Life sciences and mass spectrometry: some personal reflections.

Molecular analysis of biological systems by mass spectrometry was in focus of technological developments in the second half of the 20th century, in which the issues of chemical identification of high molecular diversity by biophysical instrumental methods appeared as a mission impossible. By developing dialogs between researchers dealing with life sciences and medicine on one side and technology developers on the other, new horizons toward deciphering, identifying and quantifying of complex systems became a reality. Contributions toward this goal can be today considered as pioneering efforts delivered by a number of researchers, including generations of motivated students and associates.

Preliminary mass spectrometry characterization studies of galectin-3 samples, prior to carbohydrate-binding studies using Affinity mass spectrometry.

RATIONALE: Investigation of non-covalent complexes of proteins using Affinity Mass Spectrometry (AMS) represents a major challenge in modern biomedical research. However, many experimental obstacles can make AMS data analysis complex. Additionally, sample purity and size of the protein may still pose significant challenges. METHODS: Matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS) was used for initial mapping of protein samples. nanoESI (electrospray ionization) quadrupole-time-of-flight (QTOF) MS was used for mapping of protein samples under native conditions and subsequent AMS studies. The human galectin-3 protein sample was expressed in E. coli. RESULTS: Full length galectin-3 was difficult to work with, due to several truncated forms observed after the purification procedures. On the other hand, galectin-3C produced excellent quality nanoESI-MS spectra. A covalent adduct of lactose was found to be located on residue Lys 176. Functional AMS control studies indicated that galectin-3 interactions with oligosaccharides may be dependent on its charge. CONCLUSIONS: Mass spectrometry represents a valuable tool that can be efficiently used for structural characterization of protein samples prior to functional analyses. By means of accurate mass measurements, many protein truncations can be identified based on mass alone. Analysis of covalent adducts is more challenging. Finally, for AMS studies, careful use of controls may reveal charge-dependence of protein-oligosaccharide interactions. Copyright © 2016 John Wiley & Sons, Ltd.

Quantitative characterization of galectin-3-C affinity mass spectrometry measurements: Comprehensive data analysis, obstacles, shortcuts and robustness.

RATIONALE: Affinity mass spectrometry (AMS) is an emerging tool in the field of the study of protein•carbohydrate complexes. However, experimental obstacles and data analysis are preventing faster integration of AMS methods into the glycoscience field. Here we show how analysis of direct electrospray ionization mass spectrometry (ESI-MS) AMS data can be simplified for screening purposes, even for complex AMS spectra. METHODS: A direct ESI-MS assay was tested in this study and binding data for the galectin-3C•lactose complex were analyzed using a comprehensive and simplified data analysis approach. In the comprehensive data analysis approach, noise, all protein charge states, alkali ion adducts and signal overlap were taken into account. In a simplified approach, only the intensities of the fully protonated free protein and the protein•carbohydrate complex for the main protein charge state were taken into account. RESULTS: In our study, for high intensity signals, noise was negligible, sodiated protein and sodiated complex signals cancelled each other out when calculating the Kd value, and signal overlap influenced the Kd value only to a minor extent. Influence of these parameters on low intensity signals was much higher. However, low intensity protein charge states should be avoided in quantitative AMS analyses due to poor ion statistics. CONCLUSIONS: The results indicate that noise, alkali ion adducts, signal overlap, as well as low intensity protein charge states, can be neglected for preliminary experiments, as well as in screening assays. One comprehensive data analysis performed as a control should be sufficient to validate this hypothesis for other binding systems as well.

Application of ion mobility tandem mass spectrometry to compositional and structural analysis of glycopeptides extracted from the urine of a patient diagnosed with Schindler disease.

RATIONALE: Schindler disease is caused by the deficient activity of α-N-acetylgalactosaminidase, which leads to an abnormal accumulation of O-glycopeptides in tissues and body fluids. In this work the Schindler condition is for the first time approached by ion mobility (IMS) tandem mass spectrometry (MS/MS), for determining urine glycopeptide fingerprints and discriminate isomeric structures. METHODS: IMS-MS experiments were conducted on a Synapt G2s mass spectrometer operating in negative ion mode. A glycopeptide mixture extracted from the urine of a patient suffering from Schindler disease was dissolved in methanol and infused into the mass spectrometer by electrospray ionization using a syringe-pump system. MS/MS was performed by collision-induced dissociation (CID) at low energies, after mobility separation in the transfer cell. Data acquisition and processing were performed using MassLynx and Waters Driftscope software. RESULTS: IMS-MS data indicated that the attachment of one or two amino acids to the carbohydrate backbone has a minimal influence on the molecule conformation, which limits the discrimination of the free oligosaccharides from the glycosylated amino acids and dipeptides. The structural analysis by CID MS/MS in combination with IMS-MS of species exhibiting the same m/z but different configurations demonstrated for the first time the presence of positional isomers for some of the Schindler disease biomarker candidates. CONCLUSIONS: The IMS-MS and CID MS/MS platform was for the first time optimized and applied to Schindler disease glycourinome. By this approach the separation and characterization of Neu5Ac positional isomers was possible. IMS CID MS/MS showed the ability to determine the type of the glycopeptide isomers from a series of possible candidates.

Water ice is a soft matrix for the structural characterization of glycosaminoglycans by infrared matrix-assisted laser desorption/ionization.

Glycosaminoglycans (GAGs) are a class of heterogeneous, often highly sulfated glycans that form linear chains consisting of up to 100 monosaccharide building blocks and more. GAGs are ubiquitous constituents of connective tissue, cartilage, and the extracellular matrix, where they have key functions in many important biological processes. For their characterization by mass spectrometry (MS) and tandem MS, the high molecular weight polymers are usually enzymatically digested to oligomers with a low degree of polymerization (dp), typically disaccharides. However, owing to their lability elimination of sulfate groups upon desorption/ionization is often encountered leading to a loss of information on the analyte. Here, we demonstrate that, in particular, water ice constitutes an extremely mild matrix for the analysis of highly sulfated GAG disaccharides by infrared matrix-assisted laser desorption/ionization (IR-MALDI) mass spectrometry. Depending on the degree of sulfation, next to the singly charged ionic species doubly- and even triply charged ions are formed. An unambiguous assignment of the sulfation sites becomes possible by subjecting sodium adducts of the GAGs to low-energy collision-induced dissociation tandem MS. These ionic species exhibit a remarkable stability of the sulfate substituents, allowing the formation of fragment ions retaining their sulfation that arise from either cross-ring cleavages or rupture of the glycosidic bonds, thereby allowing an unambiguous assignment of the sulfation sites.

MALDI Q-TOF CID MS for diagnostic ion screening of human milk oligosaccharide samples.

Human milk oligosaccharides (HMO) represent the bioactive components of human milk, influencing the infant's gastrointestinal microflora and immune system. Structurally, they represent a highly complex class of analyte, where the main core oligosaccharide structures are built from galactose and N-acetylglucosamine, linked by 1-3 or 1-4 glycosidic linkages and potentially modified with fucose and sialic acid residues. The core structures can be linear or branched. Additional structural complexity in samples can be induced by endogenous exoglycosidase activity or chemical procedures during the sample preparation. Here, we show that using matrix-assisted laser desorption/ionization (MALDI) quadrupole-time-of-flight (Q-TOF) collision-induced dissociation (CID) as a fast screening method, diagnostic structural information about single oligosaccharide components present in a complex mixture can be obtained. According to sequencing data on 14 out of 22 parent ions detected in a single high molecular weight oligosaccharide chromatographic fraction, 20 different oligosaccharide structure types, corresponding to over 30 isomeric oligosaccharide structures and over 100 possible HMO isomers when biosynthetic linkage variations were taken into account, were postulated. For MS/MS data analysis, we used the de novo sequencing approach using diagnostic ion analysis on reduced oligosaccharides by following known biosynthetic rules. Using this approach, de novo characterization has been achieved also for the structures, which could not have been predicted.

Surface-exposed glycoproteins of hyperthermophilic Sulfolobus solfataricus P2 show a common N-glycosylation profile.

Cell surface proteins of hyperthermophilic Archaea actively participate in intercellular communication, cellular uptake, and energy conversion to sustain survival strategies in extreme habitats. Surface (S)-layer glycoproteins, the major component of the S-layers in many archaeal species and the best-characterized prokaryotic glycoproteins, were shown to have a large structural diversity in their glycan compositions. In spite of this, knowledge on glycosylation of proteins other than S-layer proteins in Archaea is quite limited. Here, the N-glycosylation pattern of cell-surface-exposed proteins of Sulfolobus solfataricus P2 were analyzed by lectin affinity purification, HPAEC-PAD, and multiple mass spectrometry-based techniques. Detailed analysis of SSO1273, one of the most abundant ABC transporters present in the cell surface fraction of S. solfataricus, revealed a novel glycan structure composed of a branched sulfated heptasaccharide, Hex4(GlcNAc)2 plus sulfoquinovose where Hex is d-mannose and d-glucose. Having one monosaccharide unit more than the glycan of the S-layer glycoprotein of S. acidocaldarius, this is the most complex archaeal glycan structure known today. SSO1273 protein is heavily glycosylated and all 20 theoretical N-X-S/T (where X is any amino acid except proline) consensus sequence sites were confirmed. Remarkably, we show that several other proteins in the surface fraction of S. solfataricus are N-glycosylated by the same sulfated oligosaccharide and we identified 56 N-glycosylation sites in this subproteome.

Proteoglycans from Boswellia serrata Roxb. and B. carteri Birdw. and identification of a proteolytic plant basic secretory protein.

Water-soluble high molecular weight compounds were isolated in yields of 21-22% from the oleogum of Boswellia serrata and B. carteri. Using anion exchange chromatography and gel permeation chromatography, different proteoglycans were purified and characterized, leading to four principally different groups: (i) Hyp-/Ser-rich extensins with O-glycosidic attached arabinan side chains; (ii) Modified extensins, with arabinogalactosylated side chains containing GlA and 4-O-Me-GlcA; (iii) Glycoproteins with N-glycosidic side chains containing higher amounts of Fuc, Man and GluNH(2,) featuring a 200 kD metalloproteinase that has been de novo sequenced and is described for the first time; (iv) Type II arabinogalactans-proteins. Significant differences between the gums from the two species were observed in the protein content (6% vs 22%), offering the possibility of a quick differentiation of gums from both species for analytical quality control. The data also offer an insight into the plant response towards wound-closing by the formation of extensin and AGP-containing gum.

Elucidation of glycoprotein structures by unspecific proteolysis and direct nanoESI mass spectrometric analysis of ZIC-HILIC-enriched glycopeptides.

Protein glycosylation was explored by direct nanoESI MS and MS/MS analysis of ZIC-HILIC-enriched proteolytic glycopeptides without further separation or purification. In a previous publication, we demonstrated that a direct MS-based analysis of proteolytic glycopeptides is feasible for a number of proteins (Henning , S. J. Mass Spectrom. 2007 , 42 , 1415 - 21). This method has now been refined for two aspects: (1) separation of glycopeptides by use of ZIC-HILIC SPE and (2) the use of unspecific proteases like thermolysin, elastase, or a trypsin/chymotrypsin mixture leading per se to a mass-based separation, that is, small nonglycosylated peptides and almost exclusively glycopeptides at higher m/z values. Furthermore, the glycopeptides produced by the above proteases in general contain short peptide backbones thus improving-probably due to their higher hydrophilicity--the ZIC-HILIC-based separation. The combination of unspecific proteolysis, glycopeptide separation, and their direct MS analysis was successfully accomplished for probing glycoproteins carrying high-mannose type (ribonuclease B), neutral (asialofetuin), and acidic (haptoglobin and α1-acid glycoprotein) complex type glycans as well as for glycopeptides derived from glycoprotein mixtures and, finally, for exploring the glycosylation of a human IgG preparation. Our results show that the presented method is a fast, facile, and inexpensive procedure for the elucidation of protein N-glycosylation.

Cell type-specific and site directed N-glycosylation pattern of FcγRIIIa.

Human leukocyte receptor IIIa (hFcγRIIIa) plays a prominent role in the elimination of tumor cells by antibody-based cancer therapies. In previous studies, a major impact of the presence of carbohydrates at Asn-162 on the binding between the receptor and the Fc part of wild type fucosylated or glycoengineered nonfucosylated antibodies has been shown. In this study, we performed a site directed carbohydrate analysis at hFcγRIIIa derived from human embryonic kidney (HEK) and Chinese hamster ovary (CHO) cells, respectively. Using mass spectrometry (MS) and a multienzyme protein digest, we analyzed the proteolysis-generated glycopeptides in detail. We could show that hFcγRIIIa expressed by HEK cells was mostly bearing multifucosylated biantennary Asn162-glycans with a major fraction terminating with GalNAc residues replacing the more common Gal. We could demonstrate that the glycan antennae with terminal GalNAc could be sialylated as indicated by a novel reporter ion HexNAcHexNAcNeuAc(+) (m/z 698.28) using a source induced dissociation (SID) scan in the MS cycle. In contrast to the hFcγRIIIa Asn-162 glycosylation pattern from HEK cells, the CHO cells derived receptor contains bi- and triantennary galactosylated and highly sialylated carbohydrates. Our data suggest that the type of expression host system was a dominating factor for formation of distinct glycopatterns of hFcγRIIIa, while the protein sequence and the site of glycosylation remained unchanged for both types of cells. Using surface plasmon resonance (SPR) interaction analysis, we show that the cell type and site specific glycosylation pattern of hFcγRIIIa influences its binding behavior to immunoglobulin molecules.

Differences in CD75s- and iso-CD75s-ganglioside content and altered mRNA expression of sialyltransferases ST6GAL1 and ST3GAL6 in human hepatocellular carcinomas and nontumoral liver tissues.

The sialic acid-specific cytotoxic lectin viscumin and its recombinant equivalent rViscumin specifically bind to CD75s-gangliosides with terminal Neu5Acα6Galß4GlcNAc sequence. We, therefore, comparatively analyzed the content of CD75s-gangliosides and closely related iso-CD75s-gangliosides (terminated by Neu5Acα3Galß4GlcNAc sequence) and the gene expression of associated ß-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) and ß-galactoside α-2,3-sialyltransferase 6 (ST3GAL6), respectively, in 35 hepatocellular carcinoma (HCC) patients. Ganglioside structures were identified in lipid extracts of matched pairs of malignant and nonmalignant liver tissues by thin-layer chromatography immunodetection coupled with infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry. CD75s- and iso-CD75s-gangliosides were found to be deregulated in tumor tissues and showed an elevated occurrence in 35 and 41% of HCCs, respectively, compared with nontumoral liver tissues. Statistical analysis revealed a correlation between enhanced iso-CD75s-ganglioside amount and a poor histopathological differentiation (τ = 0.317, P = 0.045) and a significant association of CD75s- and iso-CD75s-ganglioside levels in nontumorous (τ = 0.392, P = 0.003) and in tumorous tissues (τ = 0.650, P < 0.001). Quantitative real-time polymerase chain reaction gene expression analysis of sialyltransferases exhibited no difference in ST6GAL1 expression in cancerous and adjacent noncancerous tissues. Interestingly, the ST3GAL6 expression was significantly diminished in HCCs (P = 0.003). The results indicate that the occurrence of CD75s- and iso-CD75s-gangliosides in tumor tissues is largely independent of the transcriptional expression of ST6GAL1 and ST3GAL6, respectively. Thus, further experiments are required to explore the rationale behind the differential ganglioside level and to validate the applicability of CD75s- and iso-CD75s-gangliosides as targets for individual HCC therapies.

Downregulation of the antioxidant protein peroxiredoxin 2 contributes to angiotensin II-mediated podocyte apoptosis.

Podocytes have a significant role in establishing selective permeability of the glomerular filtration barrier. Sustained renin-angiotensin-aldosterone system activation is crucial to the pathogenesis of podocyte injury, but the mechanisms by which angiotensin II modulates podocyte survival due to physiological or injurious stimuli remain unclear. Here, we used proteomic analysis to find new mediators of angiotensin II-induced podocyte injury. Antioxidant protein peroxiredoxin 2 expression was decreased in cultured podocytes stimulated with angiotensin II. Peroxiredoxin 2 was found to be expressed in podocytes in vivo, and its expression was decreased in the glomeruli of rats transgenic for angiotensin II type 1 receptors in a podocyte-specific manner, or in rats infused with angiotensin II. Downregulation of peroxiredoxin 2 in podocytes resulted in increased reactive oxygen species release, protein overoxidation, and inhibition of the Akt pathway. Both treatment with angiotensin II and downregulation of peroxiredoxin 2 expression led to apoptosis of podocytes. Thus, peroxiredoxin 2 is an important modulator of angiotensin II-induced podocyte injury.

Analysis of noncovalent chitinase-chito-oligosaccharide complexes by infrared-matrix assisted laser desorption ionization and nanoelectrospray ionization mass spectrometry.

Transferring noncovalently bound complexes from the condensed phase into the gas phase represents a challenging task due to weak intermolecular bonds that have to be maintained during the phase transition. Currently, electrospray ionization (ESI) is the standard mass spectrometric (MS) technique to analyze noncovalent complexes. Although infrared matrix-assisted laser desorption ionization (IR-MALDI)-MS also provides particular soft desorption/ionization conditions, this method has so far hardly been applied for the analysis of noncovalent complexes. In this study, we employed IR-MALDI orthogonal time-of-flight (o-TOF)-MS in combination with the liquid matrix glycerol to characterize the specific complex formation of chito-oligosaccharide (CHOS) ligands with two variants of Chitinase A (ChiA) from Serratia marcescens, the inactive E315Q mutant and the active W167A mutant, respectively. The IR-MALDI-o-TOF-MS results were compared to those obtained using nano-ESI-quadrupole (q)-TOF-MS and ultraviolet (UV)-MALDI-o-TOF-MS. Using IR-MALDI-o-TOF-MS, specific noncovalent complexes between ChiA and CHOS were detected with distributions between enzymes with bound oligosaccharides vs free enzymes that were essentially identical to those obtained by nano-ESI-q-TOF-MS. Chitinase-CHOS complexes were not detected when UV-MALDI was employed for desorption/ionization. The results show that IR-MALDI-MS can be a valuable tool for fast and simple screening of noncovalent enzyme-ligand interactions.

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.

Direct analysis of α- and ß-chains of hemoglobins from mammalian blood samples by nanoESI mass spectrometry during in-capillary proteolytic digestion.

α- and ß-chains of hemoglobins derived from several species were analyzed directly from diluted blood samples by simultaneous in-capillary proteolytic digestion and nanoESI MS and MS/MS analysis. Starting from fresh or frozen and thawed blood samples, sequence coverages of >80% were usually obtained. Only 2 h after resuspension of a dried blood spot, human origin could be demonstrated from data obtained by in-capillary tryptic digestion, nanoESI mass spectrometric analysis, and data base search. A fast and facile differentiation of closely related species by hemoglobin-derived proteolytic "marker peptides" was demonstrated for Asian (Elephas maximus) and African elephants (Loxodonta africana). Finally, amino acid sequences deduced from collision-induced dissociation experiments during in-capillary proteolytic digestion of the corresponding blood samples allowed de novo sequencing of previously unknown sequences of hemoglobin chains of the Patagonian cavy (Dolichotum patagona) and the Persian gazelle (Gazella subgutturosa subgutturosa). 100% of the α-chain sequences and more than 85% of the ß-chain sequences were covered for both the species. Additionally, sequence data derived from tandem MS experiments obtained with the Q-Tof analyzer were confirmed by high resolution Fourier-transform ion cyclotron resonance mass spectrometric experiments. Accurate protein mass determination of the intact hemoglobin chains directly from the corresponding blood samples by use of a Fourier-transform ion cyclotron resonance mass spectrometer corroborated the deduced sequences of the respective α-chains. The present study demonstrates that in-capillary digestion allows fast characterization and/or sequencing of hemoglobin chains directly from blood samples.

Discovery of a novel unfolded protein response phenotype of cancer stem/progenitor cells from the bone marrow of breast cancer patients.

Metastases arise from disseminated tumor cells (DTC) that colonize secondary organs. However, DTC survival strategies to start metastatic outgrowth are unclear. The hostile (hypoxic, hypoglycemic) microenvironmental conditions of the bone marrow serve as an ideal model environment for investigation of DTC survival strategies under environmental stress. We investigated the breast cancer DTC cell line BC-M1 established from the bone marrow of a cancer patient by 2-D DIGE and MS analysis. We observed specific overexpression of the unfolded protein response (UPR) proteins Grp78, Grp94, and protein disulfide-isomerase in breast, lung, and prostate cancer DTC cell lines from the bone marrow. The UPR contributes to survival under adverse environmental conditions including chemotherapy. We show in cellular models that Grp78 expression of the UPR is regulated by tyrosine 1248 of ErbB-2. The breast cancer DTC cell lines shared stem/progenitor cell cancer phenotypes (CD44(high)/CD24(low)). Immunocytochemical staining of bone marrow samples from breast cancer patients confirmed in situ high expression of Grp78 and Grp94 in DTC of breast cancer patients, indicating the potential of both proteins as novel markers for DTC detection. Our results suggest the presence of a previously not recognized stress resistant DTC population that combines stem/progenitor attributes with an UPR phenotype.

Separation and identification of GM1b pathway Neu5Ac- and Neu5Gc gangliosides by on-line nanoHPLC-QToF MS and tandem MS: toward glycolipidomics screening of animal cell lines.

Monosialoganglioside fraction of YAC-1 lymphoma cells was comprehensively analyzed and structurally defined by nano-high-performance liquid chromatography (nanoHPLC) in on-line conjunction with electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOF MS). An efficient separation and sensitive detection of Neu5Gc-containing gangliosides from Neu5Ac-containing analogues was for the first time accomplished in a single nanoHPLC/ESI-QTOF MS run, as demonstrated for mouse hybridoma cell GM3 fraction containing GM3(Neu5Ac) and GM3(Neu5Gc) species and further applied for the analysis of YAC-1 lymphoma cell monosialoganglioside fraction. New insights into YAC-1 monosialoganglioside mixture heterogeneity were obtained: 31 distinct species, comprising 18 Neu5Gc-containing gangliosides and 13 Neu5Ac-containing species of GM1b and GalNAc-GM1b type were found to be expressed by YAC-1 cell line. On-line structural elucidation of individually separated Neu5Ac- and Neu5Gc-containing gangliosides provided strong evidence on the "GM1b-pathway" sourcing for monosialoganglioside synthesis. Such an analytical method is documented as superior to the classical approaches by increased speed of analysis, sensitivity and level of information, being thus a viable glycolipidomic tool.

Chip-mass spectrometry for glycomic studies.

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

The alpha2beta1 integrin-specific antagonist rhodocetin is a cruciform, heterotetrameric molecule.

The integrin alpha2beta1 plays an important role in various pathophysiological processes, such as thrombosis, wound healing, inflammation, and metastasis. Rhodocetin, a constituent of the venom of the hemorrhagic Malayan pit viper (Calloselasma rhodostoma), is a specific alpha2beta1 integrin antagonist. To understand its molecular mode of action, its structure was studied by crystallography. Its quaternary structure in solution was also analyzed biochemically. Two novel subunits of rhodocetin were sequenced by mass spectrometry. Their integrin binding was measured by protein interaction ELISAs. Rhodocetin is a C-type lectin-like protein (CLP) consisting of four homologous, yet distinct, subunits, alpha, beta, gamma, and delta, the latter two of which have been unknown to date. With their CLP folds and loop-swapping motifs, the subunits alpha, beta and gamma, delta form two heterodimeric pairs. Uniquely, they arrange orthogonally and shape a cruciform molecule. Bearing a single unpaired cysteine residue, rhodocetin can only form covalent supramolecular complexes with a maximum aggregation number of 2, unlike many heterodimeric CLPs. Being the first heterotetrameric CLP to be crystallized, rhodocetin provides not only the prototypic molecular structure for heterotetrameric CLPs, but also a lead structure for pharmaceutical alpha2beta1 integrin antagonists.

IF/TA-related metabolic changes--proteome analysis of rat renal allografts.

BACKGROUND: Chronic allograft nephropathy, now more specifically termed interstitial fibrosis and tubular atrophy without evidence of any specific aetiology (IF/TA), is still an important cause of late graft loss. There is no effective therapy for IF/TA, in part due to the disease's multifactorial nature and its incompletely understood pathogenesis. METHODS: We used a differential in-gel electrophoresis and mass spectrometry technique to study IF/TA in a renal transplantation model. Dark Agouti (DA) kidneys were allogeneically transplanted to Wistar-Furth (DA-WF, aTX) rats. Syngeneic grafts (DA-DA, sTX) served as controls. Nine weeks after transplantation, blood pressure, renal function and electrolytes were studied, in addition to real-time PCR, western blot analysis, histology and immunohistochemistry. RESULTS: In contrast to sTX, the aTX developed IF/TA-dependent renal damage. Ten differentially regulated proteins were identified by 2D gel analysis and mass spectrometry, whereupon five proteins are mainly related to oxidative stress (aldo-keto reductase, peroxiredoxin-1, NAD(+)-dependent isocitrate dehydrogenase, iron-responsive element-binding protein-1 and serum albumin), two participate in cytoskeleton organization (l-plastin and ezrin) and three are assigned to metabolic functions (creatine kinase, ornithine aminotransferase and fructose-1,6-bisphosphatase). CONCLUSION: The proteins related to IF/TA and involved in oxidative stress, cytoskeleton organization and metabolic functions may correspond with novel therapeutic targets.