41 research outputs found

    Ultrasensitive Proteome Profiling for 100 Living Cells by Direct Cell Injection, Online Digestion and Nano-LC-MS/MS Analysis

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    Single-cell proteome analysis has always been an exciting goal because it provides crucial information about cellular heterogeneity and dynamic change. Here we presented an integrated proteome analysis device (iPAD) for 100 living cells (iPAD-100) that might be suitable for single-cell analysis. Once cells were cultured, the iPAD-100 could be applied to inject 100 living cells, to transform the living cells into peptides, and to produce protein identification results with total automation. Due to the major obstacle for detection limit of mass spectrometry, we applied the iPAD-100 to analyze the proteome of 100 cells. In total, 813 proteins were identified in a DLD-cell proteome by three duplicate runs. Gene Ontology analysis revealed that proteins from different cellular compartments were well-represented, including membrane proteins. The iPAD-100 greatly simplified the sampling process, reduced sample loss, and prevented contamination. As a result, proteins whose copy numbers were lower than 1000 were identified from 100-cell samples with the iPAD-100, showing that a detection limit of 200 zmol was achieved. With increased sensitivity of mass spectrometry, the iPAD-100 may be able to reveal bountiful proteome information from a single cell in the near future

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

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    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

    Periodic Mesoporous Organosilica as a Multifunctional Nanodevice for Large-Scale Characterization of Membrane Proteins

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    A versatile protocol has been developed for large-scale characterization of hydrophobic membrane proteins based on the periodic mesoporous organosilica (PMO) acting as both an extractor for hydrophobic substrate capture and a nanoreactor for efficient in situ digestion. With introduction of organic groups in the pore frameworks and the presence of hydrophilic silanol groups on the surface, PMO can be well-dispersed into not only an organic solution to concentrate the dissolved membrane proteins but also an aqueous solution containing enzymes for sequential rapid proteolysis in the nanopores. The unique amphiphilic property of PMO ensures a facile switch in different solutions to realize the processes of substrate dissolution, enrichment, and digestion effectively. Furthermore, this novel PMO-assisted protocol has been successfully applied for identification of complex membrane proteins extracted from mouse liver as proof of general applicability

    Nano-LC-MS/MS mass spectra of doubly charged O-GlcNAcylated peptides.

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    <p>FGVEAFS#DCLR [(M+2H)<sup>2+</sup> at m/z 718.87] (A) from D-beta- hydroxybutyrate dehydrogenase, and VLGTS#VESIMATEDR [(M+2H)<sup>2+</sup> at m/z 872.45] (B) from carbamoyl-phosphate synthase.</p

    Discovery and Confirmation of O-GlcNAcylated Proteins in Rat Liver Mitochondria by Combination of Mass Spectrometry and Immunological Methods

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    <div><p>O-linked β-N-acetylglucosamine (O-GlcNAc) is an important post-translational modification (PTM) consisting of a single N-acetylglucosamine moiety attached via an O-β-glycosidic linkage to serine and threonine residues. Glycosylation with O-GlcNAc occurs on myriad nuclear and cytosolic proteins from almost all functional classes. However, with respect to O-GlcNAcylated proteins special in mitochondria, little attention has been paid. In this study, we combined mass spectrometry and immunological methods to perform global exploration of O-GlcNAcylated proteins specific in mitochondria of rat liver. First, highly purified mitochondrial proteins were obviously shown to be O-GlcNAcylated by immunoblot profiling. Then, β-elimination followed by Michael Addition with Dithiothreitol (BEMAD) treatment and LC-MS/MS were performed to enrich and identify O-GlcNAcylated mitochondrial proteins, resulting in an unambiguous assignment of 14 O-GlcNAcylation sites, mapping to 11 O-GlcNAcylated proteins. Furthermore, the identified O-GlcNAcylated mitochondrial proteins were fully validated by both electron transfer dissociation tandem mass spectrometry (ETD/MS/MS) and western blot. Thus, for the first time, our study definitely not only identified but also validated that some mitochondrial proteins in rat liver are O-GlcNAcylated. Interestingly, all of these O-GlcNAcylated mitochondrial proteins are enzymes, the majority of which are involved in a wide variety of biological processes, such as urea cycle, tricarboxylic acid cycle and lipid metabolism, indicating a role for protein O-GlcNAcylation in mitochondrial function.</p> </div

    The level of O-GlcNAcylation in mitochondrial and cytosolic fractions.

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    <p>Mitochondrial and cytosolic proteins from rat livers were separated and analyzed by western blot. The membrane was probed with RL2 (A), and RL2 plus 1 M GlcNAc to exclude nonspecific binding of RL2 antibody (B).</p

    The most enriched sub-networks in IPA results.

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    <p>Gray symbols with orange rim are O-GlcNAcylated mitochondrial proteins identified in this study. Symbol ◊ is used for enzyme, □ for nuclear receptor, rectangle for ion channel, and ○ for others.</p

    Site-Specific Quantification of Protein Ubiquitination on MS2 Fragment Ion Level via Isobaric Peptide Labeling

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    Proteome-wide quantitative analysis of protein ubiquitination is important to gain insight into its various cellular functions. However, it is still challenging to monitor how ubiquitination at each individual lysine residue is independently regulated, especially the whereabouts of peptides containing more than one ubiquitination site. In recent years, isobaric peptide termini labeling has been considered a promising strategy in quantitative proteomics, benefiting from its high accuracy by quantifying with a series of b, y fragment ion pairs. Herein, we extended the concept of isobaric peptide termini labeling to large-scale quantitative analysis of protein ubiquitination. A novel MS2 fragment ion based quantitative approach was developed, allowing the quantification of ubiquitination at site level via isobaric K-ε-GG peptide labeling, which combined metabolic labeling, K-ε-GG immunoaffinity enrichment, and site-selective N-terminus dimethylation. The feasibility of this proposed strategy was demonstrated through the ubiquitin proteome analysis of differently labeled MCF-7 cell digests. As a result, 2970 unique K-ε-GG peptides of 1383 proteins containing 2874 ubiquitinated sites were confidently quantified with high accuracy and sensitivity. In addition, we demonstrated that quantification on MS2 fragment ion level makes it possible to precisely quantify each individual ubiquitinated lysine residue in 39 K-ε-GG peptides bearing two ubiquitination sites by the use of specific ubiquitinated b, y ion pairs. It is expected that this proposed approach will serve as a powerful tool to quantify ubiquitination at the site level, especially for those multiubiquitinated peptides

    Purity and integrity of mitochondrial fractions.

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    <p>Mitochondrial proteins were applied to western blot. Cytosolic proteins, nuclear proteins and total rat liver proteins were also applied to western blot. Mitochondria marker COX3, nucleus marker c-Jun, and cytosol marker GAPDH were used to detect the purity and integrity of mitochondria. PM: purified mitochondria; Cyto: cytosol; PN: purified nucleus; TL: total liver.</p

    Nano-LC-ETD/MS/MS mass spectra of O-GlcNAcylated peptides.

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    <p>VL<i>S</i>IGDGIAR [(M+3H)<sup>3+</sup> at m/z 401.89084] (A) from ATP synthase subunit alpha, and VLDA<i>S</i>WYSPGTR [(M+3H)<sup>3+</sup> at m/z 518.92157] (B) from Thiosulfate sulfurtransferase.</p