Proteomic Analysis Reveals a Role for RSK in p120-catenin Phosphorylation and Melanoma Cell-Cell Adhesion

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Highlights

• •BioID reveals the proximity partners of RSK family members.
• •All RSK isoforms associate with and phosphorylate p120ctn on Ser320.
• •RSK negatively regulates adherens junctions and reduces cell-cell adhesion.
• •p120ctn phosphorylation plays a role in the reorganization of proximity partners.

     

Improving Identification of In-organello Protein-Protein Interactions Using an Affinity-enrichable,Isotopically Coded,and Mass Spectrometry-cleavable Chemical Crosslinker

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Highlights

• •Used affinity-enrichable, isotopically coded, and MS-cleavable crosslinker.
• •Targeted acquisition strategy based on isotopic-coding described and evaluated.
• •Novel data analysis pipeline developed provides improved crosslink identification.
• •Large dataset reveals hundreds of mitochondrial protein-protein interactions.

     

Developments and Applications of Functional Protein Microarrays

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Highlights

• •Summarize the development of functional protein microarray.
• •Application of functional proteome microarray in basic research.
• •Application of functional proteome microarray in translational research.
• •Fabrication of functional membrane protein array using virion display method.

     

A Quantitative Tri-fluorescent Yeast Two-hybrid System: From Flow Cytometry to In cellula Affinities

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Highlights

• •Simultaneous quantification of Bait, Prey and Reporter at the single cell level.
• •Two hours of reaction are enough instead of 24–48 h for conventional assays.
• •Potential expression problems of the Bait and Prey can be easily detected.
• •True positive PPIs feature a distinct pattern of Reporter level versus Bait/Prey level.
• •PPIs with unknown affinities can be ranked using an affinity ladder.

     

Next-generation Interactomics: Considerations for the Use of Co-elution to Measure Protein Interaction Networks

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Highlights

• •Co-elution stands out as a global interactome mapping method.
• •Benefits include all-to-all protein analysis and measurement of interactome perturbations.
• •Different separation, quantification and bioinformatic strategies are available.
• •Design considerations depend largely on system under study.

     

Analytical Guidelines for co-fractionation Mass Spectrometry Obtained through Global Profiling of Gold Standard Saccharomyces cerevisiae Protein Complexes

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Highlights

• •Guidelines for studying protein complexes via co-fractionation mass spectrometry.
• •A novel procedure for profiling gold standard protein complexes in CF-MS data.
• •Recommendations for efficient CF-MS fractionation collection.
• •Scoring metric recommendations for precise and sensitive CF-MS data analysis.

     

MaXLinker: Proteome-wide Cross-link Identifications with High Specificity and Sensitivity

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Highlights

• •New quality assessment metrics to evaluate proteome-wide cross-linking mass spectrometry (XL-MS) data sets.
• •New “MS3-centric” cross-link search engine named MaXLinker with high sensitivity and specificity.
• •More than 9300 cross-links from our human proteome-wide XL-MS study.
• •Orthogonal experimental validation of novel interactions identified in our study.

     

AMPK Interactome Reveals New Function in Non-homologous End Joining DNA Repair

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Highlights

• •Each component of the AMPK trimeric complex was profiled by interaction proteomics.
• •The subunit composition of the AMPK complex directs interactions to distinct proteins.
• •AMPK interacts with Artemis and plays a role in Non-Homologous End Joining.

     

Proximity Dependent Biotinylation: Key Enzymes and Adaptation to Proteomics Approaches

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Highlights

• •Proximity-dependent biotinylation (PDB) approaches involve fusion of a bait with an enzyme.
• •BioID (biotin protein ligase) and APEX (peroxidase) are distinct enzymes used in PDB.
• •Past, present and future development and applications of PDB are discussed.
• •We review labeling mechanisms and kinetics to provide guidance for experimental design.
• •We discuss controls and considerations for data interpretation.

     

Characterization of Prenylated C-terminal Peptides Using a Thiopropyl-based Capture Technique and LC-MS/MS

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Highlights

• •Brain membrane protein extraction.
• •Protein prenylation.
• •Prenyl peptide capture and characterization by LC-MS/MS.
• •HCD and EThcD peptide fragmentation.

     

FYN and ABL Regulate the Interaction Networks of the DCBLD Receptor Family

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Highlights

• •FYN and ABL differentially regulate DCBLD Ser/Thr/Tyr phosphorylation.
• •DCBLD1 and DCBLD2 interactomes are modulated by FYN and ABL.
• •ABL drives a direct DCBLD2/14-3-3 interaction.

     

Characterization of Signaling Pathways Associated with Pancreatic β-cell Adaptive Flexibility in Compensation of Obesity-linked Diabetes in db/db Mice

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Highlights

• •db/db β-cells restores appropriate insulin stores and normalize secretory function.
• •Numerous changes in the phosphorylation and sialylation states by euglycemic rest.
• •Restoration of numerous dysfunctional biological processes following euglycemic rest.
• •β-cell adaptive flexibility may lead to improvement in endogenous β-cell function.

     

Arginine in C9ORF72 Dipolypeptides Mediates Promiscuous Proteome Binding and Multiple Modes of Toxicity

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Highlights

• •Quantitative proteome interactions with 5 different C9ORF72 dipolypeptides (DPRs).
• •The arg-rich DPRs promiscuously bound to the proteome compared with the other DPRs.
• •Long repeat lengths of arg-rich DPRs, but not short lengths, stalled ribosomes.
• •The arg-rich DPRs also reduced arginine methylation and actin cytoskeleton assembly.

     

Characterizing Patients with Recurrent Urinary Tract Infections in Vesicoureteral Reflux: A Pilot Study of the Urinary Proteome

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Highlights

• •Urinary proteomes of patients with recurrent UTI, renal scarring, and VUR.
• •80 proteins differentially expressed, compared to healthy controls.
• •62 proteins may be indicative of susceptibility for UTI.
• •Altered acute phase response, extracellular matrix and carbohydrate metabolism.

     

An Improved Boosting to Amplify Signal with Isobaric Labeling (iBASIL) Strategy for Precise Quantitative Single-cell Proteomics

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Highlights

• •Higher AGC significantly improves quantitation quality in single-cell analysis.
• •The boosting-to-sample ratio should be carefully evaluated and optimized.
• •iBASIL allows for precise quantitation of 1,500 proteins from 104 AML single cells.
• •iBASIL recapitulates major biological differences in different AML single cells.

     

Global Proteome and Phosphoproteome Characterization of Sepsis-induced Kidney Injury

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Highlights

• •Cecal Ligation Puncture (CLP) mouse model to study sepsis-induced kidney disease.
• •Quantitative global proteome and phosphoproteome profiling of mouse kidneys.
• •Highly significant candidate markers for onset and progression of AKI to CKD.
• •Mechanistic insights into sepsis-associated kidney injuries.

     

Proteaphagy in Mammalian Cells Can Function Independent of ATG5/ATG7

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Highlights

• •Quantitative proteomics of isolated lysosomes, autophagosomes and proteasomes.
• •Pharmacological inhibition of proteasomes leads to their accumulation within lysosomes.
• •Inhibition of classical autophagy pathways cannot completely block this process.
• •Known autophagy adaptor proteins are not involved.

     

A Novel Mechanism for NF-κB-activation via IκB-aggregation: Implications for Hepatic Mallory-Denk-Body Induced Inflammation

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Highlights

• •Liver Mallory-Denk-Body inducers elicited an IκBα-loss and NF-κB-activation.
• •IκBα-loss was due to its sequestration into insoluble cytoplasmic aggregates.
• •Four proteomic approaches identified 10 IκBα-interacting/aggregating proteins.
• •Nup153/RanBP2-aggregation prevented IκBα nuclear entry for ending NF-κB-activation.

     

Concentration Determination of >200 Proteins in Dried Blood Spots for Biomarker Discovery and Validation

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Highlights

• •Repeatable quantification of 200 proteins in dried blood spots.
• •Determined lower limit of quantification, repeatability, parallelism and stability.
• •Protein stability in DBS stored at ambient temperatures for up to 2 months.
• •Concentration ranges for 200 proteins in 20 healthy individuals.

     

Genetic Profile and Functional Proteomics of Anal Squamous Cell Carcinoma: Proposal for a Molecular Classification

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Highlights

• •Two molecular groups in anal squamous carcinoma according proteomic profile.
• •Differences in possible targeted processes such as metabolism or immune response.
• •Different percentage of tumor lymphocyte infiltration.
• •Difference in the frequency of ATM variants, related to PPAR inhibitors.