μL Of Human Serum Research Articles

Hydrophilic Peptide and Glycopeptide as Immobilized Sorbents for Glycosylation Analysis.

Hydrophilic interaction liquid chromatography (HILIC) is widely used for glycopeptide enrichment in shot-gun glycoproteomics to enhance the glycopeptide signal and minimize the ionization competition of peptides. In this work, we have developed a novel hydrophilic material (glycoHILIC) based on glycopeptides and peptides to provide hydrophilic properties. GlycoHILIC was synthesized by oxidizing cotton and then reacting the resulting aldehyde with the N-terminus of the glycopeptide or peptide by reductive amination. Due to the large amount of hydrophilic carbohydrates and hydrophilic amino acids contained in glycopeptides, glycoHILIC showed significantly better enrichment of glycopeptides than cotton itself. Our results demonstrate that glycoHILIC has high selectivity, a low detection limit, and good stability. Over 257 unique N-linked glycosylation sites in 1477 intact N-glycopeptides from 146 glycoproteins were identified from 1 μL of human serum using glycoHILIC. Serum analysis of pancreatic cancer patients found that 38 N-glycopeptides among 21 glycoproteins changed significantly, of which 7 N-glycopeptides increased and 31 N-glycopeptides decreased. These results demonstrate that glycoHILIC can be used for glycopeptide enrichment and analysis.

ImmunoDisk-A Fully Automated Bead-Based Immunoassay Cartridge with All Reagents Pre-Stored.

In this paper, we present the ImmunoDisk, a fully automated sample-to-answer centrifugal microfluidic cartridge, integrating a heterogeneous, wash-free, magnetic- and fluorescent bead-based immunoassay (bound-free phase detection immunoassay/BFPD-IA). The BFPD-IA allows the implementation of a simple fluidic structure, where the assay incubation, bead separation and detection are performed in the same chamber. The system was characterized using a C-reactive protein (CRP) competitive immunoassay. A parametric investigation on air drying of protein-coupled beads for pre-storage at room temperature is presented. The key parameters were buffer composition, drying temperature and duration. A protocol for drying two different types of protein-coupled beads with the same temperature and duration using different drying buffers is presented. The sample-to-answer workflow was demonstrated measuring CRP in 5 µL of human serum, without prior dilution, utilizing only one incubation step, in 20 min turnaround time, in the clinically relevant concentration range of 15–115 mg/L. A reproducibility assessment over three disk batches revealed an average signal coefficient of variation (CV) of 5.8 ± 1.3%. A CRP certified reference material was used for method verification with a concentration CV of 8.6%. Our results encourage future testing of the CRP-ImmunoDisk in clinical studies and its point-of-care implementation in many diagnostic applications.

Poly-l-Lysine-Modified Graphene Field-Effect Transistor Biosensors for Ultrasensitive Breast Cancer miRNAs and SARS-CoV-2 RNA Detection.

(Mi)RNAs are important biomarkers for cancers diagnosis and pandemic diseases, which require fast, ultrasensitive, and economical detection strategies to quantitatively detect exact (mi)RNAs expression levels. The novel coronavirus disease (SARS-CoV-2) has been breaking out globally, and RNA detection is the most effective way to identify the SARS-CoV-2 virus. Here, we developed an ultrasensitive poly-l-lysine (PLL)-functionalized graphene field-effect transistor (PGFET) biosensor for breast cancer miRNAs and viral RNA detection. PLL is functionalized on the channel surface of GFET to immobilize DNA probes by the electrostatic force. The results show that PGFET biosensors can achieve a (mi)RNA detection range of five orders with a detection limit of 1 fM and an entire detection time within 20 min using 2 μL of human serum and throat swab samples, which exhibits more than 113% enhancement in terms of sensitivity compared to that of GFET biosensors. The performance enhancement mechanisms of PGFET biosensors were comprehensively studied based on an electrical biosensor theoretical model and experimental results. In addition, the PGFET biosensor was applied for the breast cancer miRNA detection in actual serum samples and SARS-CoV-2 RNA detection in throat swab samples, providing a promising approach for rapid cancer diagnosis and virus screening.

Bioinspired dandelion-like silica nanoparticles modified with L-glutathione for highly efficient enrichment of N-glycopeptides in biological samples

The pretreatment of complicated biological samples to eliminate the interference of nonglycopeptides and improve the efficiency of glycopeptides detection is crucial in glycoproteomics research. Hydrophilic interaction chromatography (HILIC) has been adopted for enrichment of glycosylated peptides following identification with mass spectrometry, but it is still urgent to develop novel hydrophilic materials to save cost and improve enrichment efficiency. Scientists are pursuing to fabricate freestanding intelligent artificial materials. One promising approach is to use biomimic material. In our case, “one-pot” strategy was developed to prepare bioinspired nano-core-shell silica microspheres (CSSMs), employing tetrapropylorthosilicate as the silicon source and phenolic resin as the soft template. The pore structure of the obtained microspheres diverged from the center to the outside with diameter ranged from 150 to 340 nm, and shell layer ranged from 25 to 83 nm by adjusting the preparation parameters. Some of them showed dandelion-like morphology. After hydrophilic modification, these CSSMs exhibited great hydrophilicity and could be used as sorbents for enriching N-glycopeptides from complicated biological samples in HILIC. Up to 594 unique N-glycopeptides and 367 N-glycosylation sites from 182 N-glycoproteins were unambiguously identified from 2 μL of human serum, which was superior to the enrichment performance of many HILIC materials in reported papers, demonstrating great potential advantages in proteomic application.

Hollow MnFe2O4@C@APBA Nanospheres with Size Exclusion and pH Response for Efficient Enrichment of Endogenous Glycopeptides

Enrichment and detection of glycopeptides are an important clinical measure for the diagnosis of complex diseases. Enrichment materials play a key role in this process; they must have an effective sample-screening ability to eliminate the interference of nonglycopeptides. In this work, novel hollow MnFe2O4@C@APBA nanospheres (HMCAs) with magnetic and pH responsiveness were prepared for glycopeptide enrichment. The as-prepared composites have a suitable hollow structure and large specific surface area, and the boron hydroxyl group in their cavities can fix or disconnect the hydrophilic groups of the glycopeptides at different pH, so the glycopeptides can be adsorbed or desorbed in a controllable way. Enrichment results showed that the HMCAs exhibited an excellent enrichment performance: ultralow limit of detection (approximately 0.5 fmol μL-1), perfect size-exclusion effect (HRP/BSA, 1:800, w/w), favorable universality (HRP, IgG, and RNase B), and high binding capacity (150 mg/g). In order to verify the application of materials in practice, the HMCAs were used for the analysis of complex samples and it was found that 474 glycopeptides were identified from 210 glycoproteins in three replicate analyses of 2 μL of human serum. The results showed that the HMCAs could be used as a promising enrichment material for glycopeptide characterization in MS-based glycoproteomics and related fields.

Electrochemical immunoplatform to improve the reliability of breast cancer diagnosis through the simultaneous determination of RANKL and TNF in serum

This paper describes a dual immunosensor using neutravidin-functionalized magnetic microbeads (Neu-MBs) and dual screen-printed carbon electrodes (SPdCEs) for the simultaneous amperometric determination of two emerging biomarkers related to breast cancer (BC) and metastasis: Receptor Activator of Nuclear Factor-κB Ligand (RANKL) and Tumor Necrosis Factor alpha (TNF). In the implemented methodology, sandwich-type immunocomplexes, using biotinylated specific capture, detector antibodies and HRP-labeled secondary antibodies, are formed onto Neu-MBs. Electrochemical detection was performed by amperometry (−0.20 V vs. the Ag pseudo-reference) electrode using the H2O2/hydroquinone (HQ) system upon capturing the Neu-MBs modified with the sandwich immunocomplexes for each target biomarker on the corresponding working electrode (WE) of SPdCEs. The approach exhibits high sensitivity offering detection limits of 2.6 and 3.0 pg mL−1 for RANKL and TNF, respectively, using simple protocols and taking 90 min as assay time. The usefulness of the dual immunoplatform was tested by determining RANKL and TNF levels in 5 μL of human serum from healthy controls and BC patients diagnosed with different HER2 subtypes. Results showed a higher expression of both biomarkers in BC patients (38 and 17 % higher for RANKL and TNF, respectively) and were in agreement to those obtained using the ELISA methodologies for each target biomarker involving the same immunoreagents. The obtained results show the potential of this immunoplatform to improve the reliability of BC diagnosis using fast and cost-effective procedures.

Postsynthesis of zwitterionic hydrophilic composites for enhanced enrichment of N-linked glycopeptides from human serum.

Glycosylation of proteins plays an important role in life activities, but the concentration of naturally occurring glycopeptides is usually relatively low, and glycosylation has microfacies heterogeneity, so direct mass spectrometry is not feasible. Therefore, selective enrichment of glycopeptides before mass spectrometry has turned into an urgent problem to be resolved. Herein, the zwitterionic L-cysteine functionalized hydrophilic graphene oxide composite (GO@PDA@MIL-125-NH2 @Au@L-Cys) was prepared via a postsynthetic method. The obtained material was used for glycopeptide enrichment. The enriched peptides were then detected using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) to demonstrate the enrichment performance of the material. In the actual enrichment process, GO@PDA@MIL-125-NH2 @Au@L-Cys nanomaterials exhibited high selectivity (1:1000), outstanding sensitivity (0.5 fmol), and excellent repeatability for the enrichment of glycopeptides. In addition, the proposed material showed good performance in the enrichment of glycopeptides from complex biosamples; 56 glycopeptides were detected from 2 μL of human serum using MALDI-TOFMS. The experimental results showed that GO@PDA@MIL-125-NH2 @Au@L-Cys exhibited excellent performance on glycopeptide analysis. It has great potential in the enrichment of glycopeptides and provides new ideas for synthetic materials with better enrichment properties in the future.

Hydrophilic maltose-modified magnetic metal-organic framework for highly efficient enrichment of N-linked glycopeptides

Biomedical sciences, and in particular disease biomarker research, demand highly selective and efficient glycoproteins/peptides enrichment platforms. In this work, a facile strategy to prepare hydrophilic maltose-functionalized magnetic metal-organic framework loaded with Au nanoparticles (denoted as magMOF@Au-maltose) for highly efficient enrichment of N-linked glycopeptides. In brief, carboxyl-functional Fe3O4 nanospheres were firstly coated with a Zr-based MOF shell, the resulting MOF was then loaded with Au nanoparticles in situ and then modified with thiol-functional maltose via Au−S bonds to obtain magMOF@Au-maltose with core-shell structure. The physical property and adsorption of magMOF@Au-maltose to glycopeptides were investigated. The results showed magMOF@Au-maltose possessing the outstanding performance in glycopeptides enrichment with high selectivity (1:200, mass ratio of horseradish peroxidase to bovine serum albumin digest), a low limit of detection (10 fmol), a high recovery (over 83.3%), and a large binding capacity (83 μg•mg−1). The magMOF@Au-maltose nanocomposite can enrich 24 and 32 glycopeptides from tryptic HRP and human IgG digests, respectively. Moreover, the nanocomposite was applied to the selective enrichment of glycopeptides from the complex biological samples and a total of 123 unique N-glycosylation sites were identified from 113 glycopeptides in 1 μL of human serum, which were assigned to 46 different glycoproteins. These results showed the promising application of magMOF@Au-maltose in the detection and identification of low-abundance N-linked glycopeptides in complex biological samples.

Method development of immunoglobulin G purification from micro-volumes of human serum for untargeted and targeted proteomics-based antibody repertoire studies

Immunoglobulins (Igs) are major serum proteins which play important roles in immunity. Both untargeted and targeted proteomic workflows can be applied to investigate antigen-binding sites and the glycosylation profiles of Igs. For a more-comprehensive picture of IgG from human serum, we developed an IgG purification process and coupled the standardized method to untargeted and targeted proteomic workflows for IgG investigations. Parameters such as the type of purification beads, volume of the bead slurry, incubation conditions, and binding capacities were evaluated in this study. Only 2 μL of human serum was required for each sample. The performance of coupling the purification process to untargeted proteomics in the IgG analysis was evaluated by comparing normalized abundances of IgG subclass-specific peptides with quantification results from an ELISA. Pearson's correlation values were all >0.82. Targeted proteomic workflow was applied to serum samples from patients with autoimmune pancreatitis and from healthy controls, and the results corresponded to clinical findings that IgG4-related peptides/glycopeptides showed higher abundances in the diseased group. The developed IgG purification process is simple and requires small sample volume, and it can be coupled to targeted and untargeted proteomic workflows for clinical investigations in the future.

Phenylboronic acid functionalized C3N4 facultative hydrophilic materials for enhanced enrichment of glycopeptides

It is challenging to capture N-glycopeptides with high recovery and high specificity from complicated biosystems. Herein, we present a facile and economical procedure to generate a novel self-assembling 4-Mercaptobenzene boronic acid functionalized and Au-doped Straticulate C3N4 (MASC), with enhanced affinity capability towards glycopeptides. The materials possess low pH value adaptation, high hydrophilicity and stability, good repeatability and recyclability, and provided high selectivity (1:100), low limit of detection (0.33 fmol/μL), high enrichment efficiency (~ 80%) and high recovery rate (~ 90%) towards glycopeptides. The materials can capture glycopeptides unbiasedly, as demonstrated by the identification of 37 glycopeptides from IgG and 21 glycopeptides from horseradish peroxidase (HRP). The performance of MASC on human urine and serum glycoproteome analysis was also tested. An average of 1465 glycopeptides from 839 glycoproteins and 1553 glycopeptides from 884 glycoproteins were identified from female and male urine samples in a single mass spectrometry analysis. O-glycopeptides from human urine were also significantly enriched. Additionally, 463 glycopeptides assigned to 209 glycoproteins were identified from 5 μL of human serum. All of these results indicate that MASC presents a good performance and applicability in the field of glycoproteomic research.

High Strength and Hydrophilic Chitosan Microspheres for the Selective Enrichment of N-Glycopeptides.

Protein glycosylation is an important post-translational modification that plays a crucial role in many biological processes. Because of the low abundance of glycoproteins and high complexity of clinical samples, the development of methods to selectively capture glycoproteins/glycopeptides is crucial to glycoproteomics study. In this work, a kind of highly cross-linked chitosan microspheres (CSMs) was prepared using epichlorhydrine as a cross-linker from chitosan solution in an alkaline/urea aqueous system. The results showed that CSMs had high amino groups content, large surface area, mesoporous structure, good acidic resistance, and high strength by various tests. On the basis of hydrophilic interaction between the polar groups (amino groups and hydroxyl groups) on CSMs and glycan moieties on glycopeptides, the prepared CSMs were applied to specific capture of N-glycopeptides from standard protein digests and complex biological samples (body fluids and tissues). The CSMs exhibited high selectivity (HRP/BSA = 1:100), good sensitivity (4.5 × 10-10 M of HRP), good recovery yield (74.9-106.4%), and high binding capacity (100 mg g-1) in glycopeptides enrichment. Because of the excellent performance in glycopeptides enrichment, CSMs were applied to selectively enrich N-glycopeptides from tryptic digests of human serum and rat brain followed by nanoLC-MS/MS analysis. We identified 194 and 947 unique N-glycosylation sites from 2 μL of human serum and 0.1 mg of rat brain, respectively. Additionally, the extraction time of our method was much shorter than the previously reported methods. Therefore, the fabricated CSMs with desirable properties will find broad application in large-scale and in-depth N-glycoproteome analysis.

In-Depth Analysis of Glycoprotein Sialylation in Serum Using a Dual-Functional Material with Superior Hydrophilicity and Switchable Surface Charge.

Sialylation typically occurs at the terminal of glycans, and its aberration often correlates with diseases including neurological diseases and cancer. However, the analysis of glycoprotein sialylation in complex biological samples is still challenging due to their low abundance. Herein, a histidine-bonded silica (HBS) material with a hydrophilic interaction and switchable surface charge was fabricated to enrich sialylated glycopeptides (SGPs) from the digest of proteomics samples. High selectivity toward SGPs was obtained by combining the superior hydrophilicity and switchable-charge characteristics. During the enrichment of sialylated glycopeptides from bovine fetuin digest, seven glycopeptides were detected even at the ratio of 1:5000 with the nonsialylated glycopeptides, demonstrating the high specificity of SGP enrichment by using HBS material. Then, HBS material was further utilized to selectively enrich SGPs from the protein digest of human serum, and 487 glycosites were identified from only 2 μL of human serum; 92.0% of the glycopeptides contained at least one sialic acid, indicating good performance for SGP enrichment by using HBS material. Furthermore, the prepared HBS material also has great potential applications in the analysis of glycoprotein sialylation from other complex biological samples.

Unprecedented highly efficient capture of glycopeptides by Fe3O4@Mg-MOF-74 core-shell nanoparticles.

Magnetic Fe3O4 nanospheres coated with Mg-MOF-74 have been facilely prepared via epitaxial growth. Owing to the intrinsic hydrophilic pore surface and size-exclusive properties of Mg-MOF-74, the Fe3O4@Mg-MOF-74 core-shell nanoparticles show effective and selective enrichment of 441 N-glycosylation sites of 418 glycopeptides from 125 glycoproteins in 1 μL of human serum.

Brain tumour differentiation: rapid stratified serum diagnostics via attenuated total reflection Fourier-transform infrared spectroscopy.

The ability to diagnose cancer rapidly with high sensitivity and specificity is essential to exploit advances in new treatments to lead significant reductions in mortality and morbidity. Current cancer diagnostic tests observing tissue architecture and specific protein expression for specific cancers suffer from inter-observer variability, poor detection rates and occur when the patient is symptomatic. A new method for the detection of cancer using 1 μl of human serum, attenuated total reflection—Fourier transform infrared spectroscopy and pattern recognition algorithms is reported using a 433 patient dataset (3897 spectra). To the best of our knowledge, we present the largest study on serum mid-infrared spectroscopy for cancer research. We achieve optimum sensitivities and specificities using a Radial Basis Function Support Vector Machine of between 80.0 and 100 % for all strata and identify the major spectral features, hence biochemical components, responsible for the discrimination within each stratum. We assess feature fed-SVM analysis for our cancer versus non-cancer model and achieve 91.5 and 83.0 % sensitivity and specificity respectively. We demonstrate the use of infrared light to provide a spectral signature from human serum to detect, for the first time, cancer versus non-cancer, metastatic cancer versus organ confined, brain cancer severity and the organ of origin of metastatic disease from the same sample enabling stratified diagnostics depending upon the clinical question asked.Electronic supplementary materialThe online version of this article (doi:10.1007/s11060-016-2060-x) contains supplementary material, which is available to authorized users.

A simple integrated system for rapid analysis of sialic‐acid‐containing N‐glycopeptides from human serum

Terminal sialylation is very important in cancer biology and has been extensively investigated for the discovery of potential clinical biomarkers of cancers. In this study, we presented a novel approach, by using of Ti(IV)-IMAC, to enrich sialic-acid-containing N-glycopeptides for the analysis of terminal sialylation. Compared with conventional method using TiO2 , this approach obtained 2.5 times more glycopeptides and glycosylation sites. Then, a simple integrated system combining filter-aided sample preparation, ACN-improved digestion, and Ti(IV)-IMAC enrichment was established for efficient analysis. In this system, protein digestion, glycopeptide enrichment, and deglycosylation were integrated and were performed sequentially in a single filter unit without any need for desalting, lyophilization, or sample transfer procedures. As a result, the number of identifications was improved by 1.5-fold and the total processing time was drastically reduced to only 7-8 h. By using this system, fast and efficient analysis of human serum sialylated N-glycoproteome was achieved. From only 1 μL of human serum, 217 unique glycopeptides and 194 glycosylation sites were successfully identified.

Centrifugation Assisted Microreactor Enables Facile Integration of Trypsin Digestion, Hydrophilic Interaction Chromatography Enrichment, and On-Column Deglycosylation for Rapid and Sensitive N-Glycoproteome Analysis

Sample handling procedures including protein digestion, glycopeptide enrichment, and deglycosylation have significant impact on the performance of glycoproteome analysis. Several glycoproteomic analysis systems were developed to integrate some of these sample preparation procedures. However, no microsystem integrates all of above three procedures together. In this work, we developed a glycoproteomic microreactor enabling seamless integration of all these procedures. In this reactor, trypsin digestion was accelerated by adding acetonitrile to 80%, and after acidification of protein digest by trifluoroacetic acid (TFA), the following hydrophilic interaction chromatography (HILIC) enrichment and deglycosylation were sequentially performed without any desalting, lyophilization, or buffer exchange steps. The total processing time could be as short as 1.5 h. The detection limit of human IgG as low as 30 fmol was also achieved. When applied to human serum glycoproteome analysis, a total number of 92, 178, and 221 unique N-glycosylation sites were identified from three replicate analyses of 10 nL, 100 nL, and 1 μL of human serum, respectively. It was demonstrated that the glycoproteomic microreactor based method had very high sensitivity and was well suited for glycoproteome analysis of minute protein samples.

A New Rapid Test for the Detection of PF4:Heparin Antibodies.

AbstractAbstract 3708Heparin Induced Thrombocytopenia (HIT), the most frequent drug-induced immune-mediated type of thrombocytopenia, is an important and sometimes life-threatening complication of heparin therapy. HIT can be triggered by standard therapeutic dose heparin, low-dose (prophylactic treatment), low molecular weight heparin, unfractionated heparin and even by minute quantities given to flush intravascular catheters.HIT is a clinicopathologic syndrome cause by platelet activating antibodies that recognize complexes of platelet factor 4 and heparin (PF4-heparin). Thrombocytopenia occurring within 5 – 10 days of administration of heparin is the most common clinical effect. The most severe complication of HIT is the occurrence of both venous and arterial thrombosis. Venous thrombosis can result in limb gangrene and the need for limb amputation.The diagnosis of HIT is based on clinical abnormalities including thrombocytopenia with or without thrombosis and the detection of antibodies to the PF4-heparin complex (termed PF4 dependant antibodies). There are two major types of assays for the detection of heparin dependant antibodies: functional assays and PF4 dependant antigen immunoassays. Functional assays include the serotonin release assay (SRA) and the heparin induced platelet activation assay (HIPA). Enzyme linked immunoassays (ELISAs) are the most frequently used PF4 dependent antigen immunoassay. Functional assays are technically difficult to perform and are considered to be complex specialty assays. ELISAs while not complex are not generally available on a STAT basis. There is a need for a simpler assay which retains or improves the sensitivity and specificity of the existing immunoassays that can be available on a STAT basis.GTI Diagnostics is developing a rapid test based on the proprietary technology used in GTI's PF4 Enhanced® ELISA. The PF4 Rapid test is a single use, semi-quantitative assay contained in a cartridge. It is designed to detect anti-PF4-heparin antibodies in human serum and/or plasma with a total assay time of 15 minutes. The assay uses only 5 μL of human serum and/or plasma along with a proprietary antigen and a proprietary fluorescent secondary antibody used for detection. The assay results are read using a fluorescent reader. A method comparison study was conducted which included an evaluation of 83 serum samples obtained from patients receiving heparin of which 36 were positive and 22 were negative in the PF4 Enhanced® assay. These samples were also tested in the PF4 Rapid Test assay. In addition 25 samples from normal individuals who were not receiving heparin were tested in both assays. Analysis of the data using a 2×2 table (shown below) resulted in a sensitivity of 100%, a specificity of 93.6% and an overall agreement of 96.4% of the PF4 Rapid Test compared to the PF4 Enhanced®.PF4 Enhanced® ELISAPositiveNegativeTotalPF4 Rapid TestPositive36339Negative04444Total364783Although HIT is a true clinicopathological syndrome, its diagnosis still rests primarily on clinical grounds since laboratory tests may not be available locally or may not be available in a sufficiently timely manner. Owing to the high risk of thrombosis associated with HIT, antithrombotic therapy with alternative anticoagulants should be started immediately when serologic assays confirm clinical suspicion. Readily available results, obtained from immunoassays for rapid detection of antibodies against PF4-heparin complexes, may be combined with the pretest estimation of clinical probability in order to exclude and/or confirm the diagnosis of HIT; therefore, assisting physicians in the time-sensitive clinical management of HIT. Disclosures:Ladvienka:GTI Diagnostics, Inc: Employment. Visentin:GTI Diagnostics, Inc: Employment. Chance:GTI Diagnostics, Inc: Employment.

Human Serum Mannose-binding Lectin Senses Wall Teichoic Acid Glycopolymer of Staphylococcus aureus, Which Is Restricted in Infancy

Innate immunity is the first line of host defense against invading pathogens, and it is recognized by a variety of pattern recognition molecules, including mannose-binding lectin (MBL). MBL binds to mannose and N-acetylglucosamine residues present on the glycopolymers of microorganisms. Human serum MBL functions as an opsonin and activates the lectin complement pathway. However, which glycopolymer of microorganism is recognized by MBL is still uncertain. Here, we show that wall teichoic acid of Staphylococcus aureus, a bacterial cell surface glycopolymer containing N-acetylglucosamine residue, is a functional ligand of MBL. Whereas serum MBL in adults did not bind to wall teichoic acid because of an inhibitory effect of anti-wall teichoic acid antibodies, MBL in infants who had not yet fully developed their adaptive immunity could bind to S. aureus wall teichoic acid and then induced complement C4 deposition. Our data explain the molecular reasons of why MBL-deficient infants are susceptible to S. aureus infection.

A sensitive and reproducible fluorescent-based HPLC assay to measure the activity of acid as well as neutral β-glucocerebrosidases

The activity of lysosomal acid β-glucocerebrosidase (AGC, EC 3.2.1.45), which hydrolyzes the O-glycosidic linkage between d-glucose and ceramide of glucosylceramide (GlcCer), is a marker for the diagnosis of Gaucher disease because the disease is caused by dysfunction of AGC due to mutations in the gene. The activity of AGC is potently inhibited by conduritol B epoxide (CBE), whereas CBE-insensitive nonlysosomal neutral β-glucocerebrosidase (NGC) activities have been found in various vertebrates, including humans. We report here a new reliable method to determine AGC as well as NGC activities using normal-phase high-performance liquid chromatography (HPLC) and NBD (4-nitrobenzo-2-oxa-1,3-diazole)- or BODIPY (4,4-difluoro-4-bora-3a,4a-diaza- s-indacene)-labeled GlcCer as a substrate. The reaction products of the enzymes, C6-NBD-ceramide and C12-BODIPY-ceramide, were clearly separated from the corresponding substrates on a normal-phase column within 5 min using a different solvent system. Reaction products could be detected quantitatively at concentrations ranging from 50 fmol to 50 pmol for C6-NBD-ceramide and from 10 fmol to 5 pmol for C12-BODIPY-ceramide. V max/ K m values of human fibroblast AGC for fluorescent GlcCer were much higher than those for 4-methylumbelliferyl-β- d-glucoside (4MU-Glc), which is used prevalently for Gaucher disease diagnosis. As a result, AGC activity was detected quantitatively using fluorescent GlcCer, but not 4MU-Glc, using 5 μl of human serum or 1 × 10 4 cultured human fibroblasts. The current method clearly showed the decrease of AGC activities in fibroblasts and serum from the patient with Gaucher disease compared with normal individuals, suggesting that the method is applicable for the diagnosis of Gaucher disease. Furthermore, this method was found to be useful for measuring the activities of nonlysosomal NGC of various cells and tissues in the presence of CBE.

N-Glycosylation Site Occupancy in Serum Glycoproteins Using Multiple Reaction Monitoring Liquid Chromatography-Mass Spectrometry

Congenital disorders of glycosylation (CDGs) are a family of N-linked glycosylation defects associated with severe clinical manifestations. In CDG type-I, deficiency of lipid-linked oligosaccharide assembly leads to the underoccupancy of N-glycosylation sites on glycoproteins. Although the level of residual glycosylation activity is known to correlate with the clinical phenotype linked to individual CDG mutations, it is not known whether the degree of N-glycosylation site occupancy by itself correlates with the severity of the disease. To quantify the extent of underglycosylation in healthy control and in CDG samples, we developed a quantitative method of N-glycosylation site occupancy based on multiple reaction monitoring LC-MS/MS. Using isotopically labeled standard peptides, we directly quantified the level of N-glycosylation site occupancy on selected serum proteins. In healthy control samples, we determined 98-100% occupancy for all N-glycosylation sites of transferrin and alpha(1)-antitrypsin. In CDG type-I samples, we observed a reduction in N-glycosylation site occupancy that correlated with the severity of the disease. In addition, we noticed a selective underglycosylation of N-glycosylation sites, indicating preferential glycosylation of acceptor sequons of a given glycoprotein. In transferrin, a preferred occupancy for the first N-glycosylation site was observed, and a decreasing preference for the first, third, and second N-glycosylation sites was observed in alpha(1)-antitrypsin. This multiple reaction monitoring LC-MS/MS method can be extended to multiple glycoproteins, thereby enabling a glycoproteomics survey of N-glycosylation site occupancies in biological samples.