Mechanism of autocatalytic activation during proteasome assembly.

Many large molecular machines are too elaborate to assemble spontaneously and are built through ordered pathways orchestrated by dedicated chaperones. During assembly of the core particle (CP) of the proteasome, where protein degradation occurs, its six active sites are simultaneously activated via cleavage of N-terminal propeptides. Such activation is autocatalytic and coupled to fusion of two half-CP intermediates, which protects cells by preventing activation until enclosure of the active sites within the CP interior. Here we uncover key mechanistic aspects of autocatalytic activation, which proceeds through alignment of the ß5 and ß2 catalytic triad residues, respectively, with these triads being misaligned before fusion. This mechanism contrasts with most other zymogens, in which catalytic centers are preformed. Our data also clarify the mechanism by which individual subunits can be added in a precise, temporally ordered manner. This work informs two decades-old mysteries in the proteasome field, with broader implications for protease biology and multisubunit complex assembly.

DSBSO-Based XL-MS Analysis of Breast Cancer PDX Tissues to Delineate Protein Interaction Network in Clinical Samples.

Protein-protein interactions (PPIs) are fundamental to understanding biological systems as protein complexes are the active molecular modules critical for carrying out cellular functions. Dysfunctional PPIs have been associated with various diseases including cancer. Systems-wide PPI analysis not only sheds light on pathological mechanisms, but also represents a paradigm in identifying potential therapeutic targets. In recent years, cross-linking mass spectrometry (XL-MS) has emerged as a powerful tool for defining endogenous PPIs of cellular networks. While proteome-wide studies have been performed in cell lysates, intact cells and tissues, applications of XL-MS in clinical samples have not been reported. In this study, we adopted a DSBSO-based in vivo XL-MS platform to map interaction landscapes from two breast cancer patient-derived xenograft (PDX) models. As a result, we have generated a PDX interaction network comprising 2,557 human proteins and identified interactions unique to breast cancer subtypes. Interestingly, most of the observed differences in PPIs correlated well with protein abundance changes determined by TMT-based proteome quantitation. Collectively, this work has demonstrated the feasibility of XL-MS analysis in clinical samples, and established an analytical workflow for tissue cross-linking that can be generalized for mapping PPIs from patient samples in the future to dissect disease-relevant cellular networks.

Mapping Pregnancy-dependent Sulfhydrome Unfolds Diverse Functions of Protein Sulfhydration in Human Uterine Artery.

Uterine artery (UA) hydrogen sulfide (H2S) production is augmented in pregnancy and, on stimulation by systemic/local vasodilators, contributes to pregnancy-dependent uterine vasodilation; however, how H2S exploits this role is largely unknown. S-sulfhydration converts free thiols to persulfides at reactive cysteine(s) on targeted proteins to affect the entire proteome posttranslationally, representing the main route for H2S to elicit its function. Here, we used Tag-Switch to quantify changes in sulfhydrated (SSH-) proteins (ie, sulfhydrome) in H2S-treated nonpregnant and pregnant human UA. We further used the low-pH quantitative thiol reactivity profiling platform by which paired sulfhydromes were subjected to liquid chromatography tandem mass spectrometry-based peptide sequencing to generate site (cysteine)-specific pregnancy-dependent H2S-responsive human UA sulfhydrome. Total levels of sulfhydrated proteins were significantly greater in pregnant vs nonpregnant human UA and further stimulated by treatment with sodium hydrosulfide. We identified a total of 360 and 1671 SSH-peptides from 480 and 1186 SSH-proteins in untreated and sodium hydrosulfide-treated human UA, respectively. Bioinformatics analyses identified pregnancy-dependent H2S-responsive human UA SSH peptides/proteins, which were categorized to various molecular functions, pathways, and biological processes, especially vascular smooth muscle contraction/relaxation. Pregnancy-dependent changes in these proteins were rectified by immunoblotting of the Tag-Switch labeled SSH proteins. Low-pH quantitative thiol reactivity profiling failed to identify low abundance SSH proteins such as KATP channels in human UA; however, immunoblotting of Tag-Switch-labeled SSH proteins identified pregnancy-dependent upregulation of SSH-KATP channels without altering their total proteins. Thus, comprehensive analyses of human UA sulfhydromes influenced by endogenous and exogenous H2S inform novel roles of protein sulfhydration in uterine hemodynamics regulation.

Exploring an Alternative Cysteine-Reactive Chemistry to Enable Proteome-Wide PPI Analysis by Cross-Linking Mass Spectrometry.

The development of MS-cleavable cross-linking mass spectrometry (XL-MS) has enabled the effective capture and identification of endogenous protein-protein interactions (PPIs) and their residue contacts at the global scale without cell engineering. So far, only lysine-reactive cross-linkers have been successfully applied for proteome-wide PPI profiling. However, lysine cross-linkers alone cannot uncover the complete PPI map in cells. Previously, we have developed a maleimide-based cysteine-reactive MS-cleavable cross-linker (bismaleimide sulfoxide (BMSO)) that is effective for mapping PPIs of protein complexes to yield interaction contacts complementary to lysine-reactive reagents. While successful, the hydrolysis and limited selectivity of maleimides at physiological pH make their applications in proteome-wide XL-MS challenging. To enable global PPI mapping, we have explored an alternative cysteine-labeling chemistry and thus designed and synthesized a sulfoxide-containing MS-cleavable haloacetamide-based cross-linker, Dibromoacetamide sulfoxide (DBrASO). Our results have demonstrated that DBrASO cross-linked peptides display the same fragmentation characteristics as other sulfoxide-containing MS-cleavable cross-linkers, permitting their unambiguous identification by MSn. In combination with a newly developed two-dimensional peptide fractionation method, we have successfully performed DBrASO-based XL-MS analysis of HEK293 cell lysates and demonstrated its capability to complement lysine-reactive reagents and expand PPI coverage at the systems-level.

Yeast PI31 inhibits the proteasome by a direct multisite mechanism.

Proteasome inhibitors are widely used as therapeutics and research tools, and typically target one of the three active sites, each present twice in the proteasome complex. An endogeneous proteasome inhibitor, PI31, was identified 30 years ago, but its inhibitory mechanism has remained unclear. Here, we identify the mechanism of Saccharomyces cerevisiae PI31, also known as Fub1. Using cryo-electron microscopy (cryo-EM), we show that the conserved carboxy-terminal domain of Fub1 is present inside the proteasome's barrel-shaped core particle (CP), where it simultaneously interacts with all six active sites. Targeted mutations of Fub1 disrupt proteasome inhibition at one active site, while leaving the other sites unaffected. Fub1 itself evades degradation through distinct mechanisms at each active site. The gate that allows substrates to access the CP is constitutively closed, and Fub1 is enriched in mutant CPs with an abnormally open gate, suggesting that Fub1 may function to neutralize aberrant proteasomes, thereby ensuring the fidelity of proteasome-mediated protein degradation.

Two-Dimensional Fractionation Method for Proteome-Wide Cross-Linking Mass Spectrometry Analysis.

Cross-linking mass spectrometry (XL-MS) is an emergent technology for studying protein-protein interactions (PPIs) and elucidating architectures of protein complexes. The development of various MS-cleavable cross-linkers has facilitated the identification of cross-linked peptides, enabling XL-MS studies at the systems level. However, the scope and depth of cellular networks revealed by current XL-MS technologies remain limited. Due to the inherently broad dynamic range and complexity of proteomes, interference from highly abundant proteins impedes the identification of low-abundance cross-linked peptides in complex samples. Thus, peptide enrichment prior to MS analysis is necessary to enhance cross-link identification for proteome-wide studies. Although chromatographic techniques including size exclusion (SEC) and strong cation exchange (SCX) have been successful in isolating cross-linked peptides, new fractionation methods are still needed to further improve the depth of PPI mapping. Here, we present a two-dimensional (2D) separation strategy by integrating peptide SEC with tip-based high pH reverse-phase (HpHt) fractionation to expand the coverage of proteome-wide XL-MS analyses. Combined with the MS-cleavable cross-linker DSSO, we have successfully mapped in vitro PPIs from HEK293 cell lysates with improved identification of cross-linked peptides compared to existing approaches. The method developed here is effective and can be generalized for cross-linking studies of complex samples.

A new urinary exosome enrichment method by a combination of ultrafiltration and TiO2 nanoparticles.

Exosomes are small membrane-bound vesicles secreted by most cell types and play an important role in cell-to-cell communication. Increasing evidence shows that exosomal proteins in urine may be used as novel biomarkers for certain diseases. Purified urinary exosomes are necessary for downstream studies and application development. However, conventional methods for exosome isolation and enrichment are technically challenging and time-consuming. Poor specificity, low recovery and instrumental dependence also limit the use of these methods. It is particularly urgent to develop a rapid and efficient extraction method for basic research and clinical application. Particularly, urine is a dilute solution system with relatively low abundance of exosomes, due to which the isolation of urinary exosome requires more efficient technology. Here, we propose a new strategy for facile exosome isolation from human urine by utilizing the ultrafiltration technique and the specific interaction of TiO2 with the phosphate groups on the lipid bilayer of exosomes. Downstream characterization and proteomic analysis indicate that high-quality exosomes can be obtained from human urine by this ultrafiltration-TiO2 series method in 20 minutes, and 91.5% exosomes with an intact structure are captured from urine by this method. Moreover, 1874 protein groups have been identified through LC-MS. The results show that the protein identification of our method is 23% higher at least than those obtained by conventional strategies. We also identified 30 differential proteins by comparing the urinary exosomes from healthy male and female volunteers. These proteins are related to biological processes, such as lipid metabolism, fatty acid metabolism and nucleotide metabolism. Our analysis reveals that combining conventional ultrafiltration and TiO2-based isolation is ideal to overcome the inherent limitations of identification of exosome proteins derived from urine, and yield highly pure exosome components for downstream proteomic analysis.

A facile "one-material" strategy for tandem enrichment of small extracellular vesicles phosphoproteome.

Small extracellular vesicles (SEVs), are cell-derived, membrane-enclosed nanometer-sized vesicles that play vital roles in many biological processes. Recent years, more and more evidences proved that small EVs have close relationship with many diseases such as cancers and Alzheimer's disease. The use of phosphoproteins in SEVs as potential biomarkers is a promising new choice for early diagnosis and prognosis of cancer. However, current techniques for SEVs isolation still facing many challenges, such as highly instrument dependent, time consuming and insufficient purity. Furthermore, complex enrichment procedures and low microgram amounts of proteins available from clinical sources largely limit the throughput and the coveage depth of SEVs phosphoproteome mapping. Here, we synthesized Ti4+-modified magnetic graphene-oxide composites (GFST) and developed a "one-material" strategy for facile and efficient phosphoproteome enrichment and identification in SEVs from human serum. By taking advantage of chelation and electrostatic interactions between metal ions and phosphate groups, GFST shows excellent performance in both SEVs isolation and phosphopeptide enrichment. Close to 85% recovery is achieved within a few minutes by simple incubation with GFST and magnetic separation. Proteome profiling of the isolated serum SEVs without phosphopeptide enrichment results in 515 proteins, which is approximately one-fold more than those otained by ultracentrifugation or coprecipitation kits. Further application of GFST in one-material-based enrichment led to identification of 859 phosphosites in 530 phosphoproteins. Kinase-substrate correlation analysis reveals enriched substrates of CAMK in serum SEVs phosphoproteome. Therefore, we expect that the low instrument dependency and the limited sample requirement of this new strategy may facilitate clinical investigations in SEV-based transportation of abnormal kinases and substrates for drug target discovery and cancer monitoring.

Novel Two-Dimensional MoS2-Ti4+ Nanomaterial for Efficient Enrichment of Phosphopeptides and Large-Scale Identification of Histidine Phosphorylation by Mass Spectrometry.

Due to its key roles in regulating the occurrence and development of cancer, protein histidine phosphorylation has been increasingly recognized as an important form of post-translational modification in recent years. However, large-scale analysis of histidine phosphorylation is much more challenging than that of serine/threonine or tyrosine phosphorylation, mainly because of its acid lability. In this study, MoS2-Ti4+ nanomaterials were synthesized using a solvothermal method and taking advantage of the electrostatic adsorption between MoS2 nanosheets and Ti4+. The MoS2-Ti4+ nanomaterials have the advantage of the combined affinity of Ti4+ and Mo toward phosphorylation under medium acidic conditions (pH = 3), which is crucial for preventing hydrolysis and loss of histidine phosphorylation during enrichment. The feasibility of using the MoS2-Ti4+ nanomaterial for phosphopeptide enrichment was demonstrated using mixtures of ß-casein and bovine serum albumin (BSA). Further evaluation revealed that the MoS2-Ti4+ nanomaterial is capable of enriching synthetic histidine phosphopeptides from 1000 times excess tryptic-digested HeLa cell lysate. Application of the MoS2-Ti4+ nanomaterials for large-scale phosphopeptide enrichment results in the identification of 10 345 serine, threonine, and tyrosine phosphosites and the successful mapping of 159 histidine phosphosites in HeLa cell lysates, therefore indicating great potential for deciphering the vital biological roles of protein (histidine) phosphorylation.

A GSH Functionalized Magnetic Ultra-thin 2D-MoS2 nanocomposite for HILIC-based enrichment of N-glycopeptides from urine exosome and serum proteins.

Protein N-glycosylation plays crucial roles in many biological processes and has close association with the occurrence and development of various cancers. Therefore, it is necessary to analyze the abnormal changes of N-glycopeptides in complex biological samples for biomarker discovery. However, due to their low abundance and poor ionization, N-glycopeptides identification in complex samples by mass spectrometry (MS) is still a challenging task. In this work, a novel magnetic hydrophilic material was prepared by serial functionalization of ultra-thin two-dimensional molybdenum disulfide with Fe3O4 nanoparticles, gold nanowire and glutathione (MoS2-Fe3O4-Au/NWs-GSH) for efficient N-glycopeptides enrichment. The advantage of using the new nanocomposite is threefold. First, the introduction of magnetic Fe3O4 nanoparticles efficiently simplifies the enrichment process. Second, the gold nanowire modification enlarges the surface area of the nanocomposites to facilitate interaction with N-glycopeptides. Third, the employment of highly hydrophilic glutathione leads to specific HILIC-based retention of N-glycopeptides. Low femtomolar detection sensitivity and 1:1000 enrichment selectivity can be achieved using MoS2-Fe3O4-Au/NWs-GSH enrichment and bio-mass spectrometry analysis. Successful applications in human urine exosome and serum proteins were demonstrated by the enrichment and identification of 1250 and 489 N-glycopeptides, respectively. This remarkable data set of N-glycoproteome indicates the application potential of the novel nanocomposites for N-glycopeptides enrichment in complex biological samples and for related glycoproteome studies.

[Progress in the application of functional materials in proteomics research].

With the increasing depth of proteomic identification, quantitative accuracy and increasing analytical speed, new challenges are being encountered in the development of proteomics methods. Traditional proteomics methods are time-consuming and have low sensitivity and poor accuracy; hence, they do not satisfy the new demands in proteomics research. Preparation of novel materials with specific functions via chemical and biochemical routes or by methods based on electricity, magnetism, heat, and photoirradiation is the key to overcome the limitations of traditional analytical techniques and promote active research in the field of proteomics. This paper reviews the recent advances in the application of functional materials in proteomics researches.

[Advances in separation techniques for exosomes and their clinical applications].

Exosomes are vesicles secreted by many types of cells through exocytosis, and their sizes range from 30 to 200 nm. Exosomes consist of a lipid bilayer membrane, containing a number of bioactive molecules, e. g., proteins, ribose nucleic acid (RNA), and deoxyribo nucleic acid (DNA) derived from the cell of origin. As intercellular communication carriers, exosomes participate in many physiological and pathological processes. Because of the complexity of body fluids, as well as the small size and low density of exosomes, the isolation of exosomes is an essential and challenging step before subsequent analysis and functional studies. This review summarizes the advances in the analytical approaches, characterization methods, biological functions and clinical applications of exosomes, with particular emphasis on exosomes isolation techniques.

A novel strategy for facile serum exosome isolation based on specific interactions between phospholipid bilayers and TiO2.

Exosomes are cell-derived, phospholipid bilayer-enclosed vesicles that play important roles in intercellular interactions and regulate many biological processes. Accumulating evidence suggests that serum exosomes are potential biomarkers for the early diagnosis of cancer. To aid the downstream molecular analyses of tumour-secreted exosomes, purified exosomes are highly desirable. However, current techniques for exosome isolation are time-consuming and highly instrument-dependent, with limited specificity and recovery. Thus, rapid and efficient methods are strongly needed for both basic research and clinical applications. Here, we present a novel strategy for facile exosome isolation from human serum by taking advantage of the specific interaction of TiO2 with the phosphate groups on the lipid bilayer of exosomes. Due to their simplicity and highly affinitive binding, model exosomes can be reversibly isolated with a high recovery (93.4%). Downstream characterization and proteome profiling reveal that high-quality exosomes can be obtained from human serum by this TiO2-based isolation method in 5 min, which is a fraction of the time required for the commonly used ultracentrifugation method. We identified 59 significantly up-regulated proteins by comparing the serum exosomes of pancreatic cancer patients and healthy donors. In addition to the 30 proteins that were reported to be closely related to pancreatic cancer, we found an additional 29 proteins that had not previously been shown to be related to pancreatic cancer, indicating the potential of this novel method as a powerful tool for exosome isolation for health monitoring and disease diagnosis.

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.

Rational synthesis of MoS2-based immobilized trypsin for rapid and effective protein digestion.

In this work, a novel MoS2-based immobilized trypsin reactor was designed and prepared. Pyrene-1-butyric acid was first assembled onto MoS2 nanosheets via the strong π-π stacking and then trypsin was covalently immobilized onto the nanocomposite supports through amidation reaction. Compared with traditional in-solution digestion, higher sequence coverage (84%) and shorter time (5min) could be achieved by the novel trypsin reactor during the digestion of BSA. The excellent performances of as-prepared trypsin reactor can be mainly attributed to the designed novel structure of the composites with high surface area resulting in high enzyme loading. In addition, strong reusability, good reproducibility and long storage of the trypsin reactor were also obtained. The novel immobilized trypsin reactor was further applied in large-scale proteomics research. The proteins extracted from HeLa cells and Amygdalus Pedunculata Pall. kernels were chosen to evaluate the digestion performance for the novel MoS2-based immobilized trypsin reactor, and the experimental results showed that the number of identified proteins from complex real bio-samples with 1h immobilized tryptic digestion was slightly more than that obtained by 12h in-solution digestion. The above results demonstrated that the protein digestion with our novel MoS2-based immobilized trypsin reactor is superior to the conventional protein digestion with free trypsin. Moreover, this simple, fast tryptic digestion method can effectively reduce the levels of artifacts in detection of oxidation and deamidation of peptides from proteins of Amygdalus Pedunculata Pall. kernels. Also, results of Gene Ontology analysis give an explanation for the good survival of Amygdalus Pedunculata Pall. in harsh desert environments from proteomics points of view. Therefore, the novel 2D-MoS2-based immobilized trypsin is potentially suitable for the high throughput proteome analysis and opening up a new avenue for Molybdenum disulfide in proteomics field.

Facile synthesis of magnetic metal organic frameworks for highly efficient proteolytic digestion used in mass spectrometry-based proteomics.

Mass spectrometry (MS) based bottom-up strategy is now the first choice for proteomics analysis. In this process, highly efficient and complete enzymatic degradation of protein samples is extremely important to achieve in-depth protein coverage and high-throughput protein profiling. However, conventional in-solution digestion suffer from long digestion time and enzyme autolysis that limit the protein sample processing throughput and identification accuracy. Here, we developed a novel type of magnetic metal organic frameworks (MOFs)-based immobilized enzyme reactor, Fe3O4@DOTA-ZIF-90-trypsin. By introducing a stable chelator, 1,4,7,10-tetraazacyclododecane N,N',N″,N‴-tetraacetic acid (DOTA), onto the magnetic cores, the hybrid supporting matrix of the immobilized enzyme reactor Fe3O4@DOTA-ZIF-90 has novel characteristics that include: i) favourable magnetic response (1.01 emu g-1) that makes the operation easy and convenient, ii) ultrahigh surface area (565.21 m2 g-11) and active sites that ensure high loading amounts and covalent linkage of enzyme, and iii) excellent structural and thermal stability that endows the immobilized enzyme reactor a prolonged lifespan. The performance of the magnetic MOFs-immobilized trypsin is first investigated using the standard protein, BSA, and the results showed that the immobilized enzyme reactor exhibits satisfactory digestion efficiency within only 1 min with the sequence coverage (80%) that is comparable or even better than that (70%) of the traditional 12 h-free trypsin digestion. To test the applicability of the magnetic MOFs-based immobilized enzyme reactor, protein samples extracted from 400 oocytes in mice were digested through the new immobilized enzyme reactor. In total, 8957 peptides corresponding to 1843 protein groups are identified, which are nearly of 40% and 67% increases in the number of identified proteins and peptides compared to using in-solution digestion with free proteases. Specifically, the identification of oocyte-specific proteins was critical in the discovery of and understanding the regulation mechanism of oocyte maturation. Thus, this synthetic procedure of Fe3O4@DOTA-ZIF-90 provides a universal method for fabrication of magnetic MOFs materials, and the successful application of Fe3O4@DOTA-ZIF-90-trypsin in efficient protein digestion for deeper proteome coverage will undoubtedly enlarge the uses for MOFs.

Polymeric hydrophilic ionic liquids used to modify magnetic nanoparticles for the highly selective enrichment of N-linked glycopeptides.

The low abundance of glycopeptides in biological samples makes it necessary to enrich them before further analysis. In this study, the polymeric hydrophilic ionic liquid-modified magnetic (Fe3O4@MPS@PMAC) nanoparticles were synthesized via a one-step reflux-precipitation polymerization. Owing to the excellent hydrophilicity and strong electrostatic interaction toward glycopeptides of the polymerized hydrophilic ionic liquid, [2-(methacryloyloxy) ethyl] trimethylammonium chloride (MAC), the synthesized Fe3O4@MPS@PMAC nanoparticles exhibited outstanding performance in glycopeptide enrichment with high detection sensitivity (10 fmol), large binding capacity (100 µg mg-1) and satisfied enrichment recovery (approximately 82%). Furthermore, the newly developed Fe3O4@MPS@PMAC nanoparticles were applied for the glycopeptide enrichment of HeLa exosome proteins. A total of 1274 glycopeptides from 536 glycoproteins were identified in three replicate analyses of 50 µg of HeLa exosome proteins. These results demonstrate the potential of Fe3O4@MPS@PMAC nanoparticles for both glycoproteomic analysis and exosome research.

Ultrathin Au nanowires assisted magnetic graphene-silica ZIC-HILIC composites for highly specific enrichment of N-linked glycopeptides.

Protein glycosylation has been proven to participate in a variety of complex biological processes; however, the low abundance of glycopeptides in natural samples makes it essential to develop methods to isolate and enrich glycopeptides. In this study, a novel ultrathin Au nanowire assisted zwitterionic hydrophilic magnetic graphene oxide (GO-Fe3O4/SiO2/AuNWs/L-Cys) was synthesized with the good biocompatibility of GO, strong magnetic responses of Fe3O4, large surface area of ultrathin Au nanowires and excellent hydrophilicity of L-Cys via four simple and rapid steps. The ultrathin Au nanowires have a one-dimensional structure and were easily grafted with an abundant amount of L-Cys for the enrichment of glycopeptides. After the GO-Fe3O4/SiO2/AuNWs/L-Cys composites were applied to glycopeptide enrichment, 26 glycopeptides from a human IgG digest could be identified, with a detection limit as low as 10 fmol. Due to the abundant amount of grafted L-Cys, the composites also showed a large binding capacity (150 µg mg-1). Furthermore, the composites were applied for the analysis of real biological samples. A total of 793 glycopeptides from 467 glycoproteins were identified in three replicate analyses of 40 µg of mouse liver proteins. The results demonstrated the great potential of GO-Fe3O4/SiO2/AuNWs/L-Cys composites for the analysis of glycoproteins.

Titanium (IV) ion-modified covalent organic frameworks for specific enrichment of phosphopeptides.

To date, plenty of new alternative materials for phosphopeptides enrichment prior to mass spectrometry (MS) analysis appear, especially immobilized metal ion affinity chromatography (IMAC) materials. The variable combinations with different metal ions, chelating ligands and solid supports offer full of optionality for IMAC. However, further improvement was predicted by the tedious and complex synthetic process. In this work, a novel covalent organic framework (COF)-based IMAC material (denoted TpPa-2-Ti4+) was prepared simply by direct immobilizing Ti (IV) into TpPa-2 COFs without any extra chelating ligands. The structure and composition of as-prepared composites were confirmed by PXRD, FT-IR and XPS, and a new flower-shaped Ti4+-IMAC with regular micro-nano hierarchical structure was observed in the SEM and TEM images. The obtained titanium (IV) ion-modified covalent organic frameworks demonstrated low limit of detection (4 fmol) and largely-satisfactory selectivity (ß-casein: BSA=1:100) for phosphopeptide capturing from ß-casein. Similarly, 18 and 17 phosphopeptides could be easily detected in the tryptic digest of α-casein or the digest mixture of α-casein and BSA (1:50). They were also successfully applied for enrichment of phosphopeptides from non-fat milk and HeLa cells with high sensitivity and satisfactory selectivity. All above results showed that the new titanium (IV) ion-modified covalent organic framework is expected to be a potential IMAC for phosphopeptide enrichment in large-scale phosphoproteomics studies.

Covalent organic framework-coated magnetic graphene as a novel support for trypsin immobilization.

Deep and efficient proteolysis is the critical premise in mass spectrometry-based bottom-up proteomics. It is difficult for traditional in-solution digestion to meet the requirement unless prolonged digestion time and enhanced enzyme dosage are employed, which makes the whole workflow time-consuming and costly. The abovementioned problems could be effectively ameliorated by anchoring many proteases on solid supports. In this work, covalent organic framework-coated magnetic graphene (MG@TpPa-1) was designed and prepared as a novel enzyme carrier for the covalent immobilization of trypsin with a high degree of loading (up to 268 µg mg-1). Profiting from the advantages of magnetic graphene and covalent organic frameworks, the novel trypsin bioreactor was successfully applied for the enzymatic digestion of a model protein with dramatically improved digestion efficiency, stability, and reusability. Complete digestion could be achieved in a time period as short as 2 min. For the digestion of proteins extracted from Amygdalus pedunculata, a total of 2833 protein groups were identified, which was slightly more than those obtained by 12 h of in-solution digestion (2739 protein groups). All of the results demonstrate that MG@TpPa-1-trypsin is an excellent candidate for sample preparation in a high-throughput proteomics analysis. Graphical abstract Covalent organic frameworks-coated magnetic graphene was prepared as novel carrier for highly efficient tryptic immobilization.