Palladium-Catalyzed Hiyama Cross-Coupling Reactions of Aryl Mesylates

The combination of palladium acetate with XPhos shows high efficiency in the Hiyama cross-coupling reactions of aryl mesylates with arylsilanes. The reactions proceed smoothly to generate the corresponding biaryl compounds in good yields

Mass Spectrometry Analysis of Phosphopeptides after Peptide Carboxy Group Derivatization

A nearly 100% yield peptide carboxy group derivatization method was offered to largely enhance phosphopeptide ionization efficiency. This method, adopting 1-(2-pyrimidyl) piperazine (PP) as the derivatization reagent, shows several advantages such as good reproducibility, ease of handling, rapid reaction time, and no side reaction. PP derivatization improves the hydrophobicities, pI values, and gas-phase basicities of peptides especially those of phosphopeptides. In the matrix assisted laser desorption ionization (MALDI) source, the ionization efficiencies of four synthetic phosphopeptides were increased by 50−101 times while that of three nonphosphopeptides were 10−40-fold. In the electrospray ionization (ESI) source, PP-derivatized phosphopeptides also gave much higher ionization efficiency improvements than nonphosphopeptides. When this method was applied to much more complex mixtures, tryptic BSA digests spiked with one single phosphopeptide in different molar ratios, the signal intensity of this phosphopeptide always had the largest increment among all those peptides. Obviously, this easily manipulated as well as highly specific method provides a promising tool for high-throughput phosphoproteome research

Endoglycosidase-Mediated Incorporation of ¹⁸O into Glycans for Relative Glycan Quantitation

Stable isotopic labeling coupled with mass spectrometry analysis is a promising method of detecting quantitative variations in glycans, which may result in aberrant glycosylation in many disorders and diseases. Although various isotopic labeling methods have been used for relative glycan quantitation, enzymatic 18O labeling, which offers advantages for glycomics similar to those by protease-catalyzed 18O labeling for proteomics, has not been developed yet. In this study, endoglycosidase incorporated 18O into the N-glycan reducing end in 18O-water as N-glycans were released from glycoproteins, rendering glycan reducing-end 18O labeling (GREOL) a potential strategy for relative glycan quantitation. This proposed method provided good linearity with high reproducibility within 2 orders of magnitude in dynamic range. The ability of GREOL to quantitatively discriminate between isomeric hybrid N-glycans and complex N-glycans in glycoproteins was validated due to the distinct substrate specificities of endoglycosidases. GREOL was also used to analyze changes in human serum N-glycans associated with hepatocellular carcinoma

Synthesis of Fe₃O₄/Graphene/TiO₂ Composites for the Highly Selective Enrichment of Phosphopeptides from Biological Samples

In this work, Fe₃O₄/graphene/TiO₂ composites with a large surface area were designed and synthesized for the selective extraction and enrichment of phosphopeptides from biological samples. First, magnetic graphene was prepared according to our previous method. Next, we made the Fe₃O₄/graphene/TiO₂ composite precursor using tetrabutyl titanate. Fe₃O₄/graphene/TiO₂ composites were obtained after solvothermal and calcination treatments. We used standard protein-digestion solutions and human liver samples to test the enrichment ability of the obtained Fe₃O₄/graphene/TiO₂ composites. The experimental results demonstrate that Fe₃O₄/graphene/TiO₂ composites have a good phosphopeptide enrichment ability

Synthesis of Highly Water-Dispersible Polydopamine-Modified Multiwalled Carbon Nanotubes for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Analysis

In this work, we synthesized highly water-dispersible multiwalled carbon nanotubes@polydopamine (MWCNTs@PDA) core–shell composites by a facile in situ oxidative polymerization. The composites were successfully applied as a novel matrix for the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF–MS) analysis of various water-soluble small molecule compounds. It was found that MWCNTs@PDA composites have a higher sensitivity and peak intensities for small molecules detection

Designed Synthesis of Aptamer-Immobilized Magnetic Mesoporous Silica/Au Nanocomposites for Highly Selective Enrichment and Detection of Insulin

We designed and synthesized aptamer-immobilized magnetic mesoporous silica/Au nanocomposites (MMANs) for highly selective detection of unlabeled insulin in complex biological media using MALDI-TOF MS. The aptamer was easily anchored onto the gold nanoparticles in the mesochannels of MMANs with high capacity for highly efficient and specific enrichment of insulin. With the benefit from the size-exclusion effect of the mesoporous silica shell with a narrow pore size distribution (∼2.9 nm), insulin could be selectively detected despite interference from seven untargeted proteins with different size dimensions. This method exhibited an excellent response for insulin in the range 2–1000 ng mL^–1. Moreover, good recoveries in the detection of insulin in 20-fold diluted human serum were achieved. We anticipate that this novel method could be extended to other biomarkers of interest and potentially applied in disease diagnostics

14-3-3ε Boosts Bleomycin-induced DNA Damage Response by Inhibiting the Drug-resistant Activity of MVP

Major vault protein (MVP) is the predominant constituent of the vault particle, the largest known ribonuclear protein complex. Although emerging evidence have been establishing the links between MVP (vault) and multidrug resistance (MDR), little is known regarding exactly how the MDR activity of MVP is modulated during cellular response to drug-induced DNA damage (DDR). Bleomycin (BLM), an anticancer drug, induces DNA double-stranded breaks (DSBs) and consequently triggers the cellular DDR. Due to its physiological implications in hepatocellular carcinoma (HCC) and cell fate decision, 14-3-3ε was chosen as the pathway-specific bait protein to identify the critical target(s) responsible for HCC MDR. By using an LC–MS/MS-based proteomic approach, MVP was first identified in the BLM-induced 14-3-3ε interactome formed in HCC cells. Biological characterization revealed that MVP possesses specific activity to promote the resistance to the BLM-induced DDR. On the other hand, 14-3-3ε enhances BLM-induced DDR by interacting with MVP. Mechanistic investigation further revealed that 14-3-3ε, in a phosphorylation-dependent manner, binds to the phosphorylated sites at both Thr52 and Ser864 of the monomer of MVP. Consequently, the phosphorylation-dependent binding between 14-3-3ε and MVP inhibits the drug-resistant activity of MVP for an enhanced DDR to BLM treatment. Our findings provide an insight into the mechanism underlying how the BLM-induced interaction between 14-3-3ε and MVP modulates MDR, implicating novel strategy to overcome the chemotherapeutic resistance through interfering specific protein–protein interactions

Infrared-Assisted On-Plate Proteolysis for MALDI-TOF-MS Peptide Mapping

In this report, infrared (IR)-assisted on-plate proteolysis has been developed for rapid peptide mapping. Protein solutions containing trypsin were allowed to digest directly on the spots of matrix-assisted laser desorption/ionization (MALDI) plates under IR radiation. The feasibility and performance of the novel proteolysis approach were investigated by the digestion of bovine serum albumin (BSA) and cytochrome c (Cyt-c). It was demonstrated that IR radiation substantially enhanced the efficiency of proteolysis and the digestion time was significantly reduced to 5 min. The digests were identified by MALDI time-of-flight mass spectrometry with sequence coverages of 55 (BSA) and 75% (Cyt-c) that were comparable to those obtained by using conventional in-solution tryptic digestion. The suitability of IR-assisted on-plate proteolysis to complex proteins was demonstrated by digesting human serum and casein extracted from commercially available milk sample. The present proteolysis strategy is simple and efficient, offering great promise for high-throughput protein identification

Multifunctional Nanoreactor for Comprehensive Characterization of Membrane Proteins Based on Surface Functionalized Mesoporous Foams

An integrated protocol is proposed here for efficient analysis of membrane proteins based on surface functionalized mesoporous graphene foams (MGF). The inherent hydrophobic nature of MGF and surface modification with hydrophilic chitosan (CS) make it highly suitable for the enrichment of hydrophobic membrane proteins from organic solvent, while remaining well-dispersed in aqueous solution for subsequent proteolysis. Therefore, such a multifunctional reactor ensures a facile solvent adjustment route. Furthermore, as a chitosan modified nanoporous reactor, it also provides a biocompatible nanoenvironment that can maintain the stability and activity of enzymes to realize efficient <i>in situ</i> digestion of the enriched membrane proteins. The concept was first proved with a standard hydrophobic membrane protein, bacteriorhodopsin, where a high number of identified peptides and amino acid sequence coverage were achieved even at extremely low protein concentration. The mesoporous reaction system was further applied to the analysis of complex real-case proteome samples, where 931 membrane proteins were identified in triplicate analyses by 2D LC-MS/MS. In contrast, with in-solution proteolysis, only 73 membrane proteins were identified from the same sample by the same 2D LC-MS/MS. The identified membrane proteins by the MGF-CS protocol include many biomarkers of the cell line. These results suggest that the multifunctional MGF-CS protocol is of great value to facilitate the comprehensive characterization of membrane proteins in the proteome research

BLM-triggered association between 14-3-3ε and TAK1 correlates to the phosphorylation-dependent activation of TAK1.

<p><b>A.</b> Immunoblotting analysis of the BLM-induced interaction between 14-3-3ε and TAK1. <b>B.</b> Immunoblotting analysis of the BLM-induced interaction between 14-3-3ε and the known TAK1 binding protein TAB1. <b>C.</b> BLM-induced time-dependent phosphorylation changes of TAK1in HCC cells. The whole cell lysates were blotted against anti-phospho-TAK1 (Thr187) antibody. <b>D.</b> The change of phosphorylated TAK1 interacting with 14-3-3ε of HCC response to dose-dependent BLM stimulation.</p