A Smartphone-Based Rapid Telemonitoring System for Ebola and Marburg Disease Surveillance.

We have developed a digital and multiplexed platform for the rapid detection and telemonitoring of infections caused by Ebola and Marburg filoviruses. The system includes a flow cell assay cartridge that captures specific antibodies with microarrayed recombinant antigens from all six species of filovirus, and a smartphone fluorescent reader for high-performance interpretation of test results. Multiplexed viral proteins, which are expandable to include greater numbers of probes, were incorporated to obtain highest confidence results by cross-correlation, and a custom smartphone application was developed for data analysis, interpretation, and communication. The smartphone reader utilizes an opto-electro-mechanical hardware attachment that snaps at the back of a Motorola smartphone and provides a user interface to manage the operation, acquire test results, and communicate with cloud service. The application controls the hardware attachment to turn on LEDs and digitally record the optically enhanced images. Assay processing time is approximately 20 min for microliter amounts of blood, and test results are digitally processed and displayed within 15 s. Furthermore, a secure cloud service was developed for the telemonitoring of test results generated by the smartphone readers in the field. Assay system results were tested with sera from nonhuman primates that received a live attenuated EBOV vaccine. This integrated system will provide a rapid, reliable, and digital solution to prevent the rapid overwhelming of medical systems and resources during EVD or MVD outbreaks. Further, this disease-monitoring system will be useful in resource-limited countries where there is a need for dispersed laboratory analysis of recent or active infections.

A single arabinan chain is attached to the phosphatidylinositol mannosyl core of the major immunomodulatory mycobacterial cell envelope glycoconjugate, lipoarabinomannan.

Lipoarabinomannan (LAM) is composed of a phosphatidylinositol anchor followed by a mannan followed by an arabinan that may be capped with various motifs including oligosaccharides of mannose. A related polymer, lipomannan (LM), is composed of only the phosphatidylinositol and mannan core. Both the structure and the biosynthesis of LAM have been studied extensively. However, fundamental questions about the branching structure of LM and the number of arabinan chains on the mannan backbone in LAM remain. LM and LAM molecules produced by three different glycosyltransferase mutants of Mycobacterium smegmatis were used here to investigate these questions. Using an MSMEG_4241 mutant that lacks the α-(1,6)-mannosyltransferase used late in LM elongation, we showed that the reducing end region of the mannan that is attached to inositol has 5-7 unbranched α-6-linked-mannosyl residues followed by two or three α-6-linked mannosyl residues branched with single α-mannopyranose residues at O-2. After these branched mannosyl residues, the α-6-linked mannan chain is terminated with an α-mannopyranose at O-2 rather than O-6 of the penultimate residue. Analysis of the number of arabinans attached to the mannan core of LM in two other mutants (ΔembC and ΔMSMEG_4247) demonstrated exactly one arabinosyl substitution of the mannan core suggestive of the arabinosylation of a linear LM precursor with ∼10-12 mannosyl residues followed by additional mannosylation of the core and arabinosylation of a single arabinosyl "primer." Thus, these studies suggest that only a single arabinan chain attached near the middle of the mannan core is present in mature LAM and allow for an updated working model of the biosynthetic pathway of LAM and LM.

Novel LC-MS² product dependent parallel data acquisition function and data analysis workflow for sequencing and identification of intact glycopeptides.

Data dependent acquisition (DDA) of higher collision energy dissociation (HCD)-MS(2) followed by electron transfer dissociation (ETD)-MS(2) upon detection of glycan-specific oxonium is one of the better approaches in current LC-MS(2) analysis of intact glycopeptides. Although impressive numbers of glycopeptide identification by a direct database search have been reported, false positives remained high and difficult to determine. Even in cases when the peptide backbones were correctly identified, the exact glycan moieties were often erroneously assigned. Any attempt to fit the best glycosyl composition match by mass only is problematic particularly when the correct monoisotopic precursor cannot be determined unambiguously. Taking advantage of a new trihybrid Orbitrap configuration, we experimented with adding in a parallel ion trap collision induced dissociation (CID)-MS(2) data acquisition to the original HCD-product dependent (pd)-ETD function. We demonstrated the feasibility and advantage of identifying the peptide core ion directly from edited HCD-MS(2) data as an easy way to reduce false positives without compromising much sensitivity in intact glycopeptide positive spectrum matches. Importantly, the additional CID-MS(2) data allows one to validate the glycan assignment and provides insight into possible glycan modifications. Moreover, it is a viable alternative to deduce the glycopeptide backbone particularly in cases when the peptide backbone cannot be identified by ETD/HCD. The novel HCD-pd-CID/ETD workflow combines the best possible decision tree dependent MS(2) data acquisition modes currently available for glycoproteomics within a rapid Top Speed DDA duty cycle. Additional informatics can conceivably be developed to mine and integrate the rich information contained within for simultaneous N- and O-glycopeptide analysis.

An adaptive workflow coupled with Random Forest algorithm to identify intact N-glycopeptides detected from mass spectrometry.

MOTIVATION: Despite many attempts for algorithm development in recent years, automated identification of intact glycopeptides from LC-MS(2) spectral data is still a challenge in both sensitivity and precision. RESULTS: We implemented a supervised machine learning algorithm, Random Forest, in an automated workflow to identify N-glycopeptides using spectral features derived from ion trap-based LC-MS(2) data. The workflow streamlined high-confident N-glycopeptide spectral data and enabled adaptive model optimization with respect to different sampling strategies, training sample size and feature set. A critical evaluation of the features important for glycopeptide identification further facilitated effective feature selection for model improvement. Using split sample testing method from 577 high-confident N-glycopeptide spectral data, we demonstrated that an optimal true-positive rate, precision and false-positive rate of 73, 88 and 10%, respectively, can be attained for overall N-glycopeptide identification Availability and implementation: The workflow developed in this work and the application suite, Sweet-Heart, that the workflow supports for N-glycopeptide identification are available for download at http://sweet-heart.glycoproteomics.proteome.bc.sinica.edu.tw/.

AGO61-dependent GlcNAc modification primes the formation of functional glycans on α-dystroglycan.

Dystroglycanopathy is a major class of congenital muscular dystrophy that is caused by a deficiency of functional glycans on α-dystroglycan (α-DG) with laminin-binding activity. A product of a recently identified causative gene for dystroglycanopathy, AGO61, acted in vitro as a protein O-mannose ß-1, 4-N-acetylglucosaminyltransferase, although it was not functionally characterized. Here we show the phenotypes of AGO61-knockout mice and demonstrate that AGO61 is indispensable for the formation of laminin-binding glycans of α-DG. AGO61-knockout mouse brain exhibited abnormal basal lamina formation and a neuronal migration defect due to a lack of laminin-binding glycans. Furthermore, our results indicate that functional α-DG glycosylation was primed by AGO61-dependent GlcNAc modifications of specific threonine-linked mannosyl moieties of α-DG. These findings provide a key missing link for understanding how the physiologically critical glycan motif is displayed on α-DG and provides new insights on the pathological mechanisms of dystroglycanopathy.

BAD-lectins: boronic acid-decorated lectins with enhanced binding affinity for the selective enrichment of glycoproteins.

The weak and variable binding affinities exhibited by lectin-carbohydrate interactions have often compromised the practical utility of lectin in capturing glycoproteins for glycoproteomic applications. We report here the development and applications of a new type of hybrid biomaterial, namely a boronic acid-decorated lectin (BAD-lectin), for efficient bifunctional glycoprotein labeling and enrichment. Our binding studies showed an enhanced affinity by BAD-lectin, likely to be mediated via the formation of boronate ester linkages between the lectin and glycan subsequent to the initial recognition process and thus preserving its glycan-specificity. Moreover, when attached to magnetic nanoparticles (BAD-lectin@MNPs), 2 to 60-fold improvement on detection sensitivity and enrichment efficiency for specific glycoproteins was observed over the independent use of either lectin or BA. Tested at the level of whole cell lysates for glycoproteomic applications, three different types of BAD-lectin@MNPs exhibited excellent specificities with only 6% overlapping among the 295 N-linked glycopeptides identified. As many as 236 N-linked glycopeptides (80%) were uniquely identified by one of the BAD-lectin@MNPs. These results indicated that the enhanced glycan-selective recognition and binding affinity of BAD-lectin@MNPs will facilitate a complementary identification of the under-explored glycoproteome.

Sweet-Heart - an integrated suite of enabling computational tools for automated MS2/MS3 sequencing and identification of glycopeptides.

High efficiency identification of intact glycopeptides from a shotgun glycoproteomic LC-MS(2) dataset remains problematic. The prevalent mode of identifying the de-N-glycosylated peptides is littered with false positives and addresses only the issue of site occupancy. Here, we present Sweet-Heart, a computational tool set developed to tackle the heart of the problems in MS(2) sequencing of glycopeptide. It accepts low resolution and low accuracy ion trap MS(2) data, filters for glycopeptides, couples knowledge-based de novo interpretation of glycosylation-dependent fragmentation pattern with protein database search, and uses machine-learning algorithm to score the computed glyco and peptide combinations. Higher ranking candidates are then compiled into a list of MS(2)/MS(3) entries to drive subsequent rounds of targeted MS(3) sequencing of putative peptide backbone, allowing its validation by database search in a fully automated fashion. With additional fishing out of all related glycoforms and final data integration, the platform proves to be sufficiently sensitive and selective, conducive to novel glycosylation discovery, and robust enough to discriminate, among others, N-glycolyl neuraminic acid/fucose from N-acetyl neuraminic acid/hexose. A critical appraisal of its computing performance shows that Sweet-Heart allows high sensitivity comprehensive mapping of site-specific glycosylation for isolated glycoproteins and facilitates analysis of glycoproteomic data. BIOLOGICAL SIGNIFICANCE: The biological relevance of protein site-specific glycosylation cannot be meaningfully addressed without first defining its pattern by direct analysis of glycopeptides. Sweet-Heart is a novel suite of computational tools allowing for automated analysis of mass spectrometry-based glycopeptide sequencing data. It is developed to accept ion trap MS2/MS3 data and uses a machine learning algorithm to score and rank the candidate peptide core and glycosyl substituent combinations. By eliminating the need for manual, labor-intensive, and subjective data interpretation, it facilitates high throughput shotgun glycoproteomic data analysis and is conducive to identification of unanticipated glycosylation, as demonstrated here with a recombinant EGFR.

In vivo regulation of steroid hormones by the Chst10 sulfotransferase in mouse.

Chst10 adds sulfate to glucuronic acid to form a carbohydrate antigen, HNK-1, in glycoproteins and glycolipids. To determine the role of Chst10 in vivo, we generated systemic Chst10-deficient mutant mice. Although Chst10(-/-) mice were born and grew to adulthood with no gross defects, they were subfertile. Uteri from Chst10(-/-) females at the pro-estrus stage were larger than those from wild-type females and exhibited a thick uterine endometrium. Serum estrogen levels in Chst10(-/-) females were higher than those from wild-type females, suggesting impaired down-regulation of estrogen. Because steroid hormones are often conjugated to glucuronic acid, we hypothesized that Chst10 sulfates glucuronidated steroid hormone to regulate steroid hormone in vivo. Enzymatic activity assays and structural analysis of Chst10 products by HPLC and mass spectrometry revealed that Chst10 indeed sulfates glucuronidated estrogen, testosterone, and other steroid hormones. We also identified an HPLC peak corresponding to sulfated and glucuronidated estradiol in serum from wild-type but not from Chst10 null female mice. Estrogen-response element reporter assays revealed that Chst10-modified estrogen likely did not bind to its receptor. These results suggest that subfertility exhibited by female mice following Chst10 loss results from dysregulation of estrogen. Given that Chst10 transfers sulfates to several steroid hormones, Chst10 likely functions in widespread regulation of steroid hormones in vivo.

Identification of the Mycobacterium marinum Apa antigen O-mannosylation sites reveals important glycosylation variability with the M. tuberculosis Apa homologue.

The 45/47 kDa Apa, an immuno-dominant antigen secreted by Mycobacterium tuberculosis is O-mannosylated at multiple sites. Glycosylation of Apa plays a key role in colonization and invasion of the host cells by M. tuberculosis through interactions of Apa with the host immune system C-type lectins. Mycobacterium marinum (M.ma) a fish pathogen, phylogenetically close to M. tuberculosis, induces a granulomatous response with features similar to those described for M. tuberculosis in human. Although M.ma possesses an Apa homologue, its glycosylation status is unknown, and whether this represents a crucial element in the pathophysiology induced by M.ma remains to be addressed. To this aim, we have identified two concanavalin A-reactive 45/47 kDa proteins from M.ma, which have been further purified by a two-step anion exchange chromatography process. Advanced liquid chromatography-nanoESI mass spectrometry-based proteomic analyses of peptides, derived from either tryptic digestion alone or in combination with the Asp-N endoproteinase, established that M.ma Apa possesses up to seven distinct O-mannosylated sites with mainly single mannose substitutions, which can be further extended at the Ser/Thr/Pro rich region near the N-terminus. This opens the way to further studies focussing on the involvement and biological functions of Apa O-mannosylation using the M.ma/zebrafish model.

MS-based glycomic strategies for probing the structural details of polylactosaminoglycan chain on N-glycans and glycoproteomic identification of its protein carriers.

Most MS-based glycomic and glycoproteomic analyses focus on identifying changes in terminal glyco-epitopes represented by sialylation and fucosylation at specific positions of the terminal N-acetyllactosamine units. Much less attention was accorded to the underlying linear or branched poly-N-acetyllactosamine extension from the N-glycan trimannosyl core other than a simple inference of its presence due to mass data and hence glycosyl compositional assignment. Using the EA.hy926 cell line derived from human umbilical vein endothelial cells (HUVEC), we have systematically investigated the MALDI- and ESI-MS-based methodologies for probing the structural details of endothelial polylactosaminoglycans at both MS and MS(2) levels in conjunction with the use of endo-ß-galactosidase to identify branching motifs and initiation sites. We showed that the polylactosaminoglycan chains on the N-glycans of EA.hy926 were less sialylated and fucosylated but more extended and branched than those of human umbilical vein endothelial cells, thus demonstrating a fundamental glycomic difference. For EA.hy926 that was investigated in more details, its polylactosaminoglycan chains were shown to be not restricted to extending from a specific antenna including the biologically important 6-arm position. Finally, experimental conditions for glycopeptide enrichment by tomato lectin were further optimized, which led to identification of over 40 candidate endothelial membrane protein carriers of polylactosaminoglycans by proteomic analysis.

Glycoproteomics analysis to identify a glycoform on haptoglobin associated with lung cancer.

Glycosylation is a common protein modification that is of interest in current cancer research because altered carbohydrate moieties are often found during cancer progress. A search for biomarkers in human lung cancer serum samples using glycoproteomic approaches identified fucosylated haptoglobin (Hp) significantly increased in serum of each subtype of lung cancer compared to normal donors. In addition, MS provided evidence of an increase of Hp fucosylation; the glycan structure was determined to be an α 2,6-linked tri-sialylated triantennary glycan containing α1,3-linked fucose attached to the four-linked position of the three-arm mannose of N-linked core pentasaccharide. These preliminary findings suggest that the specific glycoform of Hp may be useful as a marker to monitor lung cancer progression.

Terminal disialylated multiantennary complex-type N-glycans carried on acutobin define the glycosylation characteristics of the Deinagkistrodon acutus venom.

Glycosylation analysis of nonmammalian sources often springs surprises and conjures up intriguing views of evolutionary adaptation. Many of the constituents of snake venoms are known to be glycosylated and yet very few were fully characterized and accorded specific functions. In the process of glycomic screening through the venoms from Asian pit vipers, a partially O-acetylated NeuAcα2-8NeuAcα2-3Galß1-4GlcNAcß1-terminal epitope was found to be the predominant glycosylation characteristic of the snake venom produced by the monotypic Deinagkistrodon acutus, with acutobin, a highly specific fibrinogenase, being identified as a primary protein carrier. Full structural definition and glycosylation site mapping were completed through advanced mass spectrometry analyses at both the glycan and glycopeptide levels in conjunction with chemical and enzymatic cleavages. Although similar occurrence of such terminal disialyl cap on the N-glycans of several mammalian glycoproteins has been implicated, most of these correspond to only minor constituents of the full glycomic heterogeneity and remain poorly characterized. In contrast, each antennae of the hybrid- and complex-type N-glycans derived from acutobin was found to be rather homogeneously disialylated. With up to eight sialic acids evenly distributed on nonextended tetraantennary core structure, these unusual N-glycans are among those of highest sialic acid density ever identified without actually carrying polysialic acid chains. It remains to be tested whether they may serve as multivalent disialyl ligands for several of the human Siglecs and thus meddle with the natural immuno-recognition systems of snakebite victims, apart from affecting the general efficacy of acutobin as anticoagulant in biomedical applications.

Selective extraction and effective separation of galactosylsphingosine (psychosine) and glucosylsphingosine from other glycosphingolipids in pathological tissue samples.

To facilitate the study of the chemical pathology of galactosylsphingosine (psychosine, GalSph) in Krabbe disease and glucosylsphingosine (GlcSph) in Gaucher disease, we have devised a facile method for the effective separation of these two glycosylsphingosines from other glycosphingolipids (GSLs) in Krabbe brain and Gaucher spleen samples. The procedure involves the use of acetone to selectively extract GalSph and GlcSph, respectively, from Krabbe brain and Gaucher spleen samples. Since acetone does not extract other GSLs except modest amounts of galactosylceramide, sulfatide, and glucosylceramide, the positively charged GalSph or GlcSph in the acetone extract can be readily separated from other GSLs by batchwise cation-exchange chromatography using a Waters Accell Plus CM Cartridge. GalSph or GlcSph enriched by this simple procedure can be readily analyzed by thin-layer chromatography or high-performance liquid chromatography.

Enabling techniques and strategic workflow for sulfoglycomics based on mass spectrometry mapping and sequencing of permethylated sulfated glycans.

Sulfate modifications on terminal epitopes of N- and O-glycans have increasingly been implicated as critical determinants mediating a diverse range of biological recognition functions. To address these low abundance but important sulfated glycans, and the sulfoglycome in general, further development of enrichment strategies and enabling mass spectrometry (MS)-based mapping techniques are needed. In this report, we demonstrate that the sulfated glycans, with and without additional sialylation, can be successfully permethylated by the sodium hydroxide slurry method and be distinguished from phosphorylated glycans by virtue of this derivatization. In conjunction with simple microscale postderivatization fractionation steps, permethyl derivatives fully retaining the negatively charged sulfate moiety and separated from the nonsulfated ones, can be efficiently detected and sequenced de novo by advanced MS/MS in the positive-ion mode. In particular, we show that the highly sequence and linkage informative high energy collision induced dissociation (CID) MS/MS afforded by MALDI-TOF/TOF can be extended to sulfoglycomic applications. The sulfated parent ion selected for CID MS/MS was found to mostly retain the sulfate moiety and therefore allow efficient fragmentation via the usual array of glycosidic, cross ring, and concerted double cleavages. Collectively, the optimized strategy enables a high sensitivity detection and critical mapping of the sulfoglycome such as the one derived from lymph node tissues or cell lines in both negative and positive-ion modes. Novel sulfated epitopes were identified from a crude mouse lymph node preparation, which fully attested to the practical utility of the methodology developed.

Characterization of a distinct arabinofuranosyltransferase in Mycobacterium smegmatis.

The D-arabinans in Mycobacterium are essential, extraordinarily complex entity comprised of d-arabinofuranose residues which are rarely found in nature. Despite the well-recognized importance of the mycobacterial arabinan, delineation of the arabinosylation process has been severely hampered due to lack of positively identified arabinosyltransferases. Identification of genes involved in arabinan biosynthesis entailed the use of ethambutol (EMB), a first-line antituberculosis agent that is known to inhibit cell wall arabinan synthesis. The three genes (embA, embB, and embC) encode novel membrane proteins, implicated as the only known mycobacterial arabinosyltransferases to this date. We have now adapted a multifaceted approach involving development of convenient arabinosyltransferase assay using novel synthetic acceptors to identify arabinosyltransferase/s that will be distinct from the Emb proteins. In our present work, Mycobacterium smegmatis mc(2) 155 (WTMsm) was used as a model to study the biosynthesis of cell wall arabinan. In an in vitro assay, we demonstrate that transfer of only alpha-Araf had occurred from decaprenylphosphoryl-D-arabinofuranose (DPA) on a newly synthesized branched acceptor [alpha-D-Araf](2)-3,5-alpha-D-Araf-(1-->5)-alpha-d-Araf-(1-->5)-alpha-D-Araf with an octyl aglycon. Higher molecular weight (up to Ara(10)) oligomers were also detected in a parallel reaction using cold phosphoribosepyrophosphate (pRpp). Matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF MS/MS) analysis of these products revealed that isomeric products were formed and initiation and elongation of arabinan can occur either on the 5-arm or 3-arm of the branched 3,5-alpha-D-Araf. Individual embA, embB, and embC knockout strains retained this alpha-1,5 arabinosyltransferase activity, and the activity was partially inhibited by ethambutol. This particular enzyme function is distinct from the function of the Emb proteins.

Analysis of protein-linked glycosylation in a sperm-somatic cell adhesion system.

Murine sperm initiate fertilization by binding to the specialized extracellular matrix of their complementary eggs, known as the zona pellucida. On the basis of data reported in this study, mouse sperm also bind to rabbit erythrocytes with higher affinity than they do to murine eggs. This unusual interaction between a germ cell and a somatic cell ("sperm-somatic cell adhesion system") is also carbohydrate dependent based on its sensitivity to mild periodate oxidation. To determine what types of carbohydrate sequences could be involved in this interaction, the protein-linked oligosaccharides of rabbit erythrocytes were sequenced using novel matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry methods that enabled the analysis of individual components up to m/z 9000. The N-glycans are primarily complex biantennary and triantennary types terminated with Galalpha1-3Gal sequences. The majority of these oligosaccharides also possess one antenna consisting of a highly branched polylactosamine-type sequence that is also associated with many glycosphingolipids that coat rabbit erythrocytes. These erythrocytes also express Core 1 and Core 2 O-glycans terminated primarily with Galalpha1-3Gal sequences and to a lesser extent sialic acid. These results confirm that rabbit erythrocytes and mouse eggs present very different types of carbohydrate sequences on their surfaces. However, oligosaccharides terminated with beta1-6-linked N-acetyllactosamine or its alpha1-3 galactosylated analog are expressed on both the mouse zona pellucida and this somatic cell type. The far more abundant presentation of such sequences on rabbit erythrocytes compared with murine eggs could explain why mouse sperm display such exceptional affinity for this somatic cell type.

Sequencing of oligoarabinosyl units released from mycobacterial arabinogalactan by endogenous arabinanase: identification of distinctive and novel structural motifs.

The mycobacterial D-arabinofuran is a common constituent of both cell wall mycolyl-arabinogalactan (AG) and the associated lipoarabinomannan (LAM), and is thus accorded critical structural and immunological roles. Despite a well-recognized importance, progress in understanding its full structural characteristics beyond the nonreducing terminal motifs has hitherto been limited by available analytical tools. An endogenous arabinanase activity recently isolated from Mycobacterium smegmatis was previously shown to be capable of releasing large oligoarabinosyl units from AG. Advanced tandem mass spectrometry utilizing both low and high energy collision induced dissociation now afforded a facile way to map and directly sequence the digestion products which were dominated by distinctive Ara18 and Ara19 structural units, together with Ara7 and lesser amount of Ara11 and Ara12. Significantly, evidence was obtained for the first time which validated the linkages and branching pattern of the previously inferred Ara22 structural motif of AG, on which the preferred cleavage sites of the novel arabinanase could be localized. The established linkage-specific MS/MS fragmentation characteristics further led to identification of a galactosamine substituent on the C2 position of a portion of the internal 3,5-branched Ara residue of the AG of Mycobacterium tuberculosis, but not that of the nonpathogenic, fast growing M. smegmatis.

Distinctive characteristics of MALDI-Q/TOF and TOF/TOF tandem mass spectrometry for sequencing of permethylated complex type N-glycans.

Concerted MALDI-MS profiling and CID MS/MS sequencing of permethylated glycans is one of the most effective approaches for high throughput glycomics applications. In essence, the identification of larger complex type N-glycans necessitates an unambiguous definition of any modification on the trimannosyl core and the complement of non-reducing terminal sequences which constitute the respective antennary structures. Permethylation not only affords analyses of both neutral and sialylated glycans at comparable ease and sensitivity but also yields more sequence-informative fragmentation pattern. Facile glycosidic cleavages directed mostly at N-acetylglucosamine under low energy CID, as implemented on a quadrupole/time-of-flight (Q/TOF) instrument, often afford multiple losses of the attached antenna resulting in characteristic ions related to the number of antennary branches on the trimannosyl core. Non-reducing terminal epitopes can be easily deduced but information on the linkage specific substituent on the terminal units is often missing. The high energy CID MS/MS afforded by TOF/TOF instrument can fill in the gap by giving an array of additional cross-ring and satellite ions. Glycosidic cleavages occurring specifically in concert with loss of 2-linked or 3-linked substituents provide an effective way to identify the branch-specific antennary extension. These characteristics are shown here to be effective in deriving the sequences of additionally galactosylated, sialylated and fucosylated terminal N-acetyllactosamine units and their antennary location. Together, a highly reproducible fragmentation pattern can be formulated to simplify spectral assignment. This work also provides first real examples of sequencing multiply sialylated complex type N-glycans by high energy CID on a TOF/TOF instrument.

Design and characterization of a multimeric DNA binding protein using Sac7d and GCN4 as templates.

The protein Sac7d belongs to a class of small chromosomal proteins from the hyperthermophilic archaeon Sulfolobus acidocaldarius. Sac7d is extremely stable to heat, acid, and chemical agents. This protein is a monomer and it binds DNA without any particular sequence preference, while inducing a sharp kink in the DNA. By appending a leucine-zipper-like helical peptide derived from the yeast transcriptional activator GCN4 to the C-terminal end of Sac7d, the modified monomers (denoted S7dLZ) are expected to interact with each other via hydrophobic force to form a parallel dimer. The recombinant S7dLZ was expressed in Escherichia coli and purified by heating and ion-exchange chromatography. The formation of dimer was detected by gel-filtration chromatography and chemical cross-link. The results of surface plasmon resonance and circular dichroism experiments showed that the DNA-binding capacity was retained. Furthermore, X-ray diffraction analysis of single crystals of S7dLZ in complex with DNA decamer CCTATATAGG showed that the leucine-zipper segments of S7dLZ were associated into an antiparallel four-helix bundle. There are two DNA fragments bound to each S7dLZ tetramer in the crystal. This model works as a successful template that endows protein a new function without losing original properties.