Testing the feasibility of fully automated chip-based nanoelectrospray ionization mass spectrometry as a novel tool for rapid diagnosis of Fabry disease.

Fabry condition, a lysosomal storage disease (LSD) is characterized by the absence or reduction of the α-galactosidase A activity. Recently, a new diagnostic method for detection of α-galactosidase activity from dried blood spots (DBS) using a chemical substrate and quantification of reaction mixture was developed. To improve this method in the terms of automation, reproducibility, sensitivity, and data reliability, we introduce here an innovative analytical approach based on chip-nanoESI MS. The α-galactosidase assay products derived from DBS of 11 healthy donors and 11 Fabry disease patients were analyzed by NanoMate robot coupled to a high-capacity ion trap MS. Confirmation and structural analysis of the reaction products was achieved by CID and electron transfer dissociation (ETD) MS/MS. The cleavage of a substrate GLA-S generated a product, GLA-P, which was quantified related to an internal standard GLA-IS. Comparative patient versus control analysis indicated a 13-fold reduction in GLA-P/GLA-IS ratio in the case of the patients. Moreover, our method provided direct data on the enzyme, from which it was for the first time possible to discriminate between the patients lacking the enzyme and those presenting a less active one. GLA-IS and GLA-P were confirmed by CID/ETD, which applied together, increased considerably the sequence coverage and provided complementary information for unambiguous product identification. The present chip-nanoESI CID and ETD MS(n) strategy introduced here for first time in LSD diagnosis, provided a maximum confidence in assay product identification, a high sensitivity, speed of analysis, and result reproducibility.

Identification of an unusually sulfated tetrasaccharide chondroitin/dermatan motif in mouse brain by combining chip-nanoelectrospray multistage MS2 -MS4 and high resolution MS.

Chondroitin sulfate (CS)/dermatan sulfate (DS) are often found in nature as hybrid glycosaminoglycan chains in various proteoglycans. In the recent years, several MS methods were developed for the determination of over-, regular-, and undersulfated CS/DS chains. In the present work, the released hybrid CS/DS isolated and purified from mouse brain were digested with chondroitin AC lyase. The depolymerized chains were separated by gel filtration chromatography. Collected tetrasaccharides were analyzed by fully automated (NanoMate robot) chip-based nanoESI high capacity ion trap multistage MS (MS(2) -MS(4) ) recently introduced in glycosaminoglycan research by our laboratory. The obtained data were confirmed by high resolution MS screening and MS/MS performed on QTOF instrument. NanoMate-high capacity ion trap MS and QTOF MS screening revealed the presence in the mixture of oversulfated tetrasaccharides bearing three and four sulfate groups as well as traces of regularly and undersulfated hexamers. Additionally, several saturated species as either tetramers or hexamers exhibiting different sulfate content were discovered in the analyzed fraction. This diversity of the sulfation status indicates that the mouse brain might contain several types of proteoglycans. The molecular ions corresponding to trisulfated-[4,5Δ-GlcA-GalNAc-IdoA-GalNAc] were subjected to multistage fragmentation by CID. Sequence analysis data allowed for the postulation of two rare structural motifs: [4,5Δ-GlcA-GalNAc(4S)-IdoA(2S,3S)-GalNAc] and [4,5Δ-GlcA-GalNAc-IdoA(2S,3S)-GalNAc(4S)], previously not reported in neural tissue.

Profiling and sequence analysis of gangliosides in human astrocytoma by high-resolution mass spectrometry.

In this preliminary investigation, a low-grade astrocytoma (AcT) is investigated by high-resolution (HR) mass spectrometry (MS) aiming at characterization of gangliosides with potential biomarker value. The research was conducted towards a comparative mapping of ganglioside expression in AcT, its surrounding tissue (ST) and a normal control brain tissue (NT). HR MS was conducted in the negative ion mode nanoelectrospray ionization (nanoESI). Fragmentation analysis was carried out by collision-induced dissociation (CID) MS(2)-MS(4.) Due to the high resolving power and mass accuracy, by comparative mapping of the ganglioside extracts from AcT, ST and NT, under identical conditions, 37 different species in AcT, 40 in ST and 56 in NT were identified. AcT and ST were found to contain 18 identical ganglioside components. Among all three specimens, ST extract presented the highest levels of sialylation, fucosylation and acetylation, a feature which might be correlated to the tumor expansion in the adjacent brain area. MS mapping indicated also that AcT, ST and NT share one doubly deprotonated molecule at m/z 1063.31, attributable to GT1(d18:1/18:0) or GT1(d18:0/18:1). CID MS(2)-MS(4) on these particular ions detected in AcT and ST provided data supporting GT1c isomer in the investigated astrocytoma tissue. Our results show that HR MS has a remarkable potential in brain cancer research for the determination of tumor-associated markers and for their structural determination.

Chip-based nanoelectrospray mass spectrometry of brain gangliosides.

In the past few years, a considerable effort was invested in interfacing mass spectrometry (MS) to microfluidics-based systems for electrospray ionization (ESI). Since its first introduction in biological mass spectrometry, chip-based 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.

Reprint of: chip-based nanoelectrospray mass spectrometry of brain gangliosides.

In the past few years, a considerable effort was invested in interfacing mass spectrometry (MS) to microfluidics-based systems for electrospray ionization (ESI). Since its first introduction in biological mass spectrometry, chip-based 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.

Analysis of oversulfation in biglycan chondroitin/dermatan sulfate oligosaccharides by chip-based nanoelectrospray ionization multistage mass spectrometry.

Biglycan (BGN) is a small proteoglycan that consists of a protein core containing leucine-rich repeat regions and two glycosaminoglycan (GAG) chains of either chondroitin sulfate (CS) or dermatan sulfate (DS) type. The development of novel, highly efficient analytical methods for structural identification of BGN-derived CS/DS motifs, possibly implicated in biological events, is currently the focus of research. In this work, an improved analytical method based on fully automated chip-nanoelectrospray ionization (nanoESI) in conjunction with high-capacity ion trap (HCT) multistage mass spectrometry (MS) by collision-induced dissociation (CID) was for the first time applied to BGN CS/DS oligosaccharide analysis. The CS/DS chains were released from transfected 293 BGN by ß-elimination. The chain was digested with AC I lyase, and the resulting mixture was purified and subsequently separated by size exclusion chromatography (SEC). Di- and tetrasaccharide fractions were pooled and characterized in detail using the developed chip-nanoESI protocol. The chip-nanoESI MS profile in the negative ion mode revealed the presence of under-, regularly, and oversulfated species in both di- and tetrasaccharide fractions. CID MS(2)-MS(3) yielded sequence patterns consistent with unusual oversulfated 4,5-Δ-GlcA(2S)-GalNAc(4S) and 4,5-Δ-GlcA(2S)-GalNAc(6S)-IdoA(2S)-GalNAc(6S) motifs.

High-performance separation techniques hyphenated to mass spectrometry for ganglioside analysis.

Gangliosides, sialic-acid-containing glycosphingolipids are involved in numerous biological processes and play essential roles in severe pathologies, with predilection in those of the central nervous system. Formerly, ganglioside composition and quantity were assessed exclusively by thin-layer chromatographic (TLC), immunochemical, and immunohistochemical methods, which have limited effectiveness being unable to detect minor components in mixtures of high heterogeneity. Increased awareness of the biological importance of gangliosides stimulated the development of analytical methods that are better amenable to complex ganglioside mixtures. More recently, MS in online conjunction with high-performance separation techniques brought a significant progress to the field. This review highlights the state-of-the-art development and application of separation methods online coupled to MS for ganglioside analysis. Most original and successful protocols based on GC-MS, LC-MS, and CE-MS are presented here together with the special instrumental and sample preparation requirements to be met for effective ganglioside separation, detection, and structural identification. Finally, the advantages and downsides of each methodology as well as the perspectives for simplification, standardization, and upgrading are assessed.

Combining size-exclusion chromatography and fully automated chip-based nanoelectrospray quadrupole time-of-flight tandem mass spectrometry for structural analysis of chondroitin/dermatan sulfate in human decorin.

Chondroitin/dermatan sulfate (CS/DS) chain of decorin (DCN) from human skin fibroblasts (HSk) was released by reductive ß-elimination reaction and digested with chondroitin AC I lyase. Enzymatic hydrolysis mixture of CS/DS chains was separated by size-exclusion chromatography (SEC). Collected octasaccharide fraction was subjected to fully automated chip-based nanoelectrospray (nanoESI) quadrupole time-of-flight (QTOF) MS and tandem MS (MS/MS). MS of human skin fibroblasts DCN CS/DS displayed a high complexity due to the large variety of glycoforms, which under chip-nanoESI MS readily ionized to form multiply charged ions. Except for the regularly tetrasulfated octasaccharide, the investigated fraction contained four additional octasaccharides of atypical sulfation status. Two new oversulfated glycoforms and two undersulfated species were identified. Remarkably, the series of decasaccharides discovered in the same SEC pool was found to encompass a trisulfated and a novel hexasulfated [4,5-Δ-GlcAGalNAc(IdoAGalNAc)4] species. MS/MS by collision-induced dissociation (CID) on the [M-4H]4 ion corresponding to the previously not reported [4,5-Δ-GlcAGalNAc(IdoAGalNAc)3](5S) corroborated for a novel motif in which three N-acetylgalactosamine (GalNAc) moieties are monosulfated, 4,5-Δ-GlcA and the first IdoA from the non-reducing end bear one sulfate group each, while the second N-acetylgalactosamine from the reducing end is unsulfated.

Modern developments in mass spectrometry of chondroitin and dermatan sulfate glycosaminoglycans.

Chondroitin sulfate (CS) and dermatan sulfate (DS) are special types of glycosaminoglycan (GAG) oligosaccharides able to regulate vital biological functions that depend on precise motifs of their constituent hexose sequences and the extent and location of their sulfation. As a result, the need for better understanding of CS/DS biological role called for the elaboration and application of straightforward strategies for their composition and structure elucidation. Due to its high sensitivity, reproducibility, and the possibility to rapidly generate data on fine CS/DS structure determinants, mass spectrometry (MS) based on either electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI) brought a major progress in the field. Here, modern developments in MS of CS/DS GAGs are gathered in a critical review covering the past 5 years. The first section is dedicated to protocols for CS/DS extraction from parent proteoglycan, digestion, and purification that are among critical prerequisites of a successful MS experiment. The second part highlights several MALDI MS aspects, the requirements, and applications of this ionization method to CS/DS investigation. An ample chapter is devoted to ESI MS strategies, which employ either capillary- or advanced chip-based sample infusion in combination with multistage MS (MS(n)) using either collision-induced (CID) or electron detachment dissociation (EDD). At last, the potential of two versatile separation techniques, capillary electrophoresis (CE), and liquid chromatography (LC) in off- and/or on-line coupling with ESI MS and MS(n), is discussed, alongside an assessment of particular buffer/solvent conditions and instrumental parameters required for CS/DS mixture separation followed by on-line mass analysis of individual components.

Effects of Anthocyanins on Vascular Health.

Cardiovascular disorders are leading mortality causes worldwide, often with a latent evolution. Vascular health depends on endothelial function, arterial stiffness, and the presence of atherosclerotic plaques. Preventive medicine deserves special attention, focusing on modifiable cardiovascular risk factors, including diet. A diet rich in fruits and vegetables has well-known health benefits, especially due to its polyphenolic components. Anthocyanins, water-soluble flavonoid species, responsible for the red-blue color in plants and commonly found in berries, exert favorable effects on the endothelial function, oxidative stress, inhibit COX-1, and COX-2 enzymes, exert antiatherogenic, antihypertensive, antiglycation, antithrombotic, and anti-inflammatory activity, ameliorate dyslipidemia and arterial stiffness. The present review aims to give a current overview of the mechanisms involved in the vascular protective effect of anthocyanins from the human diet, considering epidemiological data, in vitro and in vivo preclinical research, clinical observational, retrospective, intervention and randomized studies, dietary and biomarker studies, and discussing preventive benefits of anthocyanins and future research directions.

Oral lichen planus - case report.

This paper presents the case of a 58-year-old heavy smoker female who came to our clinic with acute pain, as well as mastication and feeding difficulties. The macroscopic examination revealed oral erosive lesions and ulcerations. The polymorphic aspect of the lesions required the differential diagnosis of oral erythroplakia or carcinoma, which were excluded by biopsy. At the same time, we assessed the expression of S100 protein, Ki67 and the cluster of differentiation (CD) 4, CD8 (T-cell) and CD20 (B-cell) immune cell markers by immunohistochemical analysis. As a result, after the clinical and pathological assessment, the diagnosis of oral lichen planus was established, and a therapy plan was conducted. We observed a favorable clinical evolution after the administration of corticosteroids and immunomodulatory agents.

Analysis of novel over- and under-sulfated glycosaminoglycan sequences by enzyme cleavage and multiple stage MS.

We report on a novel strategy for identification of specific sulfation motifs in chondroitin/dermatan sulfate (CS/DS) chain derived from decorin (Dcn), based on enzyme cleavage and multistage MS (MS(n)). Released CS/DS chains were digested with chondroitin B and in parallel with AC I lyases to obtain oligosaccharides of known hexuronic acid (HexA) epimerization. The depolymerized chains were separated by gel filtration, and collected di- and hexasaccharides were analyzed by ESI MS(n). MS(2) on bisulfated 4,5-Delta-HexAGalNAc revealed an additional sulfate ester group at 4,5-Delta-HexA. MS(2) data provided evidence upon GlcA sulfation in Dcn due to the fact that 4,5-Delta-HexA derived from GlcA after chondroitin AC I lyase treatment. Hexasaccharide screening in the MS(1) mode indicated direct correlation between the sulfate distribution and HexA epimerization. MS(n) performed on ions that, according to mass calculation, correspond to pentasulfated [4,5-Delta-HexAGalNAc(GlcAGalNAc)(2)], trisulfated [4,5-Delta-HexAGalNAc(GlcAGalNAc)(2)] with IdoA-derived 4,5-Delta-HexA at the nonreducing end, tetrasulfated [4,5-Delta-HexAGalNAc(IdoAGalNAc)(2)] and monosulfated [4,5-Delta-HexAGalNAc(IdoAGalNAc)(2)] with GlcA-derived 4,5-Delta-HexA at the nonreducing end rendered fragmentation patterns confirming the presence of over-, regular, and under-sulfated regions as well as structural motifs having both types of HexA sulfated within Dcn CS/DS.

Determination of sulfation pattern in brain glycosaminoglycans by chip-based electrospray ionization ion trap mass spectrometry.

Chondroitin sulfate (CS) and dermatan sulfate (DS) glycosaminoglycans display variability of sulfation in their constituent disaccharide repeats during chain elongation. Since a large proportion of the extracellular matrix of the central nervous system (CNS) is composed of proteoglycans, CS/DS disaccharide degree and profile of sulfation play important roles in the functional diversity of neurons, brain development, and some of its pathological states. To investigate the sulfation pattern of CS/DS structures expressed in CNS, we introduced here a novel method based on an advanced system encompassing fully automated chip nanoelectrospray ionization (nanoESI) in the negative ion mode and high capacity ion trap multistage mass spectrometry (MS(2)-MS(3)) by collision-induced dissociation (CID). This method, introduced here for the first time in glycomics of brain glycosaminoglycans, was particularly applied to structural investigation of disaccharides obtained by beta-elimination and digestion with chondroitin B and AC I lyase of hybrid CS/DS chains from wild-type mouse brain. Screening in the chip-MS mode of DS disaccharide fraction resulting after depolymerization with chondroitin B lyase revealed molecular ions assigned to monosulfated disaccharide species having a composition of 4,5-Delta-[IdoA-GalNAc]. By optimized CID MS(2)-MS(3), fragment ions supporting the localization of sulfate ester group at C4 within GalNAc were produced. Chip ESI MS profiling of CS disaccharide fraction obtained by depolymerization of the same CS/DS chain using chondroitin AC I lyase indicated the occurrence of mono- and bisulfated 4,5-Delta-[GlcA-GalNAc]. The site of oversulfation was determined by MS(2)-MS(3), which provided sequence patterns consistent with a rare GlcA-3-sulfate-GalNAc-6-sulfate structural motif. Figure Mouse brain GlcA-3-sulfate-GalNAc-6-sulfate structural motif.

Determination of ganglioside composition and structure in human brain hemangioma by chip-based nanoelectrospray ionization tandem mass spectrometry.

We report here on a preliminary investigation of ganglioside composition and structure in human hemangioma, a benign tumor in the frontal cortex (HFC) in comparison to normal frontal cortex (NFC) tissue using for the first time advanced mass spectrometric methods based on fully automated chip-nanoelectrospray (nanoESI) high-capacity ion trap (HCT) and collision-induced dissociation (CID). The high ionization efficiency, sensitivity and reproducibility provided by the chip-nanoESI approach allowed for a reliable MS-based ganglioside comparative assay. Unlike NFC, ganglioside mixture extracted from HFC was found dominated by species of short glycan chains exhibiting lower overall sialic acid content. In HFC, only GT1 (d18:1/20:0), and GT3 (d18:1/25:1) polysialylated species were detected. Interestingly, none of these trisialylated forms was detected in NFC, suggesting that such components might selectively be associated with HFC. Unlike the case of previously investigated high malignancy gliosarcoma, in HFC one modified O-Ac-GD2 and one modified O-Ac-GM4 gangliosides were observed. This aspect suggests that these O-acetylated structures could be associated with cerebral tumors having reduced malignancy grade. Fragmentation analysis by CID in MS(2) mode using as precursors the ions corresponding to GT1 (d18:1/20:0) and GD1 (d18:1/20:0) provided data corroborating for the first time the presence of the common GT1a and GT1b isomers and the incidence of unusual GT1c and GT1d glycoforms in brain hemangioma tumor.

High-throughput analysis of gangliosides in defined regions of fetal brain by fully automated chip-based nanoelectrospray ionization multi-stage mass spectrometry.

Gangliosides (GGs), a large group of sialylated glycosphingolipids, are considered biomarkers of human brain development, aging and certain diseases. Determination of individual GG components in complex mixtures extracted from a human brain represents a fundamental prerequisite for correlating their specificity with the specialized function of each brain area. In the context of modern glycomics, detailed investigation of GG expression and structure in human brain requires a continuous development and application of innovative methods able to improve the quality of data and speed of analysis. In this work, for the first time, a high-throughput mapping and sequencing of gangliosides in human fetal brain was performed by a novel mass spectrometry (MS)-based approach developed recently in our laboratory. Three GG mixtures extracted and purified from different regions of the same 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) were subjected to comparative high-throughput screening and multi-stage fragmentation by fully automated chip-based nanoelectrospray ionization (nanoESI) high capacity ion trap (HCT) MS. Using this method, in only a few minutes of signal acquisitions, over 100 GG and asialo-GG species were detected and identified in the three mixtures. Obtained data revealed for the first time that differences in GG expression in human fetal brain are dependent on phylogenetic development rather than topographic factors. While a significant variation of GG distribution in NEO36 vs FL36 was observed, no significant differences in GG expression in white matter of frontal vs occipital lobe were detected. Additionally, the largest number of species was identified in NEO36, which correlates with the functional complexity of neocortex as the newest brain region. In the last stage of analysis, using MS(2)-MS(3) molecular ion fragmentation at variable amplitudes, a NEO36-associated GD1b isomer could clearly be discriminated. Present results indicate that the combination of fully automated chipESI with HCT MS(n) is able to provide ultra-fast, sensitive and reliable analyses of complex lipid-linked carbohydrates from which the pattern of their expression and structure in a certain type of bio-matrix can be determined.

Bcl-2 expression in Hodgkin's lymphoma progression.

INTRODUCTION: Hodgkin's lymphoma study by immunohistochemical expression of Bcl-2 in Hodgkin and Reed-Sternberg cells can precise these cases evolutive way. MATERIAL AND METHODS: Sixty-three cases of classical Hodgkin's disease, hospitalized into the Hematology Department of the County Hospital No. 1 Timisoara, were studied. Histopathological diagnostic was performed using common staining methods, and for revealing the tumoral developments immunohistochemical staining was performed Bcl-2. RESULTS AND DISCUSSION: In our study, the results were noticed a direct relation between the rise of tumoral proliferation index expressions of Bcl-2 and progression of the disease (p < or = 0.001). For I and II stages Bcl-2 expression does not overcome (-/+) category while the III and IV stages, all the cases are situated in (+/-) and (+) categories. No connection we can be noticed between the histological type and Bcl-2 expression although the classic Hodgkin's lymphoma with lymphocyte depletion is considered the most aggressive histological type (p < or = 1). In our study, we found this correlation very important because the main cause of relapses is inadequate staging. In some cases, this staging is difficult; some little lymph nodes could be overlooked because they can be placed in less accessible areas and cannot be evidenced by the most imagistic methods. CONCLUSIONS: All the cases were Bcl-2 expression higher than (+/-) and are staged as I and II stages should be reinvestigated and restaged. This immunohistochemical reaction, although less used in Romania, is very accurate. That is very important because the therapeutically attitude is different in advances stages compared to earlier stages.

CD30 expression utilization for the accuracy of classical Hodgkin's lymphoma staging.

INTRODUCTION: The presence of Reed-Sternberg malignant cells is absolutely necessary for Hodgkin's lymphoma diagnostic, but it is not always sufficient because can be observed Reed-Sternberg-like cells in other malignant and benign diseases, too. The CD30 expression at Hodgkin and Reed-Sternberg level can give us supplementary information in differential diagnostic and can be used as progressive disease factor. MATERIAL AND METHODS: Our study was composed from 63 cases histopathological diagnosed with Hodgkin's lymphoma and hospitalized in Hematology Department of County Hospital Timisoara. CD30 expression was immunohistochemical semi-quantitative evaluated using clone BerH2 as primary antibody and APAAP-New Fuchsin as visualization system. RESULTS AND DISCUSSIONS: The increasing of CD30 expression occurs in the same time with advanced stages and the disease progression (p =0.001). For I and II stages CD30 expression does not overcome (-/+) category while the III and IV stages, all the cases are situated in (+/-) and (+) categories. No connection can be noticed between histological type and CD30 expression (p < or = 1). We consider that using this staining, although less used in Romania, must be done in all Hodgkin's lymphoma and Hodgkin's lymphoma-like cases. We say that because the main cause of relapses is represented by inadequate clinical staging and diagnostic. CONCLUSIONS: In our study, the increasing of CD30 expression is associated with advanced disease stage. We recommend reinvestigating and restaging all cases that was included into an incipient stages and they have a CD30 expression situated in (+/-) and (+) intervals because some lymph nodes could be overlooked.

Separation and identification of glycoforms by capillary electrophoresis with electrospray ionization mass spectrometric detection.

Capillary electrophoresis (CE) is a resourceful and versatile separation method for the analysis of complex carbohydrate mixtures. In combination with electrospray ionization (ESI) mass spectrometry (MS), CE enables fast, sensitive, and efficient separations for the accurate identification of a large variety of glycoform mixture types. In this chapter several reliable off- and on-line CE-based methods for the analysis of glycoforms with ESI MS/MS are presented. The first part of this chapter is dedicated to the application of off-line CE/ESI MS to complex mixtures of O-glycopeptides and mixtures of proteoglycan-derived O-glycans, i.e., glycosaminoglycans such as depolymerized hybrid chains of chondroitin sulfate (CS) and dermatan sulfate (DS). Procedures for off-line fractionation of these heterogeneous mixtures followed by ESI MS screening and sequencing of single glycoforms by collision-induced dissociation (CID) at low energies are also described. Ample sections are further devoted to on-line CE/ESI MS technique and its application to separation and identification of O-glycopeptides and CS/DS oligosaccharides. The concept and construction principles of two different sheathless CE/ESI MS interfaces together with the protocols to be applied for successful on-line analysis of O-glycopeptides and CS/DS oligosaccharides are presented in details in the last part of the chapter.

Brain chondroitin/dermatan sulfate, from cerebral tissue to fine structure: extraction, preparation, and fully automated chip-electrospray mass spectrometric analysis.

Chondroitin sulfate (CS) and dermatan sulfate (DS) glycosaminoglycans (GAGs) are covalently linked to proteins, building up a wide range of proteoglycans, with a prevalent expression in the extracellular matrix (ECM). In mammalian tissues, these GAG species are often found as hybrid CS/DS chains. Their structural diversity during chain elongation is produced by variability of sulfation in the repeating disaccharide units. In central nervous system, a large proportion of the ECM is composed of proteoglycans; therefore, CS/DS play a significant role in the functional diversity of neurons, brain development, and some brain diseases. A requirement for collecting consistent data on brain proteoglycan glycosylation is the development of adequate protocols for CS/DS extraction and detailed compositional and structure analysis. This chapter will present a strategy, which combines biochemical tools for brain CS/DS extraction, purification, and fractionation, with a modern analytical platform based on chip-nanoelectrospray multistage mass spectrometry (MS) able to provide information on the essential structural elements such as epimerization, chain length, sulfate content, and sulfation sites.