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.

A novel mass spectrometry method for the absolute quantification of several cytochrome P450 and uridine 5'-diphospho-glucuronosyltransferase enzymes in the human liver.

Cytochrome P450 (CYP450) and 5'-diphosphate glucuronosyltransferases (UGT) are the two major families of drug-metabolizing enzymes in the human liver microsome (HLM). As a result of their frequent abundance fluctuation among populations, the accurate quantification of these enzymes in different individuals is important for designing patient-specific dosage regimens in the framework of precision medicine. The preparation and quantification of internal standards is an essential step for the quantitative analysis of enzymes. However, the commonly employed stable isotope labeling-based strategy (QconCAT) suffers from requiring very expensive isotopic reagents, tedious experimental procedures, and long labeling times. Furthermore, arginine-to-proline conversion during metabolic isotopic labeling compromises the quantification accuracy. Therefore, we present a new strategy that replaces stable isotope-labeled amino acids with lanthanide labeling for the preparation and quantification of QconCAT internal standard peptides, which leads to a threefold reduction in the reagent costs and a fivefold reduction in the time consumed. The absolute amount of trypsin-digested QconCAT peptides can be obtained by lanthanide labeling and inductively coupled plasma-optical emission spectrometry (ICP-OES) analysis with a high quantification accuracy (%RE < 20%). By taking advantage of the highly selective and facile ICP-OES procedure and multiplexed large-scale absolute target protein quantification using biological mass spectrometry, this strategy was successfully used for the absolute quantification of drug-metabolizing enzymes. We obtained good linearity (correlation coefficient > 0.95) over concentrations spanning 2.5 orders of magnitude with improved sensitivity (limit of quantification = 2 fmol) in nine HLM samples, indicating the potential of this method for large-scale absolute target protein quantification in clinical samples. Graphical abstract.

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.

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.

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.