolacto-series gangliosides was found. Later on, by development and introduction in glycolipidomics of advanced MS methods based on ESI and nanoESI as methods complementary to TLC and immunochemical analyses, better insights into the ganglioside altered composition in neurodegenerative diseases was possible. In 2001, Vukelić et al. [30] optimized and applied for the first time nanoESI QTOF MS and tandem MS for compositional and structural identification of native gangliosides from anencephalic cerebral residue and cerebellum. By this approach it was found that the total ganglioside concentrations in the anencephalic cerebral remnant and in Intens. x104

1063.72

MS22-MS4 NeuAc – O – Gal – O – GalNAc – O – Gal – O – Glc – O – Cer (d18:1/18:0) 2.0

O NeuAc 917.60

O

1.5

NeuAc

1.0

1671.11 1259.92 1077.73

1237.90 1249.95

1279.88

1519.06 1553.07

1179.90

735.53

1653.21 1918.11

1139.01

0.5

1353.03 1375.03

931.72 836.68

1049.26

1207.01

1756.01 1544.16

1471.03

1757.51

1858.32 1885.08

1572.02

0.0 800

1000

1200

1400

1600

1800

2000 m/z

Fig. 10. Fully automated (−) nanoESI chip HCT MS of a native ganglioside mixture from glial islands of anencephalic fetus. Inset: the structure of GT1b (d18:1/18:0) ganglioside species as [M − 2H]2− at m/z 1063.34, deduced from MS2–MS4 analysis. Solvent: MeOH; sample concentration, 5 pmol/μL; acquisition time, 7 min; Chip ESI, –0.8 kV; capillary exit, –50 V (from Almeida et al. [42]).

C. Flangea et al. / Biochimica et Biophysica Acta 1811 (2011) 897–917 Table 5 Comparative overview upon gangliosides and asialo-gangliosides detected in the glial islands of fetal anencephalic brain and normal brain tissue. d = dihydroxy sphingoid base; t = trihydroxy sphingoid base; + = the species was detected; − = the species was not detected (from Almeida et al. [42]). GG species

Proposed structure

Anencephaly Normal tissue

GM1

nLM1 and/or LM1 (d18:0/16:0) nLM1 and/or LM1 (d18:1/18:0) nLM1 and/or LM1 (d18:0/20:0) (d18:1/16:0) (d18:1/18:0) (d18:1/22:0) (d18:1/14:0) or (d18:1/h14:0) or HexNAcHex2Cer (d18:1/22:4) (d18:1/16:0) (t18:1/16:0) or (d18:1/h16:0) or HexNAcHex2Cer(d18:1/24:4) (d18:1/18:0) (d18:0/18:0) (d18:1/20:0) (d18:1/22:0) (d18:0/22:0) (d18:1/24:2) (d18:0/24:0) (d18:1/24:1) (d18:1/24:0) O-Ac-GM3 (d18:1/20:0) or GM3 (18:1/23:0) O-Ac-GM3 (d18:1/22:1) or GM3 (20:1/23:1) O-Ac-GM3 (d18:1/22:0) or GM3 (20:1/23:0) O-Ac-GM3 (d18:0/22:0) or GM3 (20:0/23:0) O-Ac-GM3 (d18:1/24:2) (d18:1/20:2) (d18:1/18:0) (d18:1/20:0) (d18:1/23:0) (d18:1/24:1) (d18:0/18:0) (d18:1/18:0) (d18:1/18:1) (d18:1/24:1) (d18:1/24:0) (d18:1/20:0) (d18:0/18:0) (d18:1/16:0) (d18:0/16:0) (d18:1/18:1) (d18:1/18:0) (d18:1/24:1) (18:1/16:0) (d18:1/18:0) (d18:0/20:0) (d18:1/20:0) (d18:1/24:0) (d18:1/18:0) (d18:1/24:0) (d18:1/24:1) (d18:1/18:0) HexNAcHex2Cer (d18:0/14:0) or (d16:0/16:0) HexNAcHex2Cer (d18:0/16:0) HexNAcHex2Cer (t18:0/22:0) or (d18:0/h22:0) or (d18:2/24:4) HexHexNAcHex2Cer(d18:1/18:0)

+ + + + − − +

+ + − + + + +

− −

+ +

+ − + − + + + − − −

+ + + + + + + + + +

−

+

−

+

+

+

− − + + − + + + + + + − + − − − + + + + + + − − − + + −

+ + + + + − − + − − − + + + + + + − + + − + + + + − − +

− −

+ +

−

+

GM3

GM4 GD1

GD2

GD3

GT1

GT3

GQ1 Asialo-GG species

4.2. Primary brain tumors Primary CNS tumors account for approximately 2% of all adult malignancies and are responsible for approximately 7% of years of life lost due to malignancy prior to age of 70. In children 20% of all malignancies diagnosed before age 15 are primary CNS tumors, making them the second most frequent diagnosed childhood malignancy [78]. For

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therapy of these tumors aggressive surgical resection and chemotherapy is frequently problematic due to the sensitivity of the collateral brain tissue to disruption during excision and the toxicity accompanied by side-effects of therapeutic agents. Moreover, limitation of the tumor sample size in open microscopic neurosurgery and imageguided needle biopsies as well as the discordance between the biological behavior and tumor grade [79] necessitates identification of other indicators of tumor presence and behavior. Tumorigenesis/malignant transformation is accompanied by aberrant cell surface composition, particularly due to irregularities in glycoconjugate glycosylation pathways. Various glycosyl epitopes constitute tumor-associated antigens [80,81]. Some of them promote invasion and metastases, while some other suppress tumor progression [82]. Gangliosides are among the molecules bearing characteristic glycosyl epitopes causing such effects. Glycosphingolipid-dependent cross-talk between glycosynapses interfacing tumor cells with their host cells has been even recognized as a basis to define tumor malignancy [83]. Structural elucidation of individual ganglioside components in normal human brain as well as their spatial-temporal distribution was an essential requirement for investigation of primary brain tumors gangliosides. Specific changes of ganglioside pattern in brain tumors vs. normal brain, correlating with tumor histopathological origin, malignancy grade, invasiveness and progression have been observed [84,85]. A decrease in the regular ganglioside profile and an increase in the structures detected only in small amounts in normal brain tissue was found in primary brain tumors [86], demonstrating a direct correlation between ganglioside composition and histological type and grade of the tumors and an option to use this feature as biochemical marker in early histopathological diagnosis, grading and prognosis of tumors. Tumor cells of neuroectodermal origin may shed their gangliosides into circulation, resulting in higher ganglioside concentrations in serum [87]. This shedding of gangliosides into interstitial spaces and blood of oncological patients has been suggested to be involved in increased tumor cell growth and lack of immune cell recognition. Glycoantigens and lipoantigens have been recognized as relevant and potentially valuable diagnostic and prognostic markers and tumor molecular targets for development/production of specific anti-tumor drugs, such as GSL-based vaccines, but their investigation in this regard has been neglected comparing to proteins [88]. In the last years several biophysical methods have been developed for the investigation of ganglioside expression in severe brain tumors. Ganglioside profiling, their quantification and correlation to histomorphology and grading of human gliomas has been studied [89] using a newly developed microbore HPLC method. The use of infrared (IR) spectroscopy as an adjunct to histopathology in detecting and diagnosing human brain tumors was also demonstrated [90]. In another study [91] ganglioside expression in human glioblastoma was determined by confocal microscopy of immunostained brain sections using antiganglioside monoclonal antibodies. However, a large number of low abundant tumor-associated species could not be detected by these conventional analytical methods. Systematic studies of ganglioside composition in human brain tumors are still restricted to several major components and many less abundant species with possible biomarker values could not be structurally characterized. This emphasized the need for detailed and systematic screening and structural characterization of brain tumor glycoconjugate composition, which could adequately be achieved only combining up-to-date, ultra-sensitive, high-resolution methodological approaches of detection and sequencing of biomolecules, such as advanced MS methods based on chip nanoESI sometimes complemented by immunochemical and chromatographic techniques. First chip-based ESI MS method for ganglioside analysis from human brain malignant alterations was introduced by Vukelić et al. in 2007 [11]. The ganglioside composition and structure were characterized for human brain gliosarcoma obtained during surgical procedure, using the combination of NanoMate robot and QTOF MS.

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Fig. 11. Fully automated (−) nanoESI chip HCT multistage MS of the [M − 2H]2− at m/z 1063.34 corresponding to GT1 (d18:1/18:0) ganglioside species. a) MS3 using as a precursor 2− − the Y4α ion detected MS2; b) MS4 using as a precursor the Y4β ion detected in MS3 (from Almeida et al. [42]). Ion assignment is according to published recommendations [61,62].

C. Flangea et al. / Biochimica et Biophysica Acta 1811 (2011) 897–917 100

x2.5 1179.55

1470.97 GD3 (d18:1/18:0)

GM3 (d18:1/18:0)

x3

1552.92

x6

GD3 (d18:1/24:1)

O-Ac-GD3 -(d18:1/18:0)

GM3 (d18:1/22:0)

GM1/nLM1/LM1 (d18:1/24:0)

GD3 (d18:1/20:0)

1553.89

1540.88

GM3 (d18:1/24:0)

1472.01

1180.56

GM1/ nLM1/ LM1 (d18:1 /22:0)

GM2 (d18:1/18:0) 1526.92

1629.80

1382.15

GM2 (d18:1/16:0)

1235.49

1151.52

GM2 (d18:1/20:0)

GD2 1757.53 (d18:1 /22:0)

1600.92

1181.53

GD1 (d18:1/18:0) 1835.35 1918.26

1758.52

* 1572.90

1630.79

1919.24

1914.20

1675.66

1462.08

1354.20

GD1 (d18:1/24:0)

1512.89 1464.10

1207.48

1673.64 1674.65

1554.91 1383.16

1263.41

GD2 (d18:1/24:0)

GD2 (d18:1/18:0)

1263.44 1265.45

GD1 (d18:1/24:1)

1756.55

GD3 (d18:1 /22:0)

%

Gal-GlcNAc-nLM1 -lc(d18:1/24:1)

GD2 (d18:1/24:1)

1628.80

GD3 (d18:1 /24:0)

1264.44

GM3 (d18:1 /16:0)

x12

*GM1/nLM1/LM1 (d18:1/20:0)

O-Ac-GD3 -(d18:1/20:0) GM3 (d18:1/20:0)

913

1889.28 1920.22

1759.51 1729.58

1992.53

0 1150

1200

1250

1300

1350

1400

1450

1500

1550

1600

1650

1700

1750

1800

1850

1900

1950

2000

m/z Fig. 12. Negative ion mode nanoESI chip QTOF MS1 of the native gliosarcoma ganglioside mixture. ESI voltage, 1.60 kV; sampling cone, 80 V; acquisition, 2 min; average sample consumption, 0.5 pmol (from Vukelić et al. [11]).

290.09

100 x16

M1 = O-Ac-GD3 (d18:1/20:0) x4 M2 = GM1b, nLM1 and/or LM1 (d18:1/18:0)

x6

B1

1544.93

x10

[M2-H]-

O-Ac-group containing fragment ions 281.24

B1

Y3 B2-Ac

Hex-

NeuAc –O –NeuAc –O –Gal –O –Glc –O –Cer

581.20

O

C2-Ac

B-ions

B1

599.19

161.03

%

Y2

C-ions

B2

B2

C3/B2

A3-Ac

Ac

255.22

O,2A -H-Ac 3 701.54

-CO2

220.08

B3/B2

B3/B1

202.06

*

Y3-Ac

1249.84

Y2

702.53

Z3 Y3 1073.71 1207.74 1091.67

888.68 623.22

364.13

1540.96

582.19

419.25 437.29 537.20 290.35

[M1-H]-

Y3

Y1 726.55

179.04

Y4 1253.84

465.32

O,2A -3H-Ac 3 729.54 851.32

0 200

300

400

500

600

700

800

900

1000

1100

1200

1300

1400

1500

+ −

Fig. 13. Negative ion mode nanoESI chip QTOF MS/MS of the [M− H ] ion at m/z 1540.96 corresponding to the O-Ac-GD3 (d18:1/20:0). ESI voltage, 1000–1250 V; collision energy: 25– 40 eV; collision gas pressure, 5–10 psi.; acquisition time, 11 min; average sample consumption, 3.5 pmol. Inset, the fragmentation scheme of O-Ac-GD3 (from Vukelić et al. [11]). Nomenclature of the fragment ions is according to published recommendations [61,62].

Five microliter aliquots of the ganglioside mixture working sample solutions were loaded and submitted for MS screening in negative ion mode detection (Fig. 12). By chip-nanoESI QTOF MS more than 25 species dominated by GD3 and a high abundance of O-acetylated GD3 species could be observed. High intensity ions corresponding to GM3 and GD2 species carrying different ceramides were present as well. Several considerably abundant ions related to GM2, GM1, and/or their isomers

nLM1 and LM1, as well as to GD1 species characterized by heterogeneity in composition of their ceramide moieties, were found. To provide a consistent structural identification, several detected species were subjected to fine analysis by tandem MS. Sequencing data defined the composition and detailed structure of several gliosarcoma-associated species among which GD3 (d18:1/24:1) O-Ac-GD3 (d18:1/20:0) GD2 (d18:1/18:0), GM1a (d18:1/18:0), GM1b, nLM1 or

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Fig. 14. Fully automated nanoESI chip HCT MS1 of the native human brain hemangioma ganglioside mixture extracted from frontal lobe hemangioma tissue. Solvent, MeOH; sample concentration, 0.5 pmol/μL; acquisition time, 0.5 min; chip-ESI, − 0.85 kV; capillary exit, − 30 V (from Schiopu et al. [93]).

LM1 (d18:1/18:0). A particular attention was paid to O-Ac-GD3 molecule because this ganglioside could by itself be responsible for the protection of tumor cells from apoptosis [92]. Its sequencing pattern offered the structural support to postulate a novel O-Ac-GD3 isomer, O-acetylated at the inner Neu5Ac-residue. In Fig. 13, the Y3− ion at m/z 1249.84 represents the evidence for Ac-O-Neu5Ac-Gal-Glc-sequence carrying d18:1/20:0 ceramide. This feature confirmed that in gliosarcoma O-acetylation of GD3 occurs at the inner Neu5Ac residue. Two years later, an investigation of ganglioside composition and structure in human brain hemangioma, a benign tumor, using advanced mass spectrometry methods based on NanoMate HCT and CID MSn was carried out [93]. The obtained mass spectrum revealed 29 different ganglioside species dominated by mono- and disialylated structures (Fig. 14 and Table 6). Two acetylated species, O-Ac-GM4 (d18:0/29:0) and O-AcGD2 (d18:1/23:0), the last one correlated with the reduced malignancy grade of the cerebral tumor were discovered. For fine structural analysis of the unusual, hemangioma-asociated GT1, CID MS2 at variable RF signal amplitudes within 0.6–1.0 V was applied (Fig. 15a). Five different fragment ions supported a structure of GT1c-type bearing (d18:0/20:0) ceramide (Fig. 15a, b). To confirm this assignment, the ion corresponding to GD1b (d18:0/20:0) was submitted to CID MS2 under identical conditions. It was found that GD1b structure has the same lipid constitution as the previously sequenced GT1 however, an oligosaccharide core lacking one Neu5Ac residue. The NanoMate-based system developed and optimized for determination of ganglioside expression and structure in human brain hemangioma was able to detect an elevated number of species and, most importantly, to correlate the presence of O-Ac-GD2 with the low malignancy grade of the investigated cerebral tumor. 5. Conclusions As compared to classical ESI MS with capillary-based infusion, microfluidic-MS methodology provides higher ionization efficiency, a remarkable speed of analysis and superior sensitivity, situated in the low picomole or even femtomole range. In combination with high resolution mass spectrometers or instruments able to perform multistage fragmentation of chosen precursor ions, chip-electrospray

demonstrated its unique ability to offer structural characterization of minor ganglioside species in complex mixtures, which very often represent valuable biomarkers. This aspect is of particular importance for the applicability of chip-MS approaches in clinical investigation where only minute amounts of biological material are usually available. As presented here, in the last years, these methods started to be employed also Table 6 Assignment of the major molecular ions detected in human brain hemangioma ganglioside mixture. d = dihydroxy sphingoid base (from Schiopu et al. [93]). Type of molecular ion

m/z (monoisotopic)

Assigned structure

[M − 2H]2− [M − 2H]2− (− 2H2O) [M − H]2− (− H2O) [M − H]2− (− H2O) [M − H]2− (− H2O) [M − H]2− [M + Na − 2H]2− [M − H]2− [M + Na − 2H]2− [M + Na − 2H]2[M − H]− [M − H]− [M − H]− [M − H]− [M − 2H]2− [M − H]− [M − H]− [M + 2Na − 3H]− [M − H]− [M − H]− [M − H]− (− 2H2O) [M − H]− [M − H]− (− H2O) [M − H]− [M − H]− [M + Na − 2H]− [M − H]− [M + Na − 2H]− [M + 2Na − 3H]−

733.57 749.58 793.61 827.41 839.80 863.35 875.00 917.41 922.00 956.05 983.07 987.42 1034.08 1062.54 1077.00 1151.76 1186.26 1214.45 1216.44 1306.03 1555.98 1634.04 1698.04 1748.82 1786.28 1816.52 1858.17 1886.20 1909.31

GM2(d18:1/24:0) GM2(d18:1/29:1) GD2(d18:0/13:0) GD2(d18:1/18:0) GM1(d18:1/29:0) GD2(d18:1/22:1) GM1(d18:0/31:0) GD1(d18:1/18:0) GD1(d18:0/17:0) GD1(d18:1/22:0) GM4(d18:1/16:4) GM4(d18:1/16:0) GM4(d18:0/19:0) GM4(d18:0/21:0) GT1(d18:1/20:0) GM3(d18:0/16:1) GM3(d18:1/19:4) GM4(d18:1/29:1) GM3(d18:1/21:3) GA2(d18:1/22:2) GD2(d18:0/12:0) GM1(d18:1/27:2) GD2(d18:1/21:0) GT3(d18:1/17:0) O-Ac-GD2(d18:1/23:0) GD2(d18:1/27:3) GT3(d18:1/25:1) GD1(d18:1/20:0) GD2(d18:0/20:0)

C. Flangea et al. / Biochimica et Biophysica Acta 1811 (2011) 897–917

915

Fig. 15. Fragmentation analysis of the [M − 2H]2− at m/z 1077.00 corresponding to GT1(18:1/20:0) ganglioside species detected in human brain hemangioma ganglioside mixture. a) Fully automated nanoESI chip HCT MS2; b) fragmentation scheme corresponding to the GT1c isomer. CID at variable rf signal amplitudes within 0.6–1.0 V. Acquisition time, 0.5 min (from Schiopu et al. [93]). Nomenclature of the fragment ions is according to published recommendations [61,62].

for discovery of ganglioside markers in neurodegenerative and neurodevelopmental diseases and primary brain tumors. The highlighted accomplishments in characterization of novel structures in severe brain pathologies indicate that advanced chip-based ESI MS has real perspectives to become a routine method for early diagnosis and therapy based on discovery of ganglioside molecular fingerprints.

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