Trypsin Immobilization on Hairy Polymer Chains Hybrid Magnetic Nanoparticles for Ultra Fast, Highly Efficient Proteome Digestion, Facile ¹⁸O Labeling and Absolute Protein Quantification

In recent years, quantitative proteomic research attracts great attention because of the urgent needs in biological and clinical research, such as biomarker discovery and verification. Currently, mass spectrometry (MS) based bottom up strategy has become the method of choice for proteomic quantification. In this strategy, the amount of proteins is determined by quantifying the corresponding proteolytic peptides of the proteins, therefore highly efficient and complete protein digestion is crucial for achieving accurate quantification results. However, the digestion efficiency and completeness obtained using conventional free protease digestion is not satisfactory for highly complex proteomic samples. In this work, we developed a new type of immobilized trypsin using hairy noncross-linked polymer chains hybrid magnetic nanoparticle as the matrix aiming at ultra fast, highly efficient proteomic digestion and facile ¹⁸O labeling for absolution protein quantification. The hybrid nanoparticle is synthesized by in situ growth of hairy polymer chains from the magnetic nanoparticle surface using surface initiated atom transfer radical polymerization technique. The flexible noncross-linked polymer chains not only provide large amount of binding sites but also work as scaffolds to support three-dimensional trypsin immobilization which leads to increased loading amount and improved accessibility of the immobilized trypsin. For complex proteomic samples, obviously increased digestion efficiency and completeness was demonstrated by 27.2% and 40.8% increase in the number of identified proteins and peptides as well as remarkably reduced undigested proteins residues compared with that obtained using conventional free trypsin digestion. The successful application in absolute protein quantification of enolase from <i>Thermoanaerobacter tengcongensis</i> protein extracts using ¹⁸O labeling and MRM strategy further demonstrated the potential of this hybrid nanoparticle immobilized trypsin for high throughput proteome quantification

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A Highly Efficient and Visualized Method for Glycan Enrichment by Self-Assembling Pyrene Derivative Functionalized Free Graphene Oxide

Protein glycosylation plays key roles in many biological processes, such as cell growth, differentiation, and cell–cell recognition. Therefore, global structure profiling of glycans is very important for investigating the biological significance and roles of glycans in disease occurrence and development. Mass spectrometry (MS) is currently the most powerful technique for structure analysis of oligosaccharides, but the limited availability of glycan/glycoproteins from natural sources restricts the wide adoption of this technique in large-scale glycan profiling. Though various enrichment methods have been developed, most methods relay on the weak physical affinity between glycans and adsorbents that yields insufficient enrichment efficiency. Furthermore, the lack of monitoring the extent/completeness of enrichment may lead to incomplete enrichment unless repeated sample loading and prolonged incubation are adopted, which limits sample handling throughput. Here, we report a rapid, highly efficient, and visualized approach for glycan enrichment using 1-pyrenebutyryl chloride functionalized free graphene oxide (PCGO). In this approach, glycan capturing is achieved by reversible covalent bond formation between the hydroxyl groups of glycans and the acyl chloride groups on graphene oxide (GO) introduced by π–π stacking of 1-pyrenebutyryl chloride on the GO surface. The multiple hydroxyl groups of glycans lead to cross-linking and self-assembly of free PCGO sheets into visible aggregation within 30 s, therefore achieving simple visual monitoring of the enrichment process. Improved enrichment efficiency is achieved by the large specific surface area of free PCGO and heavy functionalization of highly active 1-pyrenebutyryl chloride. Application of this method in enrichment of standard oligosaccharides or <i>N</i>-glycans released from glycoproteins results in remarkably increased MS signal intensity (approximately 50 times), S/N, and number of glycoform identified

Two-Dimensional MoS₂‑Based Zwitterionic Hydrophilic Interaction Liquid Chromatography Material for the Specific Enrichment of Glycopeptides

Mass spectrometry (MS)-based glycoproteomics research requires highly efficient sample preparation to eliminate interference from non-glycopeptides and to improve the efficiency of glycopeptide detection. In this work, a novel MoS₂/Au-NP (gold nanoparticle)–L-cysteine nanocomposite was prepared for glycopeptide enrichment. The two-dimensional (2D) structured MoS₂ nanosheets served as a matrix that could provide a large surface area for immobilizing hydrophilic groups (such as L-cysteine) with low steric hindrance between the materials and the glycopeptides. As a result, the novel nanomaterial possessed an excellent ability to capture glycopeptides. Compared to commercial zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) materials, the novel nanomaterials exhibited excellent enrichment performance with ultrahigh selectivity and sensitivity (approximately 10 fmol), high binding capacity (120 mg g^–1), high enrichment recovery (more than 93%), satisfying batch-to-batch reproducibility, and good universality for glycopeptide enrichment. In addition, its outstanding specificity and efficiency for glycopeptide enrichment was confirmed by the detection of glycopeptides from an human serum immunoglobulin G (IgG) tryptic digest in quantities as low as a 1:1250 molar ratio of IgG tryptic digest to bovine serum albumin tryptic digest. The novel nanocomposites were further used for the analysis of complex samples, and 1920 glycopeptide backbones from 775 glycoproteins were identified in three replicate analyses of 50 μg of proteins extracted from HeLa cell exosomes. The resulting highly informative mass spectra indicated that this multifunctional nanomaterial-based enrichment method could be used as a promising tool for the in-depth and comprehensive characterization of glycoproteomes in MS-based glycoproteomics

Two-Dimensional MoS₂‑Based Zwitterionic Hydrophilic Interaction Liquid Chromatography Material for the Specific Enrichment of Glycopeptides

Mass spectrometry (MS)-based glycoproteomics research requires highly efficient sample preparation to eliminate interference from non-glycopeptides and to improve the efficiency of glycopeptide detection. In this work, a novel MoS₂/Au-NP (gold nanoparticle)–L-cysteine nanocomposite was prepared for glycopeptide enrichment. The two-dimensional (2D) structured MoS₂ nanosheets served as a matrix that could provide a large surface area for immobilizing hydrophilic groups (such as L-cysteine) with low steric hindrance between the materials and the glycopeptides. As a result, the novel nanomaterial possessed an excellent ability to capture glycopeptides. Compared to commercial zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) materials, the novel nanomaterials exhibited excellent enrichment performance with ultrahigh selectivity and sensitivity (approximately 10 fmol), high binding capacity (120 mg g^–1), high enrichment recovery (more than 93%), satisfying batch-to-batch reproducibility, and good universality for glycopeptide enrichment. In addition, its outstanding specificity and efficiency for glycopeptide enrichment was confirmed by the detection of glycopeptides from an human serum immunoglobulin G (IgG) tryptic digest in quantities as low as a 1:1250 molar ratio of IgG tryptic digest to bovine serum albumin tryptic digest. The novel nanocomposites were further used for the analysis of complex samples, and 1920 glycopeptide backbones from 775 glycoproteins were identified in three replicate analyses of 50 μg of proteins extracted from HeLa cell exosomes. The resulting highly informative mass spectra indicated that this multifunctional nanomaterial-based enrichment method could be used as a promising tool for the in-depth and comprehensive characterization of glycoproteomes in MS-based glycoproteomics

Two-Dimensional MoS₂‑Based Zwitterionic Hydrophilic Interaction Liquid Chromatography Material for the Specific Enrichment of Glycopeptides

Mass spectrometry (MS)-based glycoproteomics research requires highly efficient sample preparation to eliminate interference from non-glycopeptides and to improve the efficiency of glycopeptide detection. In this work, a novel MoS₂/Au-NP (gold nanoparticle)–L-cysteine nanocomposite was prepared for glycopeptide enrichment. The two-dimensional (2D) structured MoS₂ nanosheets served as a matrix that could provide a large surface area for immobilizing hydrophilic groups (such as L-cysteine) with low steric hindrance between the materials and the glycopeptides. As a result, the novel nanomaterial possessed an excellent ability to capture glycopeptides. Compared to commercial zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) materials, the novel nanomaterials exhibited excellent enrichment performance with ultrahigh selectivity and sensitivity (approximately 10 fmol), high binding capacity (120 mg g^–1), high enrichment recovery (more than 93%), satisfying batch-to-batch reproducibility, and good universality for glycopeptide enrichment. In addition, its outstanding specificity and efficiency for glycopeptide enrichment was confirmed by the detection of glycopeptides from an human serum immunoglobulin G (IgG) tryptic digest in quantities as low as a 1:1250 molar ratio of IgG tryptic digest to bovine serum albumin tryptic digest. The novel nanocomposites were further used for the analysis of complex samples, and 1920 glycopeptide backbones from 775 glycoproteins were identified in three replicate analyses of 50 μg of proteins extracted from HeLa cell exosomes. The resulting highly informative mass spectra indicated that this multifunctional nanomaterial-based enrichment method could be used as a promising tool for the in-depth and comprehensive characterization of glycoproteomes in MS-based glycoproteomics

Two-Dimensional MoS₂‑Based Zwitterionic Hydrophilic Interaction Liquid Chromatography Material for the Specific Enrichment of Glycopeptides

Mass spectrometry (MS)-based glycoproteomics research requires highly efficient sample preparation to eliminate interference from non-glycopeptides and to improve the efficiency of glycopeptide detection. In this work, a novel MoS₂/Au-NP (gold nanoparticle)–L-cysteine nanocomposite was prepared for glycopeptide enrichment. The two-dimensional (2D) structured MoS₂ nanosheets served as a matrix that could provide a large surface area for immobilizing hydrophilic groups (such as L-cysteine) with low steric hindrance between the materials and the glycopeptides. As a result, the novel nanomaterial possessed an excellent ability to capture glycopeptides. Compared to commercial zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) materials, the novel nanomaterials exhibited excellent enrichment performance with ultrahigh selectivity and sensitivity (approximately 10 fmol), high binding capacity (120 mg g^–1), high enrichment recovery (more than 93%), satisfying batch-to-batch reproducibility, and good universality for glycopeptide enrichment. In addition, its outstanding specificity and efficiency for glycopeptide enrichment was confirmed by the detection of glycopeptides from an human serum immunoglobulin G (IgG) tryptic digest in quantities as low as a 1:1250 molar ratio of IgG tryptic digest to bovine serum albumin tryptic digest. The novel nanocomposites were further used for the analysis of complex samples, and 1920 glycopeptide backbones from 775 glycoproteins were identified in three replicate analyses of 50 μg of proteins extracted from HeLa cell exosomes. The resulting highly informative mass spectra indicated that this multifunctional nanomaterial-based enrichment method could be used as a promising tool for the in-depth and comprehensive characterization of glycoproteomes in MS-based glycoproteomics

Two-Dimensional MoS₂‑Based Zwitterionic Hydrophilic Interaction Liquid Chromatography Material for the Specific Enrichment of Glycopeptides

Mass spectrometry (MS)-based glycoproteomics research requires highly efficient sample preparation to eliminate interference from non-glycopeptides and to improve the efficiency of glycopeptide detection. In this work, a novel MoS₂/Au-NP (gold nanoparticle)–L-cysteine nanocomposite was prepared for glycopeptide enrichment. The two-dimensional (2D) structured MoS₂ nanosheets served as a matrix that could provide a large surface area for immobilizing hydrophilic groups (such as L-cysteine) with low steric hindrance between the materials and the glycopeptides. As a result, the novel nanomaterial possessed an excellent ability to capture glycopeptides. Compared to commercial zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) materials, the novel nanomaterials exhibited excellent enrichment performance with ultrahigh selectivity and sensitivity (approximately 10 fmol), high binding capacity (120 mg g^–1), high enrichment recovery (more than 93%), satisfying batch-to-batch reproducibility, and good universality for glycopeptide enrichment. In addition, its outstanding specificity and efficiency for glycopeptide enrichment was confirmed by the detection of glycopeptides from an human serum immunoglobulin G (IgG) tryptic digest in quantities as low as a 1:1250 molar ratio of IgG tryptic digest to bovine serum albumin tryptic digest. The novel nanocomposites were further used for the analysis of complex samples, and 1920 glycopeptide backbones from 775 glycoproteins were identified in three replicate analyses of 50 μg of proteins extracted from HeLa cell exosomes. The resulting highly informative mass spectra indicated that this multifunctional nanomaterial-based enrichment method could be used as a promising tool for the in-depth and comprehensive characterization of glycoproteomes in MS-based glycoproteomics

Two-Dimensional MoS₂‑Based Zwitterionic Hydrophilic Interaction Liquid Chromatography Material for the Specific Enrichment of Glycopeptides

Mass spectrometry (MS)-based glycoproteomics research requires highly efficient sample preparation to eliminate interference from non-glycopeptides and to improve the efficiency of glycopeptide detection. In this work, a novel MoS₂/Au-NP (gold nanoparticle)–L-cysteine nanocomposite was prepared for glycopeptide enrichment. The two-dimensional (2D) structured MoS₂ nanosheets served as a matrix that could provide a large surface area for immobilizing hydrophilic groups (such as L-cysteine) with low steric hindrance between the materials and the glycopeptides. As a result, the novel nanomaterial possessed an excellent ability to capture glycopeptides. Compared to commercial zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) materials, the novel nanomaterials exhibited excellent enrichment performance with ultrahigh selectivity and sensitivity (approximately 10 fmol), high binding capacity (120 mg g^–1), high enrichment recovery (more than 93%), satisfying batch-to-batch reproducibility, and good universality for glycopeptide enrichment. In addition, its outstanding specificity and efficiency for glycopeptide enrichment was confirmed by the detection of glycopeptides from an human serum immunoglobulin G (IgG) tryptic digest in quantities as low as a 1:1250 molar ratio of IgG tryptic digest to bovine serum albumin tryptic digest. The novel nanocomposites were further used for the analysis of complex samples, and 1920 glycopeptide backbones from 775 glycoproteins were identified in three replicate analyses of 50 μg of proteins extracted from HeLa cell exosomes. The resulting highly informative mass spectra indicated that this multifunctional nanomaterial-based enrichment method could be used as a promising tool for the in-depth and comprehensive characterization of glycoproteomes in MS-based glycoproteomics

Two-Dimensional MoS₂‑Based Zwitterionic Hydrophilic Interaction Liquid Chromatography Material for the Specific Enrichment of Glycopeptides

Mass spectrometry (MS)-based glycoproteomics research requires highly efficient sample preparation to eliminate interference from non-glycopeptides and to improve the efficiency of glycopeptide detection. In this work, a novel MoS₂/Au-NP (gold nanoparticle)–L-cysteine nanocomposite was prepared for glycopeptide enrichment. The two-dimensional (2D) structured MoS₂ nanosheets served as a matrix that could provide a large surface area for immobilizing hydrophilic groups (such as L-cysteine) with low steric hindrance between the materials and the glycopeptides. As a result, the novel nanomaterial possessed an excellent ability to capture glycopeptides. Compared to commercial zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) materials, the novel nanomaterials exhibited excellent enrichment performance with ultrahigh selectivity and sensitivity (approximately 10 fmol), high binding capacity (120 mg g^–1), high enrichment recovery (more than 93%), satisfying batch-to-batch reproducibility, and good universality for glycopeptide enrichment. In addition, its outstanding specificity and efficiency for glycopeptide enrichment was confirmed by the detection of glycopeptides from an human serum immunoglobulin G (IgG) tryptic digest in quantities as low as a 1:1250 molar ratio of IgG tryptic digest to bovine serum albumin tryptic digest. The novel nanocomposites were further used for the analysis of complex samples, and 1920 glycopeptide backbones from 775 glycoproteins were identified in three replicate analyses of 50 μg of proteins extracted from HeLa cell exosomes. The resulting highly informative mass spectra indicated that this multifunctional nanomaterial-based enrichment method could be used as a promising tool for the in-depth and comprehensive characterization of glycoproteomes in MS-based glycoproteomics