Low-abundance Glycopeptides Research Articles

Oriented assembly of hydrophilic nanochains modified by porous zirconium-based coordination polymers for glycopeptides analysis

Mass spectrometry (MS)-based glycoproteomics research requires additional sample pretreatment to improve the effective identification of low-abundance glycopeptides without interference from non-glycoproteins. Herein, an attractive strategy using resorcinol-formaldehyde (RF) resin and zirconium-based coordination polymer (Zr-BCP) was established to prepare one-dimensional porous coordination polymer composites for glycopeptide enrichment before MS analysis. The obtained Fe3O4@RF@Zr-BCP nanochains feature excellent magnetic response (42.26 emu/g), high hydrophilicity (16.0°), and large specific surface area (140.84 m2/g), which provides abundant affinity sites for specific capture of glycopeptides. The materials exhibit outstanding performance in the enrichment of glycopeptides in terms of sensitivity (15 fmol/μL IgG), selectivity (1:200, molar ratio of IgG/BSA), loading capacity (200 mg/g) and recovery (106.4 ± 3.5%). In addition, the developed method based on Fe3O4@RF@Zr-BCP has been successfully applied to capture glycopeptides in tryptic digest of mouse teratoma cell extracts. It is worth emphasizing that compared with dispersed nanoparticles, the one-dimensional chain structure brings extraordinary reusability to Fe3O4@RF@Zr-BCP nanochains, which is conducive to the rapid cyclic enrichment of glycopeptides. This present work provides a potential enrichment platform for comprehensive glycoprotein analysis, and opens a new avenue for the application of oriented-assembly nanochains.

Mapping the low abundant plasma glycoproteome using Ranachrome-5 immobilized magnetic terpolymer as improved HILIC sorbent

HILIC (hydrophilic interaction liquid chromatography) materials enrich glycopeptides. The non-specific interactions because of support material and inadequate hydrophilicity render loss of less abundant glycopeptides in SPE-based enrichments. In this work, magnetic terpolymer (Fe3O4@MAA/DVB/1,2-Epoxy-5-hexene) is functionalized with Ranachrome-5 to generate enhanced hydrophilicity. Amine, carboxylic, and amide groups of ranachrome-5 provide zwitterionic chemistry. Material’s magnetic core contributes to ease of operation while higher surface area 97.0711 m2 g−1 immobilizes better quantities of Ranachrome-5. Homogeneous morphology, nano-size, and super hydrophilicity enhance enrichment. Ranachrome-5 functionalized polymeric core-shell beads enrich 25, 18 and 16 N-linked glycopeptides via SPE strategy from tryptic digests of model glycoproteins i.e., immunoglobulin G (IgG), horseradish peroxidase (HRP) and chicken avidin, respectively. Zwitterionic chemistry of ranachrome-5 helps in achieving higher selectivity (1:250, HRP / Bovine Serum Albumin), and lower detection limit (100 attomole, HRP digest) with complete glycosylation profile of each standard digest. High binding capacity (137.1 mg/g) and reuse of affinity material up to seven cycles reduce the cost and amount of affinity material for complex sample analysis. A recovery of 91.76% and relative standard deviation (RSD) values less than 1 define the application of HILIC beads for complex samples like plasma. 508 N-linked intact low abundant glycopeptides corresponding to 50 glycoproteins are identified from depleted human plasma samples via nano-Liquid Chromatography-Tandem Mass Spectrometry (nLC-MS/MS). Using Single Nucleotide Variances (BioMuta) for low abundant plasma glycoproteins, the potential association of proteins to four cancers, i.e., breast, lung, uterine, and melanoma is evaluated. Via the bottom-up approach, HILIC beads can analyze clinically important low-abundant glycoproteins.

Glycoproteomic Analyses of Influenza A Viruses Using timsTOF Pro MS.

N-Linked glycosylation in hemagglutinin and neuraminidase glycoproteins of influenza viruses affects antigenic and receptor binding properties, and precise analyses of site-specific glycoforms in these proteins are critical in understanding the antigenic and immunogenic properties of influenza viruses. In this study, we developed a glycoproteomic approach by using a timsTOF Pro mass spectrometer (MS) to determine the abundance and heterogeneity of site-specific glycosylation for influenza glycoproteins. Compared with a Q Exactive HF MS, the timsTOF Pro MS method without the hydrophilic interaction liquid chromatography column enrichment achieved similar glycopeptide coverage and quantities but was more effective in identifying low-abundance glycopeptides. We quantified the distributions of intact site-specific glycopeptides in hemagglutinin of A/chicken/Wuxi/0405005/2013 (H7N9) and A/mute swan/Rhode Island/A00325125/2008 (H7N3). Results showed that hemagglutinin for both viruses had complex N-glycans at N22, N38, N240, and N483 but only high-mannose glycans at N411 and, however, that the type and quantities of glycans were distinct between these viruses. Collisional cross section (CCS) provided by the ion mobility spectrometry from the timsTOF Pro MS data differentiated sialylation linkages of the glycopeptides. In summary, timsTOF Pro MS method can quantify intact site-specific glycans for influenza glycoproteins without enrichment and thus facilitate influenza vaccine development and production.

Design and fabrication of highly hydrophilic magnetic material by anchoring l-cysteine onto chitosan for efficient enrichment of glycopeptides

Hydrophilic interaction liquid chromatography (HILIC) has been recognized as an effective strategy for glycopeptide enrichment. Hydrophilic materials pave the way to solve the limit of low enrichment capacity and poor selectivity. The present study is the first attempt to combine chitosan (CS) and l-cysteine (l-Cys) to design a novel hydrophilic material focusing on glycopeptide enrichment. CS containing a large number of hydrophilic amino and hydroxyl groups has unique chemical properties, which makes it a very attractive biomaterial for glycopeptide enrichment. The excellent hydrophilicity of zwitterionic molecule l-Cys inspires the idea of anchoring l-Cys onto CS to design a novel hydrophilic material (named as Fe3O4@CS@Au-l-Cys) for the capture of low abundance glycopeptides. To be specific, Au nanoparticles (Au NPs) was introduced into CS-coated Fe3O4via electrostatic interaction and served as bridges to anchor l-Cys onto the surface of CS through strong Au-S bond interaction. The prepared Fe3O4@CS@Au-l-Cys exhibited strong affinity, low detection limit (0.5 fmol/µL HRP), high selectivity (HRP/BSA with a molar ratio of 1:1000) for glycopeptides. Moreover, successful application of glycopeptide enrichment in human serum and saliva by Fe3O4@CS@Au-l-Cys was achieved. A satisfactory data set indicates that Fe3O4@CS@Au-l-Cys has promising potential in the application of glycopeptide enrichment in real complex bio-samples and for related glycoproteome research.

Methods for quantification of glycopeptides by liquid separation and mass spectrometry.

Recent advances in analytical techniques provide the opportunity to quantify even low-abundance glycopeptides derived from complex biological mixtures, allowing for the identification of glycosylation differences between healthy samples and those derived from disease states. Herein, we discuss the sample preparation procedures and the mass spectrometry (MS) strategies that have facilitated glycopeptide quantification, as well as the standards used for glycopeptide quantification. For sample preparation, various glycopeptide enrichment methods are summarized including the columns used for glycopeptide separation in liquid chromatography separation. For MS analysis strategies, MS1 level-based quantification and MS2 level-based quantification are described, either with or without labeling, where we have covered isotope labeling, TMT/iTRAQ labeling, data dependent acquisition, data independent acquisition, multiple reaction monitoring, and parallel reaction monitoring. The strengths and weaknesses of these methods are compared, particularly those associated with the figures of merit that are important for clinical biomarker studies and the pathological and functional studies of glycoproteins in various diseases. Possible future developments for glycopeptide quantification are discussed.

Data-independent acquisition mass spectrometry for site-specific glycoproteomics characterization of SARS-CoV-2 spike protein.

The spike protein of SARS-CoV-2, the virus responsible for the global pandemic of COVID-19, is an abundant, heavily glycosylated surface protein that plays a key role in receptor binding and host cell fusion, and is the focus of all current vaccine development efforts. Variants of concern are now circulating worldwide that exhibit mutations in the spike protein. Protein sequence and glycosylation variations of the spike may affect viral fitness, antigenicity, and immune evasion. Global surveillance of the virus currently involves genome sequencing, but tracking emerging variants should include quantitative measurement of changes in site-specific glycosylation as well. In this work, we used data-dependent acquisition (DDA) and data-independent acquisition (DIA) mass spectrometry to quantitatively characterize the five N-linked glycosylation sites of the glycoprotein standard alpha-1-acid glycoprotein (AGP), as well as the 22 sites of the SARS-CoV-2 spike protein. We found that DIA compared favorably to DDA in sensitivity, resulting in more assignments of low-abundance glycopeptides. However, the reproducibility across replicates of DIA-identified glycopeptides was lower than that of DDA, possibly due to the difficulty of reliably assigning low-abundance glycopeptides confidently. The differences in the data acquired between the two methods suggest that DIA outperforms DDA in terms of glycoprotein coverage but that overall performance is a balance of sensitivity, selectivity, and statistical confidence in glycoproteomics. We assert that these analytical and bioinformatics methods for assigning and quantifying glycoforms would benefit the process of tracking viral variants as well as for vaccine development.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s00216-021-03643-7.

Post-synthesis of boric acid-functionalized magnetic covalent organic framework as an affinity probe for the enrichment of N-glycopeptides.

A novel type of boric acid-functionalized magnetic covalent organic framework (mCOF) with polyethyleneimine (PEI) as a linker (denoted as mCOF@PEI@B(OH)2) has beenprepared through a post-synthesis strategy, which points out an achievable path for the construction of boronic acid-functionalized COFs. Based on the boric acid chemistry, the obtained core-shell structured mCOF@PEI@B(OH)2 can selectively isolate glycopeptides through the modified boronic acid groups. The mCOF@PEI@B(OH)2 exhibits excellent performance with good reusability (ten cycles), low detection limit (0.5 fmol·μL-1), size-exclusion effect, and relatively high loading capacity (80μg·mg-1), recovery yield (94.9 ± 2.8%), and selectivity (HRP digests:BSA digests = 1:500). Detection is done bymatrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In addition, 37 endogenous glycopeptides are captured from human saliva with mCOF@PEI@B(OH)2, providing effective proofs for its capability to capture low-abundance glycopeptides from actual biological samples.

Recent advances in functionalized magnetic nanomaterials for glycoprotein and glycopeptide enrichment

蛋白质糖基化作为最重要的翻译后修饰之一,在生物体诸如细胞信号转导、蛋白质翻译调控、免疫应答等诸多生命过程中发挥重要作用。此外,蛋白质的异常糖基化还与肿瘤等疾病的发生发展密切相关,这为以糖蛋白为目标的疾病生物标志物的发现提供了可能。尽管质谱已经成为糖蛋白质组学的重要分析工具,但糖肽的低丰度和低电离效率使得其直接质谱分析仍面临挑战。在糖蛋白质组学研究中,从复杂的生物样品中富集糖蛋白和糖肽是重要的环节。磁性固相萃取(MSPE)是一种操作简单、成本低和萃取效率高的样品预处理方法。在磁性固相萃取中,磁性吸附剂是影响萃取效果的关键,将功能化磁性纳米材料作为吸附剂进行糖蛋白质组学研究已经得到广泛应用。该文综述了糖分子、离子液体、凝集素、硼酸亲和配体、金属有机框架、共价有机骨架等功能化磁性纳米材料的制备及其在糖蛋白及糖肽富集中的应用。上述功能化磁性纳米材料具有高比表面积、大量作用位点等特点,其富集机理包括亲水相互作用色谱、凝集素亲和作用色谱、硼酸化学法和肼化学法等,主要应用于血清、血浆、细胞、组织、唾液等样品的糖蛋白和糖肽的富集。该文引用了近十年来发表的约90篇源于科学引文索引(SCI)与中文核心期刊的相关论文,并于文末对磁性纳米材料在糖蛋白和糖肽富集领域的发展趋势进行了展望。

Post-synthesis modification of covalent organic frameworks for ultrahigh enrichment of low-abundance glycopeptides from human saliva and serum

In this study, a novel type of covalent organic framework (COF) material rich in boronic acid sites was prepared through post-synthesis modification (TbBD@PEI@Au@4-MPBA). The surface of COF material had abundant carboxylic acid groups, which could bind a large amount of polyethyleneimine (PEI) through electrostatic interaction. At the same time, the amino groups on the PEI can be grafted with Au nanoparticles (Au NPs) in situ, and then 4-mercaptophenylboronic acid (4-MPBA) was modified by the reaction of Au and sulfhydryl groups. The massive grafting of boronic acid groups made the material's enrichment effect on glycopeptides expected. The results of experiments indicated that the composite material has high sensitivity (5 amol μL−1) and selectivity (1:1000). In addition, the material has outstanding stability and reusability, with a load capacity of about 100 mg g−1 and a recovery of 99.3 ± 2.2%. What's more, after enriched by TbBD@PEI@Au@4-MPBA, 56 endogenous glycopeptides from fresh human saliva were detected by MALDI-TOF MS, 56 unique glycopeptides corresponding to 31 glycoproteins from human saliva and 513 unique glycopeptides corresponding to 208 glycoproteins from serum of throat cancer patient were detected by nano-LC-MS/MS, respectively, which was expected to be applied to glycoproteomics research.

共价有机骨架功能材料及其在糖肽选择性富集中的应用

蛋白质糖基化是生物体中最重要的翻译后修饰手段之一,糖蛋白/糖肽的有效分离和富集成为目前糖蛋白组学研究的首要问题。对于复杂的生物样本,糖蛋白的数量较少,酶解后大量高丰度非糖基化修饰肽的存在,使得低丰度糖肽的检测更加困难。因此,需要一些手段来有效地富集糖肽以提高其检测丰度,发展高选择性的糖肽富集材料及方法就成为在分子水平上有效地监测糖蛋白或糖肽的重要途径。相对于传统的糖肽富集材料,共价有机骨架材料具有比表面积大和可修饰位点丰富的优点,在糖肽富集领域具有很大的应用潜力。该文制备了一种新型的共价有机骨架材料(O-T-D-COFs),利用1,3,5-三(4-氨苯基)苯和2,5-二甲氧基苯-1,4-二甲醛作为反应单体通过共聚缩合反应生成的席夫碱构成了材料的框架,对合成后的中间体材料进行氧化处理,从而提高材料的富集性能。利用扫描电镜、透射电镜、红外光谱和固体核磁等表征技术对材料的结构进行了表征,并将其应用于糖肽的选择性富集。分别对富集过程的上样条件、淋洗条件、洗脱条件进行了优化,结合质谱检测技术,从人血清免疫球蛋白G酶解液中观察到32个明显的糖肽信号峰。通过模拟复杂样本体系验证材料富集选择性,在人血清免疫球蛋白G和牛血清白蛋白的酶解液混合物摩尔比达到1∶50时,该材料仍然保持了良好的选择性。此外,还考察了材料的检测限、富集容量、回收率等富集性能,及在实际样品中的应用潜力。以人血清免疫球蛋白G为评价对象,O-T-D-COFs具有较低的检测限(2.5 fmol/μL)、较高的富集容量(120 mg/g),及较好的富集回收率(103.5%±6.6%、101.5%±10.4%)。在血清样品中富集到来自53个N-糖蛋白中的86个N-糖肽序列,并鉴定到了94个N-糖基化位点。这些结果都表明,该材料在糖肽富集领域有较好的应用前景。

Quantitative clinical glycomics strategies: A guide for selecting the best analysis approach.

Glycans introduce complexity to the proteins to which they are attached. These modifications vary during the progression of many diseases; thus, they serve as potential biomarkers for disease diagnosis and prognosis. The immense structural diversity of glycans makes glycosylation analysis and quantitation difficult. Fortunately, recent advances in analytical techniques provide the opportunity to quantify even low-abundant glycopeptides and glycans derived from complex biological mixtures, allowing for the identification of glycosylation differences between healthy samples and those derived from disease states. Understanding the strengths and weaknesses of different quantitative glycomics analysis methods is important for selecting the best strategy to analyze glycosylation changes in any given set of clinical samples. To provide guidance towards selecting the proper approach, we discuss four widely used quantitative glycomics analysis platforms, including fluorescence-based analysis of released N-linked glycans and three different varieties of MS-based analysis: liquid chromatography (LC)-mass spectrometry (MS) analysis of glycopeptides, matrix-assisted laser desorption ionization-time of flight MS, and LC-ESI-MS analysis of released N-linked glycans. These methods' strengths and weaknesses are compared, particularly associated with the figures of merit that are important for clinical biomarker studies, including: the initial sample requirements, the methods' throughput, sample preparation time, the number of species identified, the methods' utility for isomer separation and structural characterization, method-related challenges associated with quantitation, repeatability, the expertise required, and the cost for each analysis. This review, therefore, provides unique guidance to researchers who endeavor to undertake a clinical glycomics analysis by offering insights on the available analysis technologies.

Facile Synthesis of Hydrophilic Magnetic Mesoporous Silica Microspheres for Selective Enrichment of Glycopeptides and Glycans

Hydrophilic magnetic mesoporous silica microspheres with core-shell structure were prepared via a facile approach and applied as an enrichment probe for low-abundance glycopeptides and glycans from complex biological samples. The prepared mesoporous silica microspheres modified with gluconamide groups at the interior surface of mesopore channels showed more prominent features with respect to high specific surface area (490 m2/g), narrow pore size distribution (2.5 nm), and high saturation magnetization (23.87 emu/g), endowing them with hydrophilicity and size-exclusion selectivity. Taking these advantages of the size-exclusion and the hydrophilic interactions, the hydrophilic mesoporous magnetic microspheres exhibited specificity for glycopeptides in a digest of horseradish peroxidase. The detection limit for horseradish peroxidase was 25 fmol. In addition, the hydrophilic mesoporous magnetic microspheres were further applied to the selective enrichment of N-linked glycans from a digest of human serum. The successful application demonstrates the potential of the hydrophilic mesoporous magnetic microspheres in glycoproteomic analysis.

Porous graphene oxide/chitosan beads with honeycomb-biomimetic microchannels as hydrophilic adsorbent for the selective capture of glycopeptides.

A porous hydrophilic affinity bead consisting of graphene oxide and chitosan (pGC) with the honeycomb-biomimetic microchannels has beensynthesized and applied as hydrophilic adsorbent for selective capture of glycopeptides. The pGC beads have open-porous structure, honeycomb-like microchannels, large interior voids, and hydrophilic property. Based on the multivalent hydrophilic interactions between glycan moieties on glycopeptides and amino groups and hydroxyl groups on chitosan, the glycopeptides were enriched and separated by pGC beads. The pGC beads exhibit high sensitivity (detection limit, 5fmol), binding capacity (111.1mg/g), enrichment selectivity (molar ratio of human IgG to BSA tryptic digests of 1:200), and recovery yield (89.78%). By combing pGC beads and nano LC-MS/MS analysis, a total of 325 N-glycosylated peptides corresponding to 152 N-glycosylated proteins were identified from 2μL human serum. These experimental results demonstrate the practical application of the method in glycoproteomics research. Graphical abstract Schematic representation of fabrication for porous hydrophilic affinity beads (pGC) with honeycomb-biomimetic microchannels based on graphene oxide (GO) and chitosan (CS). The pGC was successfully applied to capturing and identifying low-abundant glycopeptides from biological samples.

Selective enrichment of glycopeptides based on copper tetra(N-carbonylacrylic) aminephthalocyanine and iminodiacetic acid functionalized polymer monolith.

Efficient separation and enrichment of low-abundance glycopeptides from complex biological samples is the key to the discovery of disease biomarkers. In this work, a new material was prepared by coating copper tetra(N-carbonylacrylic) aminephthalocyanine and iminodiacetic acid onto poly(glycidyl methacrylate-pentaerythritol triacrylate) monolith. The monolith was applied to polymer monolithic microextraction for specific capture of glycopeptides coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The developed monolith exhibited satisfactory efficiency for glycopeptide enrichment with high selectivity and detection sensitivity. When the tryptic digest of immunoglobulin G was used as the sample, total 24 glycopeptides were identified and the detection limit was determined as 5fmol. When the approach was applied to the analysis of glycopeptides in the mixture of bovine serum albumin and immunoglobulin G (100:1, m/m) digests, 16 glycopeptides could still be observed. Moreover, the monolith was successfully applied to the selective enrichment of glycopeptides from human serum digests, exhibiting great practicability in identifying low-abundance glycopeptides in complex biological samples.

An enrichment method based on synergistic and reversible covalent interactions for large-scale analysis of glycoproteins

Protein glycosylation is ubiquitous in biological systems and essential for cell survival. However, the heterogeneity of glycans and the low abundance of many glycoproteins complicate their global analysis. Chemical methods based on reversible covalent interactions between boronic acid and glycans have great potential to enrich glycopeptides, but the binding affinity is typically not strong enough to capture low-abundance species. Here, we develop a strategy using dendrimer-conjugated benzoboroxole to enhance the glycopeptide enrichment. We test the performance of several boronic acid derivatives, showing that benzoboroxole markedly increases glycopeptide coverage from human cell lysates. The enrichment is further improved by conjugating benzoboroxole to a dendrimer, which enables synergistic benzoboroxole–glycan interactions. This robust and simple method is highly effective for sensitive glycoproteomics analysis, especially capturing low-abundance glycopeptides. Importantly, the enriched glycopeptides remain intact, making the current method compatible with mass-spectrometry-based approaches to identify glycosylation sites and glycan structures.

A designable aminophenylboronic acid functionalized magnetic Fe3O4/ZIF-8/APBA for specific recognition of glycoproteins and glycopeptides.

We fabricated a novel aminophenylboronic acid functionalized magnetic Fe3O4/zeolitic imidazolate framework-8/APBA (denoted as Fe3O4/ZIF-8/APBA). First, Fe3O4 was coated by zeolitic imidazolate framework-8 (denoted as Fe3O4/ZIF-8) using the hydrothermal method. Next, the phenylboronic acid functionalized triethoxysilane reagent was synthesized by 3-aminophenylboronic acid and 3-isocyanatopropyltriethoxysilane, which was modified on the surface of the Fe3O4/ZIF-8 nanocomposite through the sol–gel technique and electrostatic interaction as well as π–π stacking interaction. The synthetic Fe3O4/ZIF-8/APBA exhibited high adsorption capacity and good specificity toward glycoproteins. Moreover, the Fe3O4/ZIF-8/APBA possessed high saturation magnetization (51.41 emu g−1) and achieved better separation in the presence of an external magnetic field. Above all, the as-designed Fe3O4/ZIF-8/APBA was successfully used to capture glycoproteins and identify the horseradish peroxidase (HRP) tryptic digest. This study provides a facile strategy to embellish the aminophenylboronic acid onto the nanocomposite substrate and develop a new material for the specific recognition and enrichment of glycoproteins and low-abundance glycopeptides in proteomics research.

Glucose-6-Phosphate-Functionalized Magnetic Microsphere as Novel Hydrophilic Probe for Specific Capture of N-Linked Glycopeptides.

Developing cost-effective approaches based on hydrophilic interaction liquid chromatography (HILIC) has been the main tendency for low-abundance glycopeptides capture before LC-MS/MS analysis. Carbohydrates with outstanding biocompatibility and hydrophilicity are ubiquitous in the kingdoms of animal and plant and could be a wonderful choice as functional groups for glycopeptides enrichment. In this work, glucose-6-phosphate, as one of the indispensable cogs in pivotal metabolic wheels of life, was chosen as functionalized groups to be grafted onto the surface of Fe3O4 microspheres via one-step surface fabrication strategy. The acquired hydrophilic Fe3O4@G6P microspheres showed superior enrichment performance for glycopeptides with high sensitivity (0.5 fmol/μL) and high selectivity (1:100) and good repeatability (10 times at least). Furthermore, the Fe3O4@G6P microspheres also exhibited enrichment ability for glycopeptides in different biosamples. A total of 243 glycopeptides assigned to 92 glycoproteins and 183 glycopeptides corresponding to 74 different glycoproteins was identified from merely 2 μL of serum and saliva, respectively.

Efficient enrichment of glycopeptides with sulfonic acid-functionalized mesoporous silica

This work presents an efficient and selective enrichment method for glycoprotein/glycopeptides with sulfonic acid-functionalized mesoporous silica (SBA-15-SO3H), which is synthesized via simple oxidation of -SH groups with H2O2. The functionalized SBA-15 shows large surface area and accessible pores, and can selectively adsorb glycopeptides via hydrogen bond and hydrophilic interaction. Upon the selective enrichment prior to the mass spectrometric (MS) analysis, the signals of glycopeptides are significantly enhanced, which leads to the identifiable signals of 21 glycopeptides from the digest of HRP, 16 glycopeptides from the digest of human IgG, and 16 glycopeptides from the digest of chicken avidin. The SBA-15-SO3H gives significant selectivity for glycopeptides even at a low molar ratio of glycopeptides to nonglycopeptides with an enrichment time of 15min. Therefore, this work provides a powerful material for selective enrichment and identification of low abundant glycopeptides in glycoproteomic analysis.

Dipeptide-Based Carbohydrate Receptors and Polymers for Glycopeptide Enrichment and Glycan Discrimination.

Glycoproteomics identifies and catalogs protein glycosylation and explores its impact on protein conformations and biofunctions. However, these studies are restricted by the bottleneck to enrich low-abundance glycopeptides from complex biosamples and the difficulties in analyzing glycan structures by mass spectrometry. Here, we report dipeptide as a simple but promising carbohydrate binding platform to tackle these problems. We build a hydropathy-index-based strategy for sequence optimization and screen out three optimal dipeptide sequences from 54 types of dipeptides. The optimized dipeptide-based homopolymers display excellent performance (e.g., selectivity up to ∼70% for real biosamples and strong anti-interference capacity capable of resisting 1000-fold bovine serum albumin interference) in glycopeptide enrichment. Meanwhile, our polymers exhibit high-efficiency chromatographic separation toward oligosaccharides with different compositions, polymerization degrees and even their linkage isomers. This brings another attractive feature that our materials can discriminate subtly variable glycan structures of glycopeptides, especially, isomeric glycosidic linkages. These features provide a solid foundation to analyze the complex glycan structures and glycosites simultaneously, which will benefit future development of glycoproteomics and glycobiology.

Facile synthesis of carboxymethyl-β-cyclodextrin conjugated magnetic nanoparticles for selective enrichment of glycopeptides.

Selective enrichment of glycopeptides prior to mass spectrometry (MS) analysis is essential due to the low abundance of the modified glycopeptides in complex samples, ion suppression effects during MS ionization and detection caused by the co-presence of non-glycosylated peptides, etc. Among different enrichment approaches, hydrophilic interaction liquid chromatography (HILIC)-based magnetic separation has become one of the most popular methods in recent years, due to its high efficiency and selectivity for glycopeptide enrichment. Herein, novel carboxymethyl-β-cyclodextrin (CMCD)-modified magnetic nanoparticles (MNPs) were synthesized via a carbodiimide activation method. CMCD was covalently bonded with the -OH group on the surface of MNPs through carbodiimide, and the proposed procedure provides a rapid and efficient alternative for glycopeptide enrichment due to its stable interaction, time-saving, and easy operation. The prepared absorbents with a mean diameter of 15nm demonstrated a strong magnetic response to an externally applied magnetic field. The results of thermogravimetric analysis showed the content of bound CMCD was 3wt%. The outer CMCD layer conjugated on the Fe3 O4 core showed high hydrophilic surface property. In the analysis of a complex mouse liver sample, a total of 666 unique N-glycosylation sites corresponding to 494 glycosylated proteins were identified successfully. The study demonstrated an easy-to-use CMCD-modified MNPs-based approach with high selectivity and high capacity in the enrichment of low-abundance glycopeptides from complex biological samples. Copyright © 2016 John Wiley & Sons, Ltd.