עבודה זו נתמכה בחלקם על ידי קרן מחקר מחקר ופיתוח של הקרן למחקר הסרטן בישראל, מענק מהקרן הלאומית לננוטכנולוגיה וקרן הצדקה של הלמסלי עבור תחום מוקד על ננו-רפואה לתרנוסטים מותאמים אישית (VP-K) מכוני בריאות מענק R01GM076360 (ל- XC).

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מוצלח glycan ייצור microarray דורש תכנון זהיר כולל מספר שלבים חשובים בפרוטוקול. אלה כוללים: (1) תכנון פריסות הבלוק והצלחות המגדירים את כל הפרמטרים הבאים ( לדוגמה, מרחקים, ריווח, כמות הדגימות והדפסה); (2) לנקות את הסיכות ולהבטיח שלמות הדק, שהיא קריטית לשליטה בהומוגניות ספוטי?...

סיאס הם סוכרים חומציים המכסים שרשראות גליקניות על גליקופרוטאינים של תאי תאים וגליקוליפידים בחוליות. ביטוי Sia הוא שונה בתאי סרטן 1 וקושרת עם התקדמות ו / או גרורות 2 , 3 . שני של צורות סיה הגדולות היונקים הם Neu5Ac ואת טופס hydroxylated שלה, Neu5Gc 2 . בני אדם לא יכולים לסנתז Neu5Gc עקב אי פעילות מסוימת של הגן קידוד האנזים CMAH. זה סיא הלא אנושי מטבולית משלבת לתוך תאים אנושיים כמו &quot;עצמי&quot;, שמקורה מזון תזונתיים Neu5Gc עשירים ( למשל, בשר אדום) 4 , 5 . Neu5Gc נמצא ברמות נמוכות על משטחי התא של אפיתל אנושי ואנדוטליה, אך הוא נצבר במיוחד בקרצינומה. Neu5Gc מזוהה זרים על ידי מערכת החיסון האנושית הומור 2 , 6 .המורכבות האנטיגנית של Neu5Gc-glycans עשויה להתעורר ברמות רבות, כולל שינוי Neu5Gc, הצמדה, גליקנים בסיסיים ו פיגומים, ואת הצפיפות שלהם, כל לידי ביטוי על ידי המורכבות של נוגדנים נוגדן Neu5Gc בבני אדם 6 . חלק מנוגדנים אלה משמשים ביומרקרים קרצינומה פוטנציאל immunotherapeutics 7 . הופעתו של chemoenzymatic סינתזה של sialoglycans שונים 8 סללה את הדרך לניתוח מעמיק יותר של נוגדנים כאלה, בהנחיית השימוש בטכנולוגיה microiray glycan 9 , 10 . לכן, עם ההכנה להקל על מניפולציה של ספריות גדולות של פחמימות טבעיות וסינתטיות, microlyrays glycan הפכו טכנולוגיה תפוקה גבוהה עוצמה לחקר האינטראקציות של פחמימות עם מספר עצום של biomolecules 10 , 11 , </sup&gt; 12 , 13 . בתבנית מערך, כמות מינימלית של חומרים משמשים, ואת זה להציג multivalent של glycans רלוונטי מבחינה ביולוגית מאפשר חקירה של אלפי אינטראקציות מחייבות בניסוי אחד. חשוב לציין, טכנולוגיה זו יכולה להיות מיושמת גם גילוי ביולוגי ו לניטור תגובות החיסון בדגימות שונות 7 , 12 . מוצלח glycan ייצור microarray מחייב את השיקול של שלושה היבטים חשובים: סוג הרובוט מדפסת, כימיה glycan הצמידה, אופטיקה זיהוי. לגבי שיקול כלי ההדפסה, שתי טכניקות זמינות: מדפסות מגע ומגע. בדפוס מגע, 1-48 סיכות פלדה הם טבל לתוך צלחת מקור רב גם המכיל פתרון glycan והם הבחינו על שקופיות זכוכית פונקציונלי ידי מגע ישיר משטח שקופיות זכוכית. כמות הפתרון דה<html> הכבד לשקופית הוא פונקציה של משך זמן מתמשכת על משטח השקופית. בדרך כלל, הדגימות הם הראשונים מראש מנומר על בלוק זכוכית (כדי להגיע כתמים הומוגנית) לפני שהם מודפסים על משטח השקופית. במדפסות ללא מגע ( לדוגמה, מדפסת piezo אלקטרוני), גליקים מודפסים מן נימי זכוכית באמצעות אותות חשמליים מבוקרת. האות החשמלי יכול להיות מכויל היטב כדי להשיג הדפסה מדויקת יותר ביחס להדפסה של איש קשר. גודל ומורפולוגיה של כתמים הם גם יחסית הומוגנית יותר. יתרון נוסף הוא מיחזור המדגם חזרה לצלחת המקור לאחר ההדפסה. עם זאת, החיסרון העיקרי של מדפסות piezo-Electronic הוא הגבלת קצה ההדפסה (4 או 8), וכתוצאה מכך משך הדפסה ארוך מאוד, המחייב תשומת לב מיוחדת ליציבות שקופיות, טמפרטורה, לחות והתאדות מדגם. מדפסת הזרקת הדיו שאינה מזדקקת דורשת כמות גדולה יותר של מדפסות 14 . S = &quot;jove_content&quot;&gt; בניגוד לאפשרויות הזמינות המוגבלות לשיטות הדפסה, כימיה גליקן הצמידה היא שיקול מורכב יותר, עם אפשרויות רבות לבחירה. כימיה immobilization נבחר חייב בחשבון הן את הקבוצות הפעילות על glycans ואת תגובת משטח שקופיות. את glycans להיות משותקת על משטח מסוים microarray, או synthtically synthesized או מבודד באופן טבעי, כל דורשים קבוצה תגובתי זהה. בנוסף, glycans צריך להיות טהור הומוגנית. מצד שני, משטח immobilization ואת הכימיה צריכה לספק reproducibility וצפיפות מצורף אמין. שיטות רבות immobilization פותחו עם קוולנטיים או לא קוולנטי (קליטה פיזית) 10 , 11 , 12 , 13 . לקבלת מידע מפורט על מודפס טכנולוגיית microarray glycan עבור uninitiateD, עיין אלה ביקורות מצוינות 13 , 15 . חשוב לציין, המידע המינימלי האחרון הנדרש עבור ניסוי Glycomics (MIRAGE) יוזמה מתאר הנחיות להכנת המדגם 16 ולדיווח נתונים מניתוח microarray glycan 17 כדי לשפר את הסטנדרטים בתחום זה גדל. כאן, אנו מתארים פרוטוקול מפורט עבור ייצור של microarrays sialoglycan באמצעות קשר ספציפי nano מדפסת בפורמט 16-היטב. כל אחד glycans יש אמין הראשי כי לתווך קוולנטי שלהם קישור אפוקסי-מופעל שקופיות זכוכית. כמו כן, אנו מתארים את הפיתוח והניתוח של שקף אחד באמצעות דגימות שונות של סרה אנושית, נוגדנים, וסטטינים של צמחים מחברים. מבחני microarray Sialoglycan לערב כמה צעדים עיקריים הכוללים ייצור מערך, עיבוד, פיתוח וניתוח. מערך מערך דורש תכנון arRay פריסה, הכנת glycans ואת צלחת המקור, תכנות nano מדפסת, והדפסת שקופיות. לאחר מכן, השקפים מעובדים, מפותחים, ונותחו ( איור 1 ).

<table><tbody><tr><td>Primary-amine containing sialoglycans</td><td>Glycohub, Inc., Davis, CA, USA (http://www.glycohub.com/services)</td><td>Contact info@glycohubusa.com for compound requests</td><td>Printed glycans</td></tr><tr><td>Monosodium phosphate monohydrate</td><td>Sigma</td><td>S9638</td><td>Printing buffer component</td></tr><tr><td>Disodium phosphate heptahydrate</td><td>Sigma</td><td>S9390</td><td>Printing buffer component</td></tr><tr><td>Phosphate buffered saline</td><td>Hy-Labs</td><td>BP-507/500D</td><td>Printing buffer/ incubation/washing buffer</td></tr><tr><td>Tris-base</td><td>Sigma</td><td>T1503</td><td>Slide blocking reagent</td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G-7893</td><td>Printing buffer component</td></tr><tr><td>Ethanolamine</td><td>Thermo-Fisher Scientific</td><td>0700/08</td><td>Slide blocking reagent</td></tr><tr><td>Ovalbumin (Grade V)</td><td>Sigma</td><td>A5503</td><td>Slide Blocking protein</td></tr><tr><td>Tween-20</td><td>Sigma</td><td>P7949</td><td>Slide washing detergent</td></tr><tr><td>Alexa 555-Hydrazide</td><td>Thermo-Fisher Scientific</td><td>A20501MP</td><td>Marker on array</td></tr><tr><td>ChromPure Human IgG, whole molecule</td><td>Jackson Immunoresearch</td><td>009-000-003</td><td>Printing component</td></tr><tr><td>Biotinylated- SNA</td><td>Vector Laboratories</td><td>B-1305</td><td>Plant Lectin &amp;ndash; binding Sia-alpha2&amp;ndash;6-linked</td></tr><tr><td>Biotinylated-MALII</td><td>Vector Laboratories</td><td>B-1265</td><td>Plant Lectin &amp;ndash; binding Sia-alpha2&amp;ndash;3-linked</td></tr><tr><td>Chicken-anti Neu5Gc IgY</td><td>BioLegend</td><td>146903</td><td>Primary detection</td></tr><tr><td>Cy3-Streptavidin</td><td>Jackson Immunoresearch</td><td>016-160-0848</td><td>Biotin binding</td></tr><tr><td>Cy3-anti Human IgG</td><td>Jackson Immunoresearch</td><td>109-165-088</td><td>Secondary detection against human IgG</td></tr><tr><td>Cy3-anti Chicken IgY</td><td>Jackson Immunoresearch</td><td>703-165-155</td><td>Secondary detection against chicken IgY</td></tr><tr><td>Human sera samples</td><td>Israeli Blood Bank</td><td></td><td>Primary detection</td></tr><tr><td>Compressed Nitrogen (Grade 5)</td><td></td><td></td><td>General dusting/drying tool</td></tr><tr><td>Epoxy-coated slides</td><td>Corning</td><td>40044</td><td>Slides</td></tr><tr><td>Epoxy-coated slides</td><td>PolyAn</td><td>2D 104-00-221</td><td>Slides. In this type of slides the surface is more hydrophobic (compared to Coring slides) therefore the glycans Print Buffer would need to be supplemented with 0.005% Tween-20 to obtain 100 &amp;micro;m size spots.</td></tr><tr><td>384-well microtiter plate</td><td>Genetix</td><td>2070</td><td>Printing plate</td></tr><tr><td>VWR lab marker</td><td>VWR</td><td>52877-310</td><td>Slide labeling</td></tr><tr><td>Staining Tube</td><td>ArrayIt</td><td>MST</td><td>Slide developing tool</td></tr><tr><td>Staining bath</td><td>VWR</td><td>25608-904</td><td>Slide developing tool</td></tr><tr><td>Slides glass holders</td><td>VWR</td><td>631-9321</td><td>Slide developing tool</td></tr><tr><td>GenePix Scanner</td><td>Molecular devices</td><td>4000B</td><td>Slide scanner</td></tr><tr><td>LM-60 NanoPrinter</td><td>ArrayIt</td><td>LM-60</td><td>Array printer</td></tr><tr><td>Pins</td><td>ArrayIt</td><td>946MP3</td><td>Printing pins</td></tr><tr><td>ProPlate Module</td><td>Grace Bio-Labs</td><td>P37004</td><td>Slide developing module</td></tr><tr><td>Distilled water</td><td>Bio-Lab</td><td>2321020500</td><td>Required for arrayer and humidifier</td></tr><tr><td>Electronic Multi Pippete, 8 Channel , volume range 2-125 &amp;mu;L</td><td>Thermo-Fisher Scientific (Matrix)</td><td>MA-2131 Impact2 Equalizer 384</td><td>Multi pippete for sample dispansing into 384-well plate</td></tr></tbody></table>

profiling anti-neu5gc igg in human sera with a sialoglycan microarray assay

תאים מכוסים בגלימה של שרשרות פחמימות (גלייקנים) שמשתנות בדרך כלל בסרטן וכוללות וריאציות של ביטוי חומצי סיאלי (Sia). אלה הם סוכרים חומצי כי יש 9-פחמן עמוד השדרה וכי מכסה חוליות glycans על משטחי התא. שני של צורות סיה הגדולות היונקים הם N- Acetylneuraminic חומצה (Neu5Ac) ואת טופס hydroxylated שלה, N- glycolylneuraminic חומצה (Neu5Gc). בני אדם לא יכולים לייצר אנדוגניים Neu5Gc עקב השבתה של הגן קידוד cytidine 5&#39;monophosphate-Neu5Ac (CMP-Neu5Ac) hydroxylase (CMAH). זר Neu5Gc נרכש על ידי תאים אנושיים באמצעות הצריכה התזונתיים של בשר אדום ומוצרי חלב ולאחר מכן מופיע על glycans מגוונים על פני התא, צובר בעיקר על קרצינומה. כתוצאה מכך, בני אדם מפזרים נוגדנים נגד Neu5Gc שמשחקים תפקידים מגוונים בסרטן ומחלות דלקתיות כרוניות אחרות, אשר הופכים לתהליכים אבחנתיים וטיפוליים פוטנציאלייםתקציבים. כאן, אנו מתארים תפוקה גבוהה sialoglycan assay microarray להעריך נוגדנים אלה נגד Neu5Gc בסרה האדם. Neu5Gc המכילים glycans ואת זוגות מתאימים שלהם של פקדים (Neu5Ac המכילים glycans), כל אחד עם אמין העיקרי הליבה, הם מקושרים קוולנטית כדי אפוקסי מצופה זכוכית שקופיות. אנו מדגימים את הדפסת 56 שקופיות בפורמט 16-היטב באמצעות מדפסת nano ספציפית מסוגל לייצר עד 896 מערכים לכל הדפסה. כל שקופית ניתן להשתמש כדי המסך 16 שונים דגימות sera האדם להערכת אנטי Neu5Gc נוגדנים, ספציפיות וגיוון. הפרוטוקול מתאר את המורכבות של כלי זה חזק ומספק הנחיה בסיסית עבור אלה שמטרתם לחקור את התגובה Neu5Gc פחמימות תזונתיים אנטיגן בדגימות קליניות מגוונות בפורמט מערך.

מערך הדפסה, פיתוח וניתוח: הדפסת microarray sialoglycan עם דגימות glycan מרובים האדם IgG STD עקומות ב 16 בלוקים שונים דורש כיול יסודי על מנת להבטיח כי כל הדגימות מודפסים כמו אחיד ככל האפשר בכל 16 בלוקים לכל שקופית לכ...

Watch this Scientific Journal Video about Profiling Anti-Neu5Gc IgG in Human Sera with a Sialoglycan Microarray Assay at JoVE.com

Profiling Anti-Neu5Gc IgG in Human Sera with a Sialoglycan Microarray Assay

פרופילינג נגד Neu5Gc IgG ב Sera האדם עם Assay Microarray Sialoglycan

Assay microarray sialoglycan ניתן להשתמש כדי להעריך נוגדנים נוגדי Neu5Gc ב sera האדם, מה שהופך אותו assay אבחון תפוקה גבוהה פוטנציאל לסרטן ומחלות כרוניות אחרות מתווך דלקת כרונית.

Cells are covered with a cloak of carbohydrate chains (glycans) that is commonly altered in cancer and that includes variations in sialic acid (Sia) ...

profiling-anti-neu5gc-igg-human-sera-with-sialoglycan-microarray

Universidad San Sebastian

Research

JoVE Journal

Behavior

9.4K Views.  Tel Aviv University.  Assay microarray sialoglycan ניתן להשתמש כדי להעריך נוגדנים נוגדי Neu5Gc ב sera האדם, מה שהופך אותו assay אבחון תפוקה גבוהה פוטנציאל לסרטן ומחלות כרוניות אחרות מתווך דלקת כרונית. 

Video: Profiling Anti-Neu5Gc IgG in Human Sera with a Sialoglycan Microarray Assay

Mass Spectrometric Analysis of Glycosphingolipid Antigens

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes such as embryonic development, signal transduction, and immune receptor recognition1-2. They contain complex sugar moieties in the form of isomers, and lipid moieties with variations including fatty acyl chain length, unsaturation, and hydroxylation. Both carbohydrate and ceramide portions may be basis of biological significance. For example, tri-hexosylceramides include globotriaosylceramide (Gal&alpha;4Gal&beta;4Glc&beta;1Cer) and isoglobotriaosylceramide (Gal&alpha;3Gal&beta;4Glc&beta;1Cer), which have identical molecular masses but distinct sugar linkages of carbohydrate moiety, responsible for completely different biological functions3-4. In another example, it has been demonstrated that modification of the ceramide part of alpha-galactosylceramide, a potent agonist ligand for invariant NKT cells, changes their cytokine secretion profiles and function in animal models of cancer and auto-immune diseases5. The difficulty in performing a structural analysis of isomers in immune organs and cells serve as a barrier for determining many biological functions6.
Here, we present a visualized version of a method for relatively simple, rapid, and sensitive analysis of glycosphingolipid profiles in immune cells7-9. This method is based on extraction and chemical modification (permethylation, see below Figure 5A, all OH groups of hexose were replaced by MeO after permethylation reaction) of glycosphingolipids10-15, followed by subsequent analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and ion trap mass spectrometry. This method requires 50 million immune cells for a complete analysis. The experiments can be completed within a week. The relative abundance of the various glycosphingolipids can be delineated by comparison to synthetic standards. This method has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 2 fmol of total iGb3/Gb3 mixture is present9.
Ion trap mass spectrometry can be used to analyze isomers. For example, to analyze the presence of globotriaosylceramide and isoglobtriaosylceramide in the same sample, one can use the fragmentation of glycosphingolipid molecules to structurally discriminate between the two (see below Figure 5). Furthermore, chemical modification of the sugar moieties (through a permethylation reaction) improves the ionization and fragmentation efficiencies for higher sensitivity and specificity, and increases the stability of sialic acid residues. The extraction and chemical modification of glycosphingolipids can be performed in a classic certified chemical hood, and the mass spectrometry can be performed by core facilities with ion trap MS instruments.

A specific and sensitive method to gain insight into the expression profile of glycosphingolipid antigens in immune organs and cells is described. The method takes advantage of the ion trap mass spectrometry allowing step-wise fragmentation of glycosphingolipid molecules for structural analysis in comparison to chemically synthesized standards.

ניתוח ספקטרומטריה של אנטיגנים Glycosphingolipid

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes ...

Immunology and Infection

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are commonly found on human glycoproteins. To achieve the desired structural diversity, the strategy began with the chemo-enzymatic synthesis of three kinds of oligosaccharyl fluoride modules, followed by their stepwise &#945;-selective glycosylations at the 3-O and 6-O positions of the mannose residue of the common core trisaccharide having a crucial &#946;-mannoside linkage. We further attached the N-glycans to the surface of an aluminum oxide-coated glass (ACG) slide to create a covalent mixed array for the analysis of hetero-ligand interaction with an HIV antibody. In particular, the binding behavior of a newly isolated HIV-1 broadly neutralizing antibody (bNAb), PG9, to the mixture of closely spaced Man5GlcNAc2 (Man5) and 2,6-di-sialylated bi-antennary complex type N-glycan (SCT) on an ACG array, opens a new avenue to guide the effective immunogen design for HIV vaccine development. In addition, our ACG array embodies a powerful tool to study other HIV antibodies for hetero-ligand binding behavior.

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

A modular approach to the synthesis of N-glycans for attachment to an aluminum oxide-coated glass slide (ACG slide) as a glycan microarray has been developed and its use for the profiling of an HIV broadly neutralizing antibody has been demonstrated.

כימותרפיה-אנזימטי סינתזה של N- glycans עבור מערך פיתוח נוגדן HIV פרופיל

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are ...

Chemistry

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

Glycosylation is a vital modification found in proteins. N-glycan profiling of glycoproteins is required to detect novel biomarker candidates and determine glycan alterations in diseases. Most commercially available biopharmaceutical proteins are glycoproteins. The efficacy of these drugs is affected by glycosylation patterns. Therefore, an in-depth characterization method for the N-glycans is necessary. Here, we present a comprehensive approach for qualitative and quantitative analysis of N-glycans using hydrophilic interaction liquid chromatography equipped with fluorescence detection and tandem mass spectrometry (HILIC-FLD-MS/MS). N-glycans were released from glycoproteins with a facile method and labeled by a procainamide fluorophore tag in the strategy. Subsequently, the procainamide labeled N-glycans were analyzed by a HILIC-FLD-MS/MS technique. In this approach, N-glycan structures were confirmed by the tandem mass spectrometric analysis, whereas fluorescence detection was used for the quantitative analysis. An application for data analysis of the detected N-glycan peaks is described in the study. This protocol can be applied to any glycoprotein extracted from various species.

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

N-glycan profiling of glycoproteins is essential for discovering novel biomarkers and understanding glycan functions in cellular events. Additionally, N-glycan analysis of protein biopharmaceuticals is very important for human use. In this current article, a high-throughput strategy for identifying and quantifying N-glycan structures was presented using the HILIC-FLD-MS/MS technique.

Glycosylation is a vital modification found in proteins. N-glycan profiling of glycoproteins is required to detect novel biomarker candidates and ...

Biochemistry

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

Assay Microarray מיניאטורי glycan להערכת הלהיטות וספציפיות של Hemagglutinins וירוס שפעת A

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing termini of oligo- and polysaccharides, as well as its unique chemical characteristics, sialic acid is involved in a multitude of different receptor-ligand interactions. By modifying the expression of sialic acid on the cell surface, sialic acid-dependent interactions will consequently be influenced. This can be helpful to investigate sialic acid-dependent interactions and has the potential to influence certain diseases in a beneficial way. Via metabolic glycoengineering (MGE), the expression of sialic acid on the cell surface can be modulated. Herein, cells, tissues, or even entire animals are treated with C2-modified derivatives of N-acetylmannosamine (ManNAc). These amino sugars act as sialic acid precursor molecules and therefore are metabolized to the corresponding sialic acid species and expressed on glycoconjugates. Applying this method produces intriguing effects on various biological processes. For example, it can drastically reduce&#160;the expression of polysialic acid (polySia) in treated neuronal cells and thus affects neuronal growth and differentiation. Here, we show the chemical synthesis of two of the most common C2-modified N-acylmannosamine derivatives, N-propionylmannosamine (ManNProp) as well as N-butanoylmannosamine (ManNBut), and further show how these non-natural amino sugars can be applied in cell culture experiments. The expression of modified sialic acid species is quantified by high performance liquid chromatography (HPLC) and further analyzed via mass spectrometry. The effects on polysialic acid expression are elucidated via Western blot using a commercially available polysialic acid antibody.

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

Sialic acid is a typical monosaccharide-unit found in glycoconjugates. It is involved in a plethora of molecular and cellular interactions. Here we present a method to modify cell surface sialic acid expression using metabolic glycoengineering with N-acetylmannosamine derivatives.

Glycoengineering מטבולית של חומצה Sialic באמצעות N-acyl השומן-שונה Mannosamines

Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing ...

Bioengineering

<meta charset="utf8"></head><body>פרופיל גליקן אוטומטי מבוסס לקטין באמצעות מערך חרוזים בקצה יחיד (BIST)

Rapid Glyco-Qualitative Assessment of Recombinant Proteins Using a Fully Automated System

Protein glycosylation, a critical post-translational modification, influences the stability, efficacy, and immunogenicity of recombinant proteins, including biopharmaceuticals. Glycan structures exhibit significant heterogeneity, varying with production cell types, culture conditions, and purification methods. Consequently, monitoring and evaluating the glycan structures of recombinant proteins is vital, particularly in biopharmaceutical production. The lectin microarray, a technique complementary to mass spectrometry, boasts high sensitivity and ease of use. However, it typically requires more than a day to yield results. To adapt it to non-glycoscience research or drug product process development, an automated, high-throughput alternative is needed. Therefore, the world's first fully automated lectin-based glycan profiling system was developed, utilizing the "bead array in a single tip (BIST)" technology concept. This system allows for the preparation and storage of lectin-immobilized beads in units of 1,000, with customizable parallel insertion orders for various purposes. This article presents a practical protocol for research involving "glyco-qualified" recombinant proteins. After testing their reactivity against 12 polyacrylamide-glycan conjugates, 15 lectins were selected to increase the system's versatility. In addition, the sample labeling process was optimized by switching from Cy3 to biotin, reducing the overall processing time by 30 min. For immediate data qualification, lectin-binding signals are displayed as a dotcode on the top monitor. The system's reliability was confirmed through day-to-day reproducibility tests, repeatability tests, and long-term storage tests, with a coefficient of variation of &lt;10%. This user-friendly and rapid glyco-analyzer has potential applications in the quality monitoring of endogenous glycoproteins for biomarker evaluation and validation. This method facilitates analysis for those new to glycoscience, thereby broadening its practical utility.

<meta charset="utf8"></head><body>מחבר זרקור: קידום מחקר גליקוזילציה של חלבונים באמצעות מערכת אוטומטית לחלוטין

A system for the automated and rapid analysis of glycans on proteins was previously developed. This article presents the protocol for "glyco-qualitative analysis," optimized for a broad spectrum of users, such as those engaged in analyzing glycan structures in biopharmaceuticals and other glycoconjugate materials.

הערכה גליקולית-איכותית מהירה של חלבונים רקומביננטיים באמצעות מערכת אוטומטית לחלוטין

Protein glycosylation, a critical post-translational modification, influences the stability, efficacy, and immunogenicity of recombinant proteins, ...

Printed Glycan Array: A Sensitive Technique for the Analysis of the Repertoire of Circulating Anti-carbohydrate Antibodies in Small Animals

The repertoire of circulating anti-carbohydrate antibodies of a given individual is often associated with its immunological status. Not only the individual immune condition determines the success in combating internal and external potential threat signals, but also the existence of a particular pattern of circulating anti-glycan antibodies (and their serological level variation) could be a significant marker of the onset and progression of certain pathological conditions. Here, we describe a Printed Glycan Array (PGA)-based methodology that offers the opportunity to measure hundreds of glycan targets with very high sensitivity; using a minimal amount of sample, which is a common restriction present when small animals (rats, mice, hamster, etc.) are used as models to address aspects of human diseases. As a representative example of this approach, we show the results obtained from the analysis of the repertoire of natural anti-glycan antibodies in BALB/c mice. We demonstrate that each BALB/c mouse involved in the study, despite being genetically identical and maintained under the same conditions, develops a particular pattern of natural anti-carbohydrate antibodies. This work claims to expand the use of PGA technology to investigate repertoire (specificities) and the levels of circulating anti-carbohydrates antibodies, both in health and during any pathological condition.

This work shows the potential of printed glycan array (PGA) technology for the analysis of circulating anti-carbohydrate antibodies in small animals.

מערך Glycan המודפס: טכניקה רגישה לניתוח של הרפרטואר של מחזורי נוגדנים אנטי-פחמימות בבעלי חיים קטנים

The repertoire of circulating anti-carbohydrate antibodies of a given individual is often associated with its immunological status. Not only the ...

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

נוגדן microarray כימי חסומים פרופיל תפוקה גבוהה מרובב של Glycosylation חלבון מסוים בדגימות מורכבות

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

פרופילינג נגד Neu5Gc IgG ב Sera האדם עם Assay Microarray Sialoglycan

Summary

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ניתוח ספקטרומטריה של אנטיגנים Glycosphingolipid

כימותרפיה-אנזימטי סינתזה של N- glycans עבור מערך פיתוח נוגדן HIV פרופיל

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

Assay Microarray מיניאטורי glycan להערכת הלהיטות וספציפיות של Hemagglutinins וירוס שפעת A

Glycoengineering מטבולית של חומצה Sialic באמצעות N-acyl השומן-שונה Mannosamines

הערכה גליקולית-איכותית מהירה של חלבונים רקומביננטיים באמצעות מערכת אוטומטית לחלוטין

הערכה גליקולית-איכותית מהירה של חלבונים רקומביננטיים באמצעות מערכת אוטומטית לחלוטין

הערכה גליקולית-איכותית מהירה של חלבונים רקומביננטיים באמצעות מערכת אוטומטית לחלוטין

מערך Glycan המודפס: טכניקה רגישה לניתוח של הרפרטואר של מחזורי נוגדנים אנטי-פחמימות בבעלי חיים קטנים

נוגדן microarray כימי חסומים פרופיל תפוקה גבוהה מרובב של Glycosylation חלבון מסוים בדגימות מורכבות