material preparation and automatic glycan profiling of proteins

<meta charset="utf8"></head><body>使用单吸头中的微珠阵列 （BIST） 进行基于凝集素的聚糖自动化分析

Protein glycosylation is a crucial post-translational modification that needs to be assessed in biopharmaceuticals. The glycan profiles of proteins can vary based on culture conditions, purification processes, and host cells1. Simple instruments are required to qualify glycosylation within the bioprocess pipeline. It is estimated that over 50% of secreted and membrane proteins in vivo are modified with multiple glycans, which change according to cell lineage, developmental stage, and disease status, such as the onset of malignancy2. Monitoring glycan profiles holds significant potential for identifying unique diagnostic markers and drug targets. Automated instruments capable of rapidly measuring large-scale sample sizes are in high demand to verify and validate such aberrant glycosylation from hundreds of patient samples in the discovery pipeline.
Microarray technology has been incorporated into glycomics for evaluating the glycan profiling of glycoproteins3. In this method, several lectins, which are glycan-binding proteins, are immobilized on a surface such as a glass slide. This interaction-based glyco-analysis technology does not necessitate releasing glycans from core proteins beforehand, simplifying the process for researchers new to glycotechnology. Despite its widespread use, for industrial applications such as bioproduction, an automated system capable of quickly and easily monitoring glycans for a larger number of analysis targets was required. To address this, an automated glycan profiling system based on a unique concept termed &#34;bead array in a single tip&#34; (BIST), initially developed for genotyping, was previously reported. This system simplifies the process with a one-box type high-throughput auto-instrument4. Using tips in which various lectin-fixed beads are arranged in parallel4,5, a method for analyzing glycan structures modified in glycoproteins was established and named GlycoBIST (hereafter referred to as &#34;automatic glycan profiling system&#34;) (Figure 1A). Lectins can be fixed on 1,000 beads and dried to maintain activity for a year, both before and after packing in a tip. Once tips and cartridges containing reagents such as HRP-labeled anti-streptavidin antibody (SA-HRP) are set in the measuring prototype instrument (an automated reaction measurement device, see Table of Materials), the tip functions as an autopipette. A chemiluminescence detection scanner at the instrument&#39;s interior rear quantifies the signals of eight tips simultaneously. The quantitative data from these eight tips are compactly and concurrently displayed as dot codes on the instrument&#39;s touchscreen for quick confirmation of measurement results. Furthermore, the value represented as the maximum of the measured peak is transported from the instrument as raw data, and it allows for graphing by individual researchers (Figure 1A, bottom panel).
In this article, the authors describe an improved method of biotin-labeling proteins, reducing processing time to 30 min. Target proteins are biotinylated in advance and detected by SA-HRP (Figure 1B). A standard GlycoBIST tip (specialized tip for automatic glycan profiling) with 15 selected lectins was constructed to achieve the versatile glycoprotein glycan profiling for analytical comprehensiveness.

<meta charset="utf8"></head><body>作者聚焦：使用全自动系统推进蛋白质糖基化研究

使用全自动系统对重组蛋白进行快速糖质定性评估

Our scope is protein glycosylation. Glycan structures are involved in changes in the stability and function of biopharmaceuticals and are altered along with host cell species and culture conditions. In some production conditions, allergenic glycan would be contaminated to a certain degree. Our goal is to make user-friendly tools for such glyco-qualification.Mostly, LC and Mass spectrometry. But, for simple testing, lectin-based technologies are applicable. Researchers have available many types of lectins with different specificities and each lectin binds to some specific glycans. So, we can estimate glycan structures using multiuse of lectin-by-lectin blotting, lectin ELISA, and lectin microarray.Glycosylation occurs in functional proteins in our body. Glycan structure is altered along with diseases. Nowadays, we have used several disease-specific glycosylations as diagnostic biomarkers, such as CA 19-9 and AFP-L3. Notably, new biomarker candidates have been discovered using lectin-based technologies. For their validation, we need glyco-qualification system.Glyco-qualification is hard work. We need to analyze hundreds or thousands of numbers in a short time. However, there is no system to meet the needs. Our analytical method is the only one with a full-automatic measurement process. It drastically reduces manpower and enables us to conduct effective validation.Our work aims to further improve our rapid instrument, especially for quantitative measurement. After this, we expect to realize a unique diagnostic tool with "glycobiomarker panel" measured by our multilectin system. Hence, we are focusing on the discovery of disease-specific glycobiomarkers.

蛋白质的材料制备和自动聚糖分析

material-preparation-and-automatic-glycan-profiling-of-proteins

Research

JoVE Journal

Biochemistry

Material Preparation and Automatic Glycan Profiling of Proteins

74 次观看. 首先，将装有冷冻干燥凝集素固定珠的密封袋在室温下的长凳上。将防静电布放在桌子上，然后将便携式静电消除器面向工作区放置。使用抗静电镊子，将磁珠插入空尖端，使两个间隔珠放置在凝集素固定磁珠之间。用钳子轻轻挤压吸头顶部并关闭开口。如果尖端弯曲，请手动拉直尖端。然后将其储存在装有干燥剂的密封袋中，在 4 摄氏度下，...

视频: 蛋白质的材料制备和自动聚糖分析

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.

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.

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

科研

生物化学

To begin, place a sealed bag containing frozen dried lectin fixed beads on a bench at room temperature. Place an antistatic cloth on the desk and then position the portable static eliminator facing the workspace. Using antistatic tweezers, insert the beads into an empty tip such that two spacer beads are placed in between the lectin fixed beads.Use nippers to gently squeeze the top of the tip and close the opening. Manually straighten the tip if it is bent. Then store it in a sealed bag with a desiccant at four degrees Celsius until needed.Add about 10 microliters of the analyte protein solution to a tube containing 10 micrograms of dried biotinylation reagent. Vortex the mixture to mix well. Then centrifuge it at 1000 G for five seconds at room temperature.Incubate the tubes for one hour at room temperature away from light. Now add 90 microliters of probing buffer to the samples to dilute it tenfold before incubating. Open the front door of the automated reaction measurement device.Transfer the liquid reservoirs, the prepared tips and the analytes into the designated positions in the device. Turn on the power switch of the main unit of an automated reaction measurement device. Touch the Start Diagnosis button displayed on the screen.Press Assay on the home screen to select the protocol. Now select the BAssaySTD option of the assay method and press Next. Enter the sample names and press Next.Then enter the tip names and press Next again. Confirm the notes on the right, then press Start to begin measurement. Confirm the screen switch to the running mode and wait until the Finalize arrow is highlighted.The screen will change into result mode when the measurement is complete. Insert a USB memory stick into the port on the side of the main unit. Press Export in the result mode to save the data as a CSV file for further data analysis and individual graphing in Excel.A distinct difference in the binding pattern of the lectins against the 12 polyacrylamide sugars was seen. Purified protein products that were subjected to the tip measurement showed increased reactivity of certain lectins to the saccharides.