Enrichment Of Glycopeptides Research Articles

Oriental covalent immobilization of N-glycan binding protein via N-terminal selective modification

Lectin affinity chromatography is one of powerful tools for the study of protein glycosylation. Different lectin proteins can recognize different structures of monosaccharides or oligosaccharide units, allowing for the selective separation of glycopeptides or glycoproteins containing different polysaccharide structures. However, the N-glycans were only partially captured by most of common lectins, reducing the coverage rate of identifying N-glycoconjugates. Recently, it has been reported that the engineering variant of glycan binding protein Fbs1 has a high affinity for innermost Man3GlcNAc2 structure and is able to bind diverse types of N-glycans, which can be suitable for the analysis of protein N-glycosylation. However, efficient immobilization of protein to separation matrix is particularly challenging as it requires the functionality and integrity of the protein to be preserved. Herein, we describe a simple and robust strategy for oriental covalent immobilization of proteins on magnetic nanoparticles by N-terminal selective labeling techniques. We inserted the enterokinase cleavage site to produce the specific N- terminal glycine of protein. Under physiological conditions, the protein was immobilized on the surface of the MNPs by this glycine tag, and the enrichment process could be completed within 30 min. A whole enrichment and purification of glycan and glycopeptides were completed and analyzed by MALDI TOF-MS. The functional materials achieved stable enrichment of glycan structure in different enzyme digestion systems or complex samples, showing excellent anti-interference and applicability.

Rapid and large-scale glycopeptide enrichment strategy based on chemical ligation.

Protein glycosylation, the most universal post-translational modification, is thought to play a crucial role in regulating multiple essential cellular processes. However, the low abundance of glycoproteins and the heterogeneity of glycans complicate their comprehensive analysis. Here, we develop a rapid and large-scale glycopeptide enrichment strategy via bioorthogonal ligation and trypsin cleavage. The enrichment process is performed in one tube to minimize sample loss and time costs. This method combines convenience and practicality, identifying over 900 O-GlcNAc sites from a 500μg sample. Surprisingly, it allows simultaneous identification of N-glycosites, O-GlcNAc sites, O-GalNAc sites and N-glycans via a two-step enzymatic release strategy. Combined with quantitative analysis, it reveals the distinct O-GlcNAcylation patterns in different compartments during oxidative stress. In summary, our study offers a convenient and robust tool for glycoproteome and glycome profiling, facilitating in-depth analysis to elucidate the biological functions of glycosylation.

Hydrophilic materials based on hydrogel polymers for enrichment of glycopeptides from serum of colorectal cancer patients.

In this work, a composite hydrogel material consisting of chitosan-based composite hydrogel was prepared by a simple and rapid synthetic method and will be named three-dimensional (3D)-IL-COF-1@CS hydrogel. Possessing a stable 3D network structure and outstanding hydrophilicity, the novel hydrogel is capable of capturing glycopeptides. The 3D-IL-COF-1@CS hydrogel showed good sensitivity (0.1 fmol/µL) and selectivity (1:2000). In addition, 19 glycopeptides were captured in standard samples. In the analysis of human serum, 148 glycopeptides assigned to 72 glycoproteins were assayed in the serum of normal individuals, and 245 glycopeptides corresponding to 100 glycoproteins were found in the serum of colorectal cancer (CRC) patients. More importantly, several functional programs based on Gene Ontology analysis supported molecular biological processes that may be relevant to the pathogenesis of CRC, including aging, fibrinogen complex, and arylesterase activity. The low cost, simplicity, rapid synthesis, and good enrichment performance have a great future in glycoproteomics analysis and related diseases.

Advanced functional materials as reliable tools for capturing food-derived peptides to optimize the peptidomics pre-treatment enrichment workflow.

Peptidomics strategies with high throughput, sensitivity, and reproducibility are key tools for comprehensively analyzing peptide composition and potential functional activities in foods. Nevertheless, complex signal interference, limited ionization efficiency, and low abundance have impeded food-derived peptides' progress in food detection and analysis. As a result, novel functional materials have been born at the right moment that could eliminate interference and perform efficient enrichment. Of note, few studies have focused on developing peptide enrichment materials for food sample analysis. This work summarizes the development of endogenous peptide, phosphopeptide, and glycopeptide enrichment utilizing materials that have been employed extensively recently: organic framework materials, carbon-based nanomaterials, bio-based materials, magnetic materials, and molecularly imprinted polymers. It focuses on the limitations, potential solutions, and future prospects for application in food peptidomics of various advanced functional materials. The size-exclusion effect of adjustable aperture and the modification of magnetic material enhanced the sensitivity and selectivity of endogenous peptide enrichment and aided in streamlining the enrichment process and cutting down on enrichment time. Not only that, the immobilization of metal ions such as Ti4+ and Nb5+ enhanced the capture of phosphopeptides, and the introduction of hydrophilic groups such as arginine, L-cysteine, and glutathione into bio-based materials effectively optimized the hydrophilic enrichment of glycopeptides. Although a portion of the carefully constructed functional materials currently only exhibit promising applications in the field of peptide enrichment for analytical chemistry, there is reason to believe that they will further advance the field of food peptidomics through improved pre-treatment steps.

One-step fabrication of dipeptide-based bifunctional polymer for individual enrichment of glycopeptides and phosphopeptides from serum

A dipeptide-based bifunctional material immobilized with Ti4+ (denoted as APE-MBA-VPA-Ti4+) was developed using precipitation polymerization. This polymer combines hydrophilic interaction liquid chromatography (HILIC) and immobilized metal affinity chromatography (IMAC) enrichment strategies, allowing for the individual and simultaneous enrichment of glycopeptides and phosphopeptides. It demonstrated high sensitivity (0.1 fmol μL−1 for glycopeptides, 0.005 fmol μL−1 for phosphopeptides), strong selectivity (molar ratio HRP: BSA = 1:1000, β-casein: BSA = 1:2500), consistent reusability (10 cycles) and satisfactory recovery rate (93.5 ± 1.8 % for glycopeptides, 91.6 ± 0.6 % for phosphopeptides) in the individual enrichment. Utilizing nano LC-MS/MS technology, the serum of liver cancer patients was analyzed after enrichment individually, resulting in the successful capture of 333 glycopeptides covering 262 glycosylation sites, corresponding to 131 glycoproteins, as well as 67 phosphopeptides covering 57 phosphorylation sites, related to 48 phosphoproteins. In comparison, the serum of normal healthy individuals yielded a total of 283 glycopeptides covering 244 glycosylation sites corresponding to 126 glycoproteins, as well as 66 phosphopeptides covering 56 phosphorylation sites related to 37 phosphoproteins. Label-free quantification identified 10 differentially expressed glycoproteins and 8 differentially expressed phosphoproteins in the serum of liver cancer patients. Among them, glycoproteins (HP, BCHE, AGT, C3, and PROC) and phosphoproteins (ZYX, GOLM1, GP1BB, CLU, and TNXB) showed upregulation and displayed potential as biomarkers for liver cancer.

Data Independent Acquisition Mass Spectrometry Enhanced Personalized Glycosylation Profiling of Haptoglobin in Hepatocellular Carcinoma.

Aberrant glycosylation has gained significant interest for biomarker discovery. However, low detectability, complex glycan structures, and heterogeneity present challenges in glycoprotein assay development. Using haptoglobin (Hp) as a model, we developed an integrated platform combining functionalized magnetic nanoparticles and zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) for highly specific glycopeptide enrichment, followed by a data-independent acquisition (DIA) strategy to establish a deep cancer-specific Hp-glycosylation profile in hepatitis B virus (HBV, n = 5) and hepatocellular carcinoma (HCC, n = 5) patients. The DIA strategy established one of the deepest Hp-glycosylation landscapes (1029 glycopeptides, 130 glycans) across serum samples, including 54 glycopeptides exclusively detected in HCC patients. Additionally, single-shot DIA searches against a DIA-based spectral library outperformed the DDA approach by 2-3-fold glycopeptide coverage across patients. Among the four N-glycan sites on Hp (N-184, N-207, N-211, N-241), the total glycan type distribution revealed significantly enhanced detection of combined fucosylated-sialylated glycans, which were the most dominant glycoforms identified in HCC patients. Quantitation analysis revealed 48 glycopeptides significantly enriched in HCC (p < 0.05), including a hybrid monosialylated triantennary glycopeptide on the N-184 site with nearly none-to-all elevation to differentiate HCC from the HBV group (HCC/HBV ratio: 2462 ± 766, p < 0.05). In summary, DIA-MS presents an unbiased and comprehensive alternative for targeted glycoproteomics to guide discovery and validation of glyco-biomarkers.

Glycan-Tailored Glycoproteomic Analysis Reveals Serine is the Sole Residue Subjected to O-Linked Glycosylation in Acinetobacter baumannii.

Protein glycosylation is a ubiquitous process observed across all domains of life. Within the human pathogen Acinetobacter baumannii, O-linked glycosylation is required for virulence; however, the targets and conservation of glycosylation events remain poorly defined. In this work, we expand our understanding of the breadth and site specificity of glycosylation within A. baumannii by demonstrating the value of strain specific glycan electron-transfer/higher-energy collision dissociation (EThcD) triggering for bacterial glycoproteomics. By coupling tailored EThcD-triggering regimes to complementary glycopeptide enrichment approaches, we assessed the observable glycoproteome of three A. baumannii strains (ATCC19606, BAL062, and D1279779). Combining glycopeptide enrichment techniques including ion mobility (FAIMS), metal oxide affinity chromatography (titanium dioxide), and hydrophilic interaction liquid chromatography (ZIC-HILIC), as well as the use of multiple proteases (trypsin, GluC, pepsin, and thermolysis), we expand the known A. baumannii glycoproteome to 33 unique glycoproteins containing 42 glycosylation sites. We demonstrate that serine is the sole residue subjected to glycosylation with the substitution of serine for threonine abolishing glycosylation in model glycoproteins. An A. baumannii pan-genome built from 576 reference genomes identified that serine glycosylation sites are highly conserved. Combined this work expands our knowledge of the conservation and site specificity of A. baumannii O-linked glycosylation.

A Type of Ferrocene-Based Derivative FE-1 COF Material for Glycopeptide and Phosphopeptide Selective Enrichment.

In this work, a new type of FE-1 COF material is prepared by a reversible imine condensation reaction with diaminoferrocene and diaminodiformaldehyde as materials. The material is connected by imine bonds to form a COF skeleton, and the presence of plenty of nitrogen-containing groups gives the material good hydrophilicity; the presence of metal Fe ions provides the material application potential in the enrichment of phosphopeptides. According to the different binding abilities of N-glycopeptide and phosphopeptide on FE-1 COF, it can simultaneously enrich N-glycopeptide and phosphopeptide through different elution conditions to realize its controllable and selective enrichment. Using the above characteristics, 18 phosphopeptides were detected from α-casein hydrolysate, 8 phosphopeptides were detected from β-casein hydrolysate and 21 glycopeptides were detected from IgG hydrolysate. Finally, the gradual elution strategy was used; 16 phosphopeptides and 19 glycopeptides were detected from the α-casein hydrolysate and IgG hydrolysate. The corresponding glycopeptides and phosphopeptides were identified from the human serum. It proves that the FE-1 COF material has a good enrichment effect on phosphopeptides and glycopeptides.

Defining albumin as a glycoprotein with multiple N-linked glycosylation sites

BackgroundGlycosylation is an enzyme-catalyzed post-translational modification that is distinct from glycation and is present on a majority of plasma proteins. N-glycosylation occurs on asparagine residues predominantly within canonical N-glycosylation motifs (Asn-X-Ser/Thr) although non-canonical N-glycosylation motifs Asn-X-Cys/Val have also been reported. Albumin is the most abundant protein in plasma whose glycation is well-studied in diabetes mellitus. However, albumin has long been considered a non-glycosylated protein due to absence of canonical motifs. Albumin contains two non-canonical N-glycosylation motifs, of which one was recently reported to be glycosylated.MethodsWe enriched abundant serum proteins to investigate their N-linked glycosylation followed by trypsin digestion and glycopeptide enrichment by size-exclusion or mixed-mode anion-exchange chromatography. Glycosylation at canonical as well as non-canonical sites was evaluated by liquid chromatography–tandem mass spectrometry (LC–MS/MS) of enriched glycopeptides. Deglycosylation analysis was performed to confirm N-linked glycosylation at non-canonical sites. Albumin-derived glycopeptides were fragmented by MS3 to confirm attached glycans. Parallel reaction monitoring was carried out on twenty additional samples to validate these findings. Bovine and rabbit albumin-derived glycopeptides were similarly analyzed by LC–MS/MS.ResultsHuman albumin is N-glycosylated at two non-canonical sites, Asn68 and Asn123. N-glycopeptides were detected at both sites bearing four complex sialylated glycans and validated by MS3-based fragmentation and deglycosylation studies. Targeted mass spectrometry confirmed glycosylation in twenty additional donor samples. Finally, the highly conserved Asn123 in bovine and rabbit serum albumin was also found to be glycosylated.ConclusionsAlbumin is a glycoprotein with conserved N-linked glycosylation sites that could have potential clinical applications.

A Fully Automated Online Enrichment and Separation System for Highly Reproducible and In-Depth Analysis of Intact Glycopeptide.

A fully automated online enrichment and separation system for intact glycopeptides, named AutoGP, was developed in this study by integrating three different columns in a nano-LC system. Specifically, the peptide mixture from the enzymatic digestion of a complex biological sample was first loaded on a hydrophilic interaction chromatography (HILIC) column. The nonglycopeptides in the sample were washed off the column, and the glycopeptides retained by the HILIC column were eluted to a C18 trap column to achieve an automated glycopeptide enrichment. The enriched glycopeptides were further eluted to a C18 column for separation, and the separated glycopeptides were eventually analyzed by using an orbitrap mass spectrometer (MS). The optimal operating conditions for AutoGP were systemically studied, and the performance of the fully optimized AutoGP was compared with a conventional manual system used for glycopeptide analysis. The experimental evaluation shows that the total number of glycopeptides identified is at least 1.5-fold higher, and the median coefficient of variation for the analyses is at least 50% lower by using AutoGP, as compared to the results acquired by using the manual system. In addition, AutoGP can perform effective analysis even with a 1-μg sample amount, while a 10-μg sample at least will be needed by the manual system, implying an order of magnitude better sensitivity of AutoGP. All the experimental results have consistently proven that AutoGP can be used for much better characterization of intact glycopeptides.

O-linked glycosylations in human milk casein and major whey proteins during lactation

As glycosylations are difficult to analyze, their roles and effects are poorly understood. Glycosylations in human milk (HM) differ across lactation. Glycosylations can be involved in antimicrobial activities and may serve as food for beneficial microorganisms. This study aimed to identify and analyze O-linked glycans in HM by high-throughput mass spectrometry. 184 longitudinal HM samples from 66 donors from day 3 and months 1, 2, and 3 postpartum were subjected to a post-translational modification specific enrichment-based strategy using TiO2 and ZrO2 beads for O-linked glycopeptide enrichment. β-CN was found to be a major O-linked glycoprotein, additionally, αS1-CN, κ-CN, lactotransferrin, and albumin also contained O-linked glycans. As glycosyltransferases and glycosidases are involved in assembling the glycans including O-linked glycosylations, these were further investigated. Some glycosyltransferases and glycosidases were found to be significantly decreasing through lactation, including two O-linked glycan initiator enzymes (GLNT1 and GLNT2). Despite their decrease, the overall level of O-linked glycans remained stable in HM over lactation. Three different motifs for O-linked glycosylation were enriched in HM proteins: Gly-Xxx-Xxx-Gly-Ser/Thr, Arg-Ser/Thr and Lys-Ser/Thr. Further O-linked glycan motifs on β-CN were observed to differ between intact proteins and endogenous peptides in HM.

Global O-glycoproteome enrichment and analysis enabled by a combinatorial enzymatic workflow

A comprehensive analysis of site-specific protein O-glycosylation is hindered by the absence of a consensus O-glycosylation motif, the diversity of O-glycan structures, and the lack of a universal enzyme that cleaves attached O-glycans. Here, we report the development of a robust O-glycoproteomic workflow for analyzing complex biological samples by combining four different strategies: removal of N-glycans, complementary digestion using O-glycoprotease (IMPa) with/without another protease, glycopeptide enrichment, and mass spectrometry with fragmentation of glycopeptides using stepped collision energy. Using this workflow, we cataloged 474 O-glycopeptides on 189 O-glycosites derived from 79 O-glycoproteins from human plasma. These data revealed O-glycosylation of several abundant proteins that have not been previously reported. Because many of the proteins that contained unannotated O-glycosylation sites have been extensively studied, we wished to confirm glycosylation at these sites in a targeted fashion. Thus, we analyzed selected purified proteins (kininogen-1, fetuin-A, fibrinogen, apolipoprotein E, and plasminogen) in independent experiments and validated the previously unknown O-glycosites.

The potential of polyaniline-coated stationary phase in hydrophilic interaction liquid chromatography-based solid-phase extraction for glycopeptide enrichment

Glycopeptide enrichment is a crucial step in glycoproteomic analysis, often achieved through solid-phase extraction (SPE) on polar stationary phases in hydrophilic interaction liquid chromatography (HILIC). This study explores the potential of polyaniline (PANI)-coated silica gel for enriching human immunoglobulin G (IgG). Experimental conditions were varied to assess their impact on glycopeptide enrichment efficiency, comparing PANI-cotton wool SPE with conventional cotton wool as SPE sorbents. Two formic acid concentrations (0.1% and 1%) in elution solvent were tested, revealing that higher concentrations led to earlier elution of studied glycopeptides, especially for sialylated glycopeptides. Substituting formic acid with acetic acid increased the interaction of neutral glycopeptides with the PANI-modified sorbent, while sialylated glycopeptides showed no significant change in enrichment efficiency. Acetonitrile concentration in the elution solvent (5%, 10%, and 20%) affected the enrichment efficiency with most glycopeptides eluting at the lowest acetonitrile concentration. The acetonitrile concentration in conditioning and washing solutions (65%, 75%, and 85%) played a crucial role; at 65% acetonitrile, glycopeptides were least retained on the stationary phase, and neutral glycopeptides were even detected in the flow-through fraction. This study shows the potential of in-house-prepared PANI-modified sorbents for SPE-HILIC glycopeptide enrichment, highlighting the crucial role of tuning experimental conditions in sample preparation to enhance enrichment efficiency and selectivity.

IgG glycopeptide enrichment using hydrophilic interaction chromatography-based solid-phase extraction on anaminopropyl column.

The sample preparation step is pivotal in glycoproteomic analysis. An effective approach in glycoprotein sample preparation involves enriching glycopeptides by solid-phase extraction (SPE) using polar stationary phases in hydrophilic interaction liquid chromatography (HILIC) mode. The aim of this work is to show how different experimental conditions influence the enrichment efficiency of glycopeptides from human immunoglobulin G (IgG) on an aminopropyl-modified SPE column. Different compositions of the elution solvent (acetonitrile, methanol, and isopropanol), along with varying concentrations of elution solvent acidifiers (formic and acetic acid), and different concentrations of acetonitrile for the conditioning and washing solvents (65%, 75%, and 85% acetonitrile) were tested to observe their effects on the glycopeptide enrichment process. Isopropanol proved less effective in enriching glycopeptides, while acetonitrile was the most efficient, with methanol in between. Higher formic acid concentrations in the elution solvent weakened the ionic interactions, particularly with sialylated glycopeptides. Substituting formic acid with acetic acid led to earlier elution of more glycopeptides. The acetonitrile concentration in conditioning and washing solutions played a key role; at 65% acetonitrile, glycopeptides were not retained on the SPE column and were detected in the flow-through fraction. Ultimately, it was proven that the enrichment method was applicable to human plasma samples, resulting in a significant decrease in the abundances of non-glycosylated peptides. To the best of our knowledge, this study represents the first systematic investigation into the impact of the mobile phase on glycopeptide enrichment using an aminopropyl-modified SPE column in HILIC mode. This study demonstrates the substantial impact of even minor variations in experimental conditions, which havenot yet beenconsidered in the literature, on SPE-HILIC glycopeptide enrichment. Consequently, meticulous optimization of these conditions is imperative to enhance the specificity and selectivity of glycoproteomic analysis, ensuring accurate and reliable quantification.

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.

Comparative Analysis of Site-Specific N-glycosylation of LAMP1 from Breast Cancer Tissues.

Glycosylation changes in cancer proteins have been associated with malignant transformation. However, techniques for analyzing site-specific glycosylation changes in target proteins obtained from clinical tissue samples are insufficient. To overcome these problems, we developed a targeted N-glycoproteomic approach consisting of immunoprecipitation, glycopeptide enrichment, LC/MS/MS and structural assignment using commercially available analytical software followed by manual confirmation. This approach was applied to the comparative site-specific glycosylation analysis of lysosome-associated membrane glycoprotein 1 (LAMP1) between breast cancer (BC) tumors and normal tissues adjacent to tumors. Extensive determination of glycan heterogeneity from four N-glycosylation sites (Asn84/103/249/261) in LAMP1 identified 262 glycoforms and revealed remarkable diversity in tumor glycan structures. A significant increase in N-glycoforms with multiple fucoses and sialic acids at Asn84/249 and high-mannose-type glycans at Asn103/261 were observed in the tumor. Principal component analysis revealed that tumors of different subtypes have independent distributions. This approach enables site-specific glycopeptide analysis of target glycoprotein in breast cancer tissue and become a powerful tool for characterizing tumors with different pathological features by their glycan profiles.

Construction of a hydrophilic porphyrin-based MOF@COF hybrid via post-synthetic modification for N-glycopeptides analysis in human serum.

A hybrid material, M@C-Br@GMA@Glu, consisting of a porphyrin-based metal-organic framework-covalent organic framework (MOF@COF), has been meticulously synthesized using a post-synthetic modification approach. This advanced material has demonstrated exceptional effectiveness in glycopeptide enrichment, characterized by an impressively low detection limit (0.2 fmol μL-1), high selectivity (1 : 2000), and loading capacity for glycopeptides (100 mg g-1). In practical applications with complex biological samples, 210 glycopeptides associated with 87 glycoproteins from the serum of the healthy group, and 156 glycopeptides related to 85 glycoproteins from the serum of cervical cancer were identified after enrichment with M@C-Br@GMA@Glu. Subsequent genetic ontology analysis has elucidated the relationship between cervical cancer and glycosylation, focusing on biological processes such as complement activation, innate immune response mechanisms, and the structural dynamics of the extracellular matrix. The collective findings not only validate the material's proficiency in the sensitive and selective enrichment of glycopeptides, but also underscore its potential in biomarker discovery applications.

Effective enrichment of glycated proteome using ultrasmall gold nanoclusters functionalized with boronic acid.

Glycated proteins play a crucial role in various biological pathways and the pathogenesis of human diseases. A comprehensive analysis of glycated proteins is essential for understanding their biological significance. However, their low abundance and heterogeneity in complex biological samples necessitate an enrichment procedure prior to their detection. Current enrichment strategies primarily rely on the boronic acid (BA) affinity method combined with functional nanoparticles; however, the effectiveness of these approaches is often suboptimal. In this study, a novel nanocluster (NC)-based enrichment material was synthesized for the first time, characterized as Au22SG18 functionalized with 24 BA groups, in which SG is glutathione. The functionalized BA established a reversible covalent bond with the cis-dihydroxy group through pH adjustment, enabling selective enrichment of glycated peptides. After the optimization of the enrichment protocol, we demonstrated highly sensitive and selective enrichment of standard glycopeptides using the NC-based enrichment material, exhibiting excellent reusability. Efficient enrichment was also demonstrated for the glycated proteome from human serum. These results highlight the potential of the atomically well-defined ultrasmall Au NCs as a powerful tool for high-throughput analysis of glycated peptides.

Identification of Core-Fucosylated Glycoproteins by Single-Step Truncation of N-Glycans.

Alpha-1,6 core fucosylation (CF) is a unique glycoform of N-glycans, and studies showed that CF modifications are involved in the occurrence and progression of various diseases and may provide potential disease biomarkers. Current strategies for the CF glycoproteome are often based on multistep enrichment of glycoproteins or glycopeptides and sequential cleavage with different glycosidases to truncate the N-glycans. Although the detection ability of low-abundance glycoproteins is improved, sample loss, high cost, and the time-consuming multistep operation also affect the reproducibility of results and the practicality of the method. Here we developed a single-step truncation (SST) strategy and evaluated its potential for the CF glycoproteome of human serum. The SST strategy has the advantages of fewer operational steps, lower cost, higher number of identifications, and better quantitative stability compared with previous approaches and provides an efficient solution for large-scale quantitative analysis of the CF glycoproteome. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Single-step truncation strategy for core fucosylation glycoproteome analysis in human serum Basic Protocol 2: Liquid chromatography-tandem mass spectrometry quantification of site-specific core fucosylation glycopeptides Alternate Protocol: Pretreatment of cellular samples of core fucosylation glycoproteome with single-step truncation strategy.

Facile preparation of nitrogen/titanium-rich porous organic polymers for specific enrichment of N-glycopeptides and phosphopeptides.

The comprehensive investigation of protein phosphorylation and glycosylation aids in the discovery of novel biomarkers as well as the understanding of the pathophysiology of illness. In this work, a nitrogen/titanium-rich porous organic polymer was developed by copolymerizing carbohydrazide (CH) and 2,3-dihydroxyterephthalaldehyde (2,3-Dha) and modifying with Ti4+ (CH-Dha-Ti4+). The adequate nitrogen contributes to the enrichment of glycopeptides via HILIC, while titanium benefits from capturing phosphopeptides through IMAC. The proposed method exhibits excellent selectivity (1 : 1000, both for glycopeptides and phosphopeptides), LOD (for glycopeptides: 0.05 fmol μL-1, for phosphopeptides: 0.2 fmol), loading capacity (for glycopeptides: 100 mg g-1, for phosphopeptides: 125 mg g-1) and size-exclusion effect (1 : 10 000, both for glycopeptides and phosphopeptides). Furthermore, CH-Dha-Ti4+ was applied to capture glycopeptides and phosphopeptides from human serum; 205 glycopeptides and 45 phosphopeptides were detected in the serum of normal controls; and 294 glycopeptides and 63 phosphopeptides were found in the serum of uremia patients after being analyzed by nano LC-MS/MS. The discovered glycopeptides and phosphopeptides were involved in several molecular biological processes and activities, according to a gene ontology study.