Search

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 HEK 293 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.

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.

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.

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.

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 modificati...

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.

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.

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 TiO

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, MoS

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, an...

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 (5 min) 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 1 h immobilized tryptic digestion was slightly more than that obtained by 12 h 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.

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 (β-casein: BSA=1:100) for phosphopeptide capturing from β-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.

In “bottom-up” proteomics, proteins are first digested into peptides and then characterized by chromatographic separation and mass spectrometry analysis, in which proteolysis has an appreciable influence on the repeatability and reliability of analytical results. To improve recovery and enzymatic efficiency, proteins are often denatured and solubilized with chemical agents such as sodium dodecyl sulphate (SDS), urea (UA), etc. However, surfactant like SDS is difficult to remove from the reacting system and may interfere with protease activity, chromatographic separation of peptides and mass spectrometry analysis. To this end, we have prepared an immobilized surfactant which can preserve the solubilization and denaturation abilities of a surfactant and has the advantage of being easily separated from samples. The denaturation and solubilization capabilities of the newly developed immobilized surfactant were evaluated by comparing the enzymatic hydrolysis efficiencies in the pretreatment of protein samples treated with immobilized surfactant and other common methods. The results showed that the average protein sequence coverage was 42.1% (digested for 1 h), which was 76.89% higher than that of a control experiment (23.8%) in the digestion of a standard protein (BSA) and reached the same denaturation ability as that of heating at 95 °C. Moreover, the immobilized surfactant was further tested in the extraction and digestion of the total protein of HeLa cells. The number of identified proteins resulted in similar levels to those exploited with 0.1% SDS and 8 M UA, both of which are frequently used in lysis buffer, indicating that the immobilized surfactant had a good ability to denature and solubilize proteins. In brief, the immobilized surfactant avoids the shortcomings of UA and SDS and can be utilized in the pretreatment of complex biological samples.

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.

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.

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 Fe

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.

Tryptic digestion before identification and quantification by mass spectrometry is an indispensable process for most proteomics studies. Conventional in-solution digestion process always suffers from incomplete digestion and is time-consuming and expensive. Development of a novel proteolytic digestion method with fast, high efficiency and reusability of the enzyme is still an urgent task. In this study, new hollow silica bubbles-based immobilized trypsin (trypsin@SiB) was developed via brushes structured glycidyl methacrylate (GMA) grafted on the silica bubbles (SiB) by the reversible addition–fragmentation chain transfer polymerization (RAFT) technique followed by trypsin's immobilization on the microsphere. The new immobilized trypsin not only showed high efficiency and stability but also reusability. Due to the low density (0.6 g cm−3) of SiB, it can float over the solution for several minutes so that the immobilized trypsin can be separated and recovered from the system easily. Highly efficient digestion of BSA was achieved with this trypsin@SiB within 1 min and the obtained sequence coverage (92%) was better than that from conventional in-solution digestion for more than 12 h (76%). To further confirm the efficiency of trypsin@SiB for complex proteomic analysis, the protein extracted from human urine was analyzed as a real sample. Within 10 min digestion, 510 protein groups were identified with the LC-MS/MS analysis and database searching, whereas the number of identified proteins after 12 h in-solution digestion was 493 with the same identification conditions. The successful application of trypsin@SiB demonstrated its potential as a high efficient digestion method for future proteomics analysis.

Phosphopeptide enrichment is critical for the deep analysis of the phosphoproteome, and many strategies have been developed to improve the coverage of identified phosphopeptides. However, the isolation of multiple phosphorylated peptides is still limited due to their distinct affinity capability to various materials. For example, titanium dioxide is preferred to isolate mono-phosphopeptides owing to its very strong binding of multiple phosphorylated peptides. Here, a new set of different metal ion (Ti4+, Zr4+, Fe3+, Tb3+, Tm3+, Ho3+)-immobilized magnetic nanoparticles, Fe3O4@TCPP-DOTA-Ms, were prepared, which possess the advantages of a stable metal complex, a large binding capacity and easy manual operation by introducing TCPP (tetrakis(4-carboxylphenyl) porphyrin), DOTA (1,4,7,10-tetraazacyclododecane N,N′,N′′,N′′′-tetraacetic acid) and various metal ions onto the magnetic nanoparticles. For the model protein α-casein tryptic digest, 15 phosphopeptides were identified with Fe3O4@TCPP-DOTA-Tb or Ti, and 46.67% of the enriched phosphopeptides were multiple phosphorylated peptides, indicating that the metal ion Tb3+ and Ti4+-immobilized materials have excellent enrichment efficiency and stronger adsorption for multiple phosphorylated peptides than other metal ion immobilized magnetic nanoparticles. Even in the tryptic digest of α-casein and BSA (1 : 50), 14–15 phosphopeptides were easily detected with Fe3O4@TCPP-DOTA-Tb/Ti, revealing that the novel materials possess strong selectivity for phosphopeptide enrichment. Additionally, Fe3O4@TCPP-DOTA-Tb/Ti was utilized to capture phosphopeptides in the tryptic digest of an extract of HeLa cells. In total, 13 450 phosphopeptides corresponding to 2965 phosphoproteins were identified in mass spectrometric analyses. The specificity for phosphopeptide enrichment was as high as 94%. More than half of the identified unique phosphopeptides were multiple phosphorylated peptides, which was much higher than that identified by the DHB/TiO2 (13.39%) method, making these materials a good choice for highly selective and global phosphopeptide enrichment in phosphoproteome analysis.

Exosomes can be efficiently isolated in a short period of time by the specific interaction of titanium dioxide with the phosphate groups on the surface of phospholipid bilayer., 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.

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 MoS

A key step in the workflow of bottom-up proteomics is the proteolysis of proteins into peptides with trypsin. In addition, enzyme-catalytic (18)O labeled peptides as internal standards coupled with multiple reaction monitoring mass spectrometry (MRM MS) for the absolute quantitation of the target proteome is commonly used for its convenient operation and low cost. However, long digestion and labeling times, incomplete digestion and (18)O to (16)O back exchange limit its application, therefore, we developed a rapid and efficient digestion method based on a high ratio of trypsin to protein. In addition, after separation of the digested samples using pipette tips packed with reversed-phase packing materials in house, the trypsin can be separated, collected and reused at least four times. Based on this approach, a novel protein quantification method using (18)O-labeled QconCAT peptides as internal standards combined with MRM MS for the absolute quantitation of a target proteome is established. Experimental results showed that the novel method had high digestion and (18)O labeling efficiencies, and no (18)O to (16)O back-exchange occurred. A linear range covering 2 orders of magnitude and a limit of quantification (LOQ) as low as 5 fmol were achieved with an RSD below 10%. Then, the quantitative method is used for the absolute quantitation of drug metabolizing enzymes in human liver microsomes. The results are in good agreement with the previously reported data, which demonstrates that the novel method can be used for absolute quantitative analyses of target proteomes in complex biological samples.

A refined ionization model of small molecules was developed based on a unit of classic models. The ionization process was divided into three sub-processes of diffusion, charge transfer, and ion evaporation. Based on the equations, the impact of a single process and multiprocesses on response was verified by employing special samples, and the relative evaporation energies of several pair of samples were calculated. This model is sensitive to slight differences in ionization energy and allows distinction of the ionization differences during each sub-process.

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, Fe 3 O 4 @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 Fe 3 O 4 @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 m 2 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 Fe 3 O 4 @DOTA-ZIF-90 provides a universal method for fabrication of magnetic MOFs materials, and the successful application of Fe 3 O 4 @DOTA-ZIF-90-trypsin in efficient protein digestion for deeper proteome coverage will undoubtedly enlarge the uses for MOFs.

In this work, a novel MoS

A new type of mixed lanthanides-immobilized (Tb(3+) , Tm(3+) , Ho(3+) , Lu(3+) ) magnetic nanoparticles, Fe3 O4 @TCPP-DOTA-M(3+) , was prepared with a particle size of approximately 30 nm. A model protein, α-casein, and a protein mixture of α-casein and BSA (1:100) were first used to test the phosphopeptide enrichment efficiency of the newly developed magnetic nanoparticles. For the model protein α-casein, 19 phosphopeptides were identified with the newly developed materials. Even in the tryptic digest of α-casein and BSA (1:100), 16 phosphopeptides were easily detected, suggesting that the novel materials possess high selectivity in phosphopeptide enrichment. To evaluate the phosphopeptide enrichment efficiency in a real biological sample, the materials were used to capture phosphopeptides in the tryptic digests of an extract of HeLa cells. In total, 9048 phosphopeptides corresponding to 2103 phosphoproteins were identified in a single mass spectrometric analysis, indicating the great potential of the new materials for practical applications. Compared with metal oxide-based enrichment methods, the newly developed materials are convenient to prepare and easy to handle, and they save time in the phosphopeptide enrichment procedure, making these materials a good choice for highly selective and sensitive phosphopeptide enrichment in future phosphoproteome analyses.

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.

Currently, the separation targets of preparative electrophoresis range from milligrams to micrograms of proteins. However, most commercially available preparative electrophoretic instruments function at the milligram level. Although some preparative electrophoretic apparatuses operated at the microgram level, the fractionation results are often unsatisfying because they suffer from low resolution, poor recovery, or a long fractionation time. To address these issues, we developed a novel microscale preparative electrophoresis system that consists of a separation apparatus and an elution apparatus. Protein samples are first loaded onto the separation apparatus and separated over the gel according to the molecular weight of each protein. Then the separation gel is transferred to the elution apparatus and the proteins on the gel are eluted through the thickness of the gel. This system offers the following advantages: (1) high resolution: almost no overlap between the adjacent fractions; (2) a short recovery time: fractionation was performed in 2 hours including separation in 100 min and elution in 20 min and (3) high recovery: recovery was as high as 91.8%.

A new type of mixed lanthanides-immobilized (Tb(3+) , Tm(3+) , Ho(3+) , Lu(3+) ) magnetic nanoparticles, Fe3 O4 @TCPP-DOTA-M(3+) , was prepared with a particle size of approximately 30 nm. A model protein, α-casein, and a protein mixture of α-casein and BSA (1:100) were first used to test the phosphopeptide enrichment efficiency of the newly developed magnetic nanoparticles. For the model protein α-casein, 19 phosphopeptides were identified with the newly developed materials. Even in the tryptic digest of α-casein and BSA (1:100), 16 phosphopeptides were easily detected, suggesting that the novel materials possess high selectivity in phosphopeptide enrichment. To evaluate the phosphopeptide enrichment efficiency in a real biological sample, the materials were used to capture phosphopeptides in the tryptic digests of an extract of HeLa cells. In total, 9048 phosphopeptides corresponding to 2103 phosphoproteins were identified in a single mass spectrometric analysis, indicating the great potential of the new materials for practical applications. Compared with metal oxide-based enrichment methods, the newly developed materials are convenient to prepare and easy to handle, and they save time in the phosphopeptide enrichment procedure, making these materials a good choice for highly selective and sensitive phosphopeptide enrichment in future phosphoproteome analyses.

Metal-organic frameworks (MOFs) are a class of crystalline materials built from organic binding ligands and metal ions through self-assembly. Currently, MOFs have drawn a growing interest among the scientific teams of various fields. Compared with conventional inorganic porous materials, MOFs possess larger specific surface areas, higher porosity and diversity of structures and functions, thus many potential applications have been proposed in the domains of gas adsorption and separation, sensors, drug delivery, catalysis or others. The combinations of MOFs and other materials such as graphene oxide, magnetic nanoparticles have obvious advantages in adsorption and separation. The appearance of novel materials greatly promotes interdisciplinary developments such as organic chemistry, inorganic chemistry, coordination chemistry, materials chemistry, life science and computer science. This article reviews the progress of MOFs in recent years, including the characteristics of MOFs, advances at home and abroad, applications, central issues of compound MOFs and the prospects in the future.