Search

N-glycopeptide is considered as one of significant biomarkers which provide guidance for the diagnosis and drug design of diseases. However, the direct analysis of N-glycopeptides is nearly impracticable mainly owing to their extremely low abundance and grave signal suppression from other interfering substances in the bio-samples. In this research, a multiply-mesoporous hydrophilic TiO 2 nanohybrid (mM-TiO 2 @Cys) was synthesized by immobilizing Cys on a TiO 2 substrate with hierarchical mesopores to achieve the highly-performed enrichment of N-glycopeptides. With the advantages of superior hydrophilicity and multiply-mesoporous structure, the obtained material exhibited an excellent selectivity (IgG digests and BSA digests at the molar ratio of 1/500), a high sensitivity (1 fmol μL -1 for IgG digests) and a good size-exclusion ability (IgG digests, IgG and BSA at the molar ratio of 1/500/500) in the enrichment of N-glycopeptides from IgG digests. As a result, 281 N-glycopeptides corresponded with 109 glycoproteins were identified from 2 μL serum digests of the patients with nasopharyngeal carcinoma, and 181 N-glycopeptides corresponded with 78 glycoproteins were identified from 2 μL serum digests of the healthy volunteers, revealing the potential application value of mM-TiO 2 @Cys in glycoproteomics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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 Fe 3 O 4 @RF@Zr-BCP nanochains feature excellent magnetic response (42.26 emu/g), high hydrophilicity (16.0°), and large specific surface area (140.84 m 2 /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 Fe 3 O 4 @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 Fe 3 O 4 @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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Considering the importance of glycopeptides in the clinical diagnosis of cancer and some serious diseases, the identification of glycopeptides from complex biological samples has attracted considerable attention. Effective pre-enrichment before mass spectrometry analysis plays an important role. In this work, a kind of hydrophilic two-dimensional composites (denoted as GO@MPDA@Arg) based on mesoporous polydopamine-graphene oxide were used to selectively enrich glycopeptides in biological samples. The mesoporous polydopamine (MPDA) layer self-assembled with template Pluronic F127 provided more binding sites to load arginine, and bound arginine enhanced the hydrophilicity of the material. As a result, GO@MPDA@Arg composites exhibited excellent enrichment performance for glycopeptides, containing good selectivity (IgG digests : BSA digests = 1:50, molar ratio), low detection limit for IgG digests (10 fmol μL -1 ), high loading capacity for IgG digests (200 μg mg -1 ), and good size exclusion (IgG digests : IgG : BSA = 1:100:100, mass ratio). In addition, mouse brain tissue was selected as the actual biological sample to further study the enrichment effect of GO@MPDA@Arg composites. In three parallel experiments, a total of 401 glycopeptides belonging to 233 glycoproteins were enriched from 200 μg digestion of mouse brain extract. The enrichment results demonstrate that GO@MPDA@Arg composites have application potential for glycopeptides enrichment in protein post-translational modification research., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Protein glycosylation is among the most important post-translational modifications in living organisms and the research in the field of protein glycosylation continues to garner attention. Currently, the efficient separation and enrichment of glycoproteins and glycopeptides is the primary challenge of glycoproteomics research. The number of glycoproteins is small in complex biological samples. Moreover, the presence of highly-abundant, non-glycosylated, and modified peptides makes the detection of low-abundance glycopeptides more difficult. Therefore, efficient glycopeptide enrichment methods are required to improve the detection of these compounds. The development of highly selective glycopeptide enrichment tools is important to efficiently capture glycoproteins or glycopeptides at the molecular level. Compared with traditional glycopeptide-enriched materials, covalent organic framework materials have the advantages of large specific surface area and rich modification sites, thereby exhibiting great application potential in the field of glycopeptide enrichment. In this study, a novel covalent organic framework material (O-T-D-COFs) was prepared and applied for selective glycopeptide enrichment. We applied the solvothermal method, using 2,5-dimethoxy benzene-1,4-2 formaldehyde and 1,3,5-Tris(4-aminophenyl) benzene, to synthesize imino-based COFs. The Schiff base generated via copolymerization condensation reaction constitutes the framework of the material. Next, the synthesized intermediate material was oxidized to improve the enrichment performance of the material. The functional, specific glycopeptide-binding groups were modified on the COF channels and the structure of the material was characterized using scanning and transmission electron microscope, as well as infrared spectrum and solid-state nuclear magnetic resonance. The enrichment conditions comprised the loading and elution steps, including the optimization of the elution conditions. We could observe the clear profile of 32 glycopeptides derived from human serum immunoglobulin G (IgG) tryptic digests with a significantly improved signal-to-noise ( S/N ) ratio. We applied a complex sample system to verify the enrichment selectivity of the material when the molar ratios of the IgG and bovine serum albumin (BSA) tryptic digest mixtures reached 1∶50. In addition, we investigated the enrichment performance of the detection limit, enrichment capacity, recovery rate of the material, and the application potential in glycopeptides enrichment using real samples. The material showed a good detection limit (2.5 fmol/μL), an ideal enrichment capacity (120 mg/g), and enrichment recovery (103.5%±6.6% and 101.5%±10.4%). We identified a total of 86 glycopeptides derived from 53 glycoproteins with 94 N -glycosylation sites from only 1 μL human serum. The O-T-D-COFs exhibited a good glycopeptide separation and enrichment potential, indicating that the COF material has promising application potential in glycoproteomics.

Glycosylation is one of the most important post-translational modifications of proteins, and plays an important role in the structure and function of proteins. However, due to the diversity of glycopeptide forms and their low abundance, it is extraordinarily challenging to capture and separate glycopeptides with high selectivity from complex biological samples with mass spectrometric analysis. Here, we synthesized a new type of hydrophilic composite based on electrostatic interactions, which has been proven to be effective in immobilizing cationic cellulose on graphene oxide-dopamine carriers (expressed as GO-DA-JR), for highly specific enrichment of N-glycopeptides. The introduction of cationic cellulose provides not only a perfect surface charge for the composite but also a greater ability to enrich glycosylated peptides. Thirty-two glycopeptides from human serum immunoglobulin G (IgG) tryptic digests were observed with a greatly improved signal-to-noise ratio (S/N) and also presented high performance in anti-interfering enrichment of glycopeptides from complex samples containing 100-fold bovine serum albumin tryptic digests. In addition, GO-DA-JR has higher sensitivity (1 fmol/μL IgG) and better enrichment capacity (up to 150 mg/g). Moreover, the results of glycopeptide enrichment and glycosylation analysis from human serum also show egood enrichment selectivity from real biological samples. This work exhibits high selectivity, high sensitivity, good stability and operability, indicating its potential for applications of glycopeptides enrichment in post-translational modification proteomics., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In view of the size and hydrophilicity of glycopeptides, materials having suitable channels (size-exclusion) and strong hydrophilic surface (hydrophilic interaction) are preferred to enrich the glycopeptides in biological samples. Metal-organic frameworks (MOFs) are good candidates. However, their smaller microporous channels and low chemical stability have limited the application. Herein, a facile strategy was established to construct hydrophilic mesoporous MOF via synergistic etching and surface functionalization by using phytic acid (PA). Besides, polyvinylpyrrolidone (PVP) was added during MOF synthesis to enhance the water stability of the MOF. Owing to the expanded hydrophilic mesoporous channels, the PA-modified Ce-MOF effectively and selectively captured 422 glycopeptides from 155 glycoproteins in tryptic digests of human serum (2 μL). The present work sheds light on the easy fabrication of hydrophilic mesoporous materials, and this established material holds unique advantages for glycopeptides analysis in biological samples.

A hydrophilic interaction liquid chromatography (HILIC) material with application in glycoproteomics was obtained by sequential deposition of polyethyleneimine (PEI) and hyaluronic acid (HA) on a negatively charged substrate by means of electrostatic self-assembly. This kind of surface modification endows the material with excellent hydrophilicity and warrants efficient glycopeptides enrichment. The feasibility of this enrichment was verified by using dendritic mesoporous silica nanoparticles (DMSNs) and magnetic graphene oxide (MagG) as negatively charged substrates for PEI and HA adhesion. The two final products (DMSNs@PEI@HA and MagG@PEI@HA) exhibit high enrichment selectivity (molar ratios of IgG and BSA digests = 1:500 and 1:1000), sensitivity (detection limit, 2 fmol/μL), recovery (>90%) and enrichment capacity (300 mg/g). When using DMSNs@PEI@HA, 419 N-glycopeptides derived from 105 glycoproteins were identified. When using MagG@PEI@HA, 376 N-glycopeptides derived from 102 glycoproteins were identified, both from a 2 μL serum sample. This is better than by methods described in previous reports. Graphical abstract Schematic representation of hydrophilic modification of negatively charged nanomaterial substrates by electrostatic self-assembly techniques to obtain hydrophilic interaction liquid chromatography (HILIC) materials for enrichment of N-glycopeptides.

In this work, magnetic mesoporous silica microspheres (Mag-MSMs) with ordered radial mesochannels were fabricated by a self-assembly synthesis in chlorobenzene-water mixed system. Then, the obtained Mag-MSMs were modified with polyethyleneimine (PEI), phytic acid (PA) and Ti 4+ (denoted as Mag-MSMs@PEI-PA-Ti 4+ ) via layer by layer (LbL) assembly. Due to the excellent hydrophilicity of PEI and PA and the large amount of Ti 4+ , the Mag-MSMs@PEI-PA-Ti 4+ possessed combined properties of hydrophilic interaction liquid chromatography (HILIC)- and immobilized metal ion affinity chromatography (IMAC)-based materials, which could be used as a dual-purpose material for N-glycopeptides or phosphopeptides enrichment. The proposed Mag-MSMs@PEI-PA-Ti 4+ exhibited an outstanding performance for N-glycopeptides enrichment (selectivity: IgG/BSA = 1:1000; sensitivity: 0.5 fmol/μL IgG) and phosphopeptides enrichment (selectivity: α-casein/BSA=1:5000; sensitivity: 0.2 fmol/μL α-casein). Furthermore, after enrichment with Mag-MSMs@PEI-PA-Ti 4+ , a total of 276 N-glycopeptides assigned to 132 glycoproteins were identified from 2 μL human serum and 1645 phosphopeptides corresponding to 704 phosphoproteins were identified from 200 μg HeLa cell extracts., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A hydrophilic nanocomposite was synthesized by an easy route to improve glycopeptides enrichment efficiency. This new composite, prepared with a method based on electrostatic interaction, was demonstrated to be efficient for immobilization of carrageenan on graphene oxide/poly(ethylenimine) support (denoted as GO-PEI-Carr). Carrageenan, which has a large number of hydroxyl groups and is fully negatively charged, is a new modified phase of hydrophilic materials in glycoproteomics. The introduction of carrageenan provided the composite not only a perfect surface charge but also a greater ability to enrich glycosylated peptides. Thirty-four glycopeptides from human serum immunoglobulin G (IgG) tryptic digests were obviously observed with greatly improved signal-to-noise (S/N) ratio. A good selectivity was still kept even when the molar ratio of IgG and bovine serum albumin (BSA) tryptic digest mixtures reached to 1:500. Meanwhile, 76 glycopeptides derived from 56 glycoproteins with 83 N-glycosylation sites were identified from human serum and 149 glycopeptides derived from 129 glycoproteins with 157 N-glycosylation sites were identified from mouse liver tissues, which showed the ability to enrich glycopeptides from complex biological samples. In addition, GO-PEI-Carr exhibited a unique repeatability and stability even after enrichment of glycopeptides for 20 times. It also performed a higher sensitivity (1 fmol/μL IgG), a better enrichment capacity (up to ∼300 mg/g), and an ideal enrichment recovery (90.8% and 109.5%) for glycopeptides enrichment, indicating a great potential for the application of glycoproteomic research.

In this work, we presented a facile elution-free method for ultrasensitive enrichment of glycopeptides using two kinds of novel Ce-metal-organic frameworks (Ce-MOF) post-modified with hyaluronic acid (Ce-MOF@HA) and glutamic acid (Ce-MOF@Glu). Both of the synthesized materials remained stable in the loading buffer to enrich glycopeptides selectively and degrade in the eluent to release captured glycopeptides. Due to the dissolution of materials, the elution step of the enrichment process is omitted, resulting in an extremely high sensitivity (detection limit, 0.5 fmol/μL). Meanwhile, Ce-MOF@HA and Ce-MOF@Glu also possessed excellent selectivity with molar ratios of IgG and BSA digests being 1:1000 and 1:500, respectively. Noticeably, the practical applicability of the obtained materials was inspected by analyzing the glycopeptides enriched from human serum (2 μL) by nano-LC-MS, in which 434 N-glycopeptides from 182 N-glycoproteins (by Ce-MOF@HA) and 328 N-glycopeptides from 135 N-glycoproteins (by Ce-MOF@Glu) were detected, respectively. This work provides a new method to simplify the process of glycopeptides enrichment and also paves a novel way for the enrichment of trace targets from complex matrices., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In this work, multifunctional Ti 4+ -immobilized phytic acid-modified magnetic graphene (denoted as MagG@PEI@PA-Ti 4+ ) nanocomposites were fabricated through a facile route for simultaneous/respective enrichment of N-glyco- and phosphopeptides. Phytic acid (PA), with six phosphate groups, possesses excellent hydrophilicity and metal ion coordination ability, which endowed the MagG@PEI@PA-Ti 4+ with combined properties of immobilized metal ion affinity chromatography (IMAC)- and hydrophilic interaction liquid chromatography (HILIC)-based materials. On the basis of the different binding ability of N-glyco- and phosphopeptides on MagG@PEI@PA-Ti 4+ , the MagG@PEI@PA-Ti 4+ nanocomposites could enrich N-glyco- and phosphopeptides simultaneously or respectively by using different enrichment conditions, achieving controllable selective enrichment of N-glyco- and phosphopeptides. The proposed nanocomposites demonstrated an outstanding performance for selective enrichment of N-glycopeptides (selectivity, 1:1000 molar ratios of IgG/BSA; sensitivity, 0.5 fmol/μL IgG; loading capacity, 300 mg g -1 ; recovery, >90%) and phosphopeptides (selectivity, 1:5000 molar ratios of α-casein/BSA; sensitivity, 0.1 fmol/μL α-casein; loading capacity, 100 mg g -1 ; recovery, >90%). Taking advantage of these merits, a total of 393 N-glycopeptides derived from 259 glycoproteins and 574 phosphopeptides derived from 341 phosphoproteins were identified from 200 μg of HeLa cell extracts through a single-step enrichment using MagG@PEI@PA-Ti 4+ .

The development of facilely synthetic materials acts an essential role in glycoproteome analysis, especially for the highly efficient enrichment of N-linked glycopeptides. In this work, a facile and timesaving route was introduced in which COF TP-TAPT served as a carrier and poly (ethylenimine) (PEI) and carrageenan (Carr) were successively coated on the surface via electrostatic interaction. The resultant COF TP-TAPT @PEI@Carr showed remarkable performance in glycopeptide enrichment with high sensitivity (2 fmol μL−1), high selectivity (1:800, molar ratio of human serum IgG to BSA digests), large loading capacity (300 mg g−1), satisfactory recovery (102.4 ± 6.0%) and reusability (at least eight times). Due to the brilliant hydrophilicity and electrostatic interactions between COF TP-TAPT @PEI@Carr and positively charged glycopeptides, the prepared materials could be applied in the identification and analysis in the human plasma of healthy subjects and patients with nasopharyngeal carcinoma. As a result, 113 N-glycopeptides with 141 glycosylation sites corresponding to 59 proteins and 144 N-glycopeptides with 177 glycosylation sites corresponding to 67 proteins were enriched from 2 μL plasma trypsin digests of the control groups and patients with nasopharyngeal carcinoma, respectively. 22 glycopeptides were identified only from the normal controls and 53 glycopeptides were detected only from the other set. The results demonstrated that this hydrophilic material was promising on a large scale and further N-glycoproteome research. [Display omitted] • The stable hydrophilic porous COFs were designed for glycopeptide enrichment. • The hydrophilicity and stability of COFs in acidic solutions were greatly improved. • COFs showed great enrichment performance via HILIC and electrostatic interactions. • COFs materials were successfully applied for human plasma glycoproteomic analysis. [ABSTRACT FROM AUTHOR]

Protein glycosylation plays a vital role in many physiological activities in organisms. Due to the low abundance of glycopeptides and the interference of numerous non-glycopeptides in biological samples, selective enrichment of glycopeptides is of great significance for their successful identification. Metal organic frameworks (MOFs) materials are appropriate for glycopeptides enrichment by virtue of their large specific surface area and outstanding hydrophilic properties. However, the instability of hydrophilic MOFs in acidic solutions have severely limited their applications. In this work, a rational facile strategy was established to synthesize a stable hydrophilic hierarchical porous MOF (denoted as HP-MOF-Arg@mSiO 2). This new material improved the selectivity and sensitivity of enrichment for glycopeptides via modification of arginine groups. More importantly, the mesoporous silica layer was introduced to enhance the stability of MOFs in aqueous solution and achieve the size exclusion effect of large-size proteins in complex samples. Overall, owing to the unique hierarchical porous and the hydrophilic modification, the synthesized HP-MOF-Arg@mSiO 2 materials showed excellent hydrophilicity and hydrolytic stability, resulting in outstanding specific separation capacity in glycopeptides enrichment. A total of 521 and 342 glycopeptides were respectively captured from 2 μL human serum digests and mouse testis tissue digests, revealing the potential of the materials in the study of glycoproteomics in complex biological samples. [Display omitted] • A novel stable hydrophilic hierarchical porous MOFs was designed for glycopeptide enrichment. • The hydrophilicity and hydrolytic stability of MOFs in acidic solutions were greatly improved. • MOFs materials showed outstanding enrichment specificity and sensitivity. • MOFs materials were applied to the detection of glycopeptides from biosamples by nano-LC-MS/MS. [ABSTRACT FROM AUTHOR]

Glycosylation and phosphorylation are two of the most common and important post-translational modifications (PTMs) of proteins, which play critical roles in regulating a variety of complex biological processes and involvement in many diseases. Due to the low abundance of phosphopeptides and glycopeptides, highly selective enrichment methods are crucial to the identification of protein phosphorylation and glycosylation by mass spectrometry (MS). Here, monodisperse uniform Al3+-doping-TiO 2 mixed oxide microspheres were easily synthesized. The morphology was controlled by a sol-gel method, during the hydrothermal treatment. The obtained microspheres with uniform particle size distribution (about 1–2 μm)，high surface area and improved pore structures, were characterized by SEM, TEM, XRD and N 2 adsorption-desorption isotherms. Al3+-doping-TiO 2 was applied in enriching glycopeptides and phosphopeptides respectively or simultaneously by using different enrichment conditions, achieving selective enrichment of glycopeptides and phosphopeptides. 20 glycopeptides and 25 phosphopeptides enriched from the tryptic digest mixtures of human serum immunoglobulin G (IgG) and α-casein (molar ratio of 1:1) were obviously observed with greatly improved signal-to-noise (S/N) ratio. Meanwhile, the enrichment results of non-fat milk and human serum also show the enrichment selectivity from complex biological samples. This study will provide a novel insight for selective enrichment of glycopeptides and phosphopeptides in post-translational modification proteomics research. Image 1 • Monodisperse uniform Al3+-doping-TiO 2 microspheres with high surface area and improved pore structures were synthesized. • Al3+-doping-TiO 2 microspheres were exhibited high selectivity and sensitivity to phosphopeptide and glycopeptide enrichment. • Al3+-doping-TiO 2 microspheres can be used for glycopeptides and phosphopeptide enrichment simultaneously in one enrichment process. • Al3+-doping-TiO 2 microspheres can identify low-abundance phosphopeptides from complex biological samples. [ABSTRACT FROM AUTHOR]