Benefits of Collisional Cross Section Assisted Precursor Selection (caps-PASEF) for Cross-linking Mass Spectrometry

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Highlights

• •Cross-linked peptides are physically separated from mono-linked peptides in the gas-phase by TIMS ion mobility.
• •Development of a novel data acquisition routine that a-priori distinguishes cross-linked from mono-linked peptides called caps-PASEF.
• •First application of PhoX-driven cross-linking mass spectrometry on the timsTOF Pro.
• •Application of cross-linking mass spectrometry to medium to high complexity samples.

     

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.

     

Single-cell Proteomics: Progress and Prospects

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Highlights

• •Label-free and isobaric labeling approaches for in-depth profiling of single cells.
• •Miniaturization and simplification of sample processing reduce surface losses.
• •Nanoflow separations enhance ionization efficiency and reduced chemical noise.
• •Ultrasensitive mass spectrometry and gas-phase separation add selectivity.

     

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.

     

ProAlanase is an Effective Alternative to Trypsin for Proteomics Applications and Disulfide Bond Mapping

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Highlights

• •ProAlanase is a powerful protease for efficient low pH disulfide bond mapping.
• •High suitability for analysis of histone family members and their PTMs.
• •Accurate phosphorylation profiling in proline-rich proteins.
• •Sequence coverage increase and full de novo sequencing in combination with trypsin.

     

Profiling Cell Signaling Networks at Single-cell Resolution

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Highlights

• •Signaling networks can be highly heterogeneous across cells in a tissue.
• •Various technologies allow analyzing signaling networks at single-cell resolution.
• •The advantages and limitations of each single-cell approach are summarized.
• •Confounding factors in single-cell signaling network analysis are discussed.

     

Agonists of Orally Expressed TRP Channels Stimulate Salivary Secretion and Modify the Salivary Proteome

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Highlights

• •Salivary secretion was increased by mouth rinsing with TRP channel agonists.
• •The salivary proteome varied over time and was changed by TRP channel stimulation.
• •Immunoreactive Cystatin S was increased in saliva after TRPV1 stimulation.

     

Organellar Maps Through Proteomic Profiling – A Conceptual Guide

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Highlights

• •Organelle profiling maps capture localizations of 1000s of proteins in one experiment.
• •Comparing maps +/− perturbation reveals disease mechanisms & cellular responses.
• •A conceptual guide to planning and interpreting organellar profiling experiments.
• •A cross-study consensus set of human organellar marker proteins.

     

A Cross-linking Mass Spectrometry Approach Defines Protein Interactions in Yeast Mitochondria

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Highlights

• •XL-MS reveals new PPIs in yeast mitochondria under glycerol and glucose condition.
• •Significant but limited results from quantitative XL-MS experiments.
• •Ndi1 participates in a CIII₂CIV₂ respiratory supercomplex.
• •Min8 promotes assembly of Cox12 into an intermediate complex IV.

     

An Interaction Network of RNA-Binding Proteins Involved in Drosophila Oogenesis

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Highlights

• •Label-free and dimethyl labeling MS analysis of 6 RBPs from Drosophila ovaries.
• •Functionally related RBPs show overlapping proteomes.
• •Selective co-purification of splicing factors and translational regulators.
• •Validation of 26 novel interactions by co-immunoprecipitation.

     

Molecular Dynamics Simulation-assisted Ionic Liquid Screening for Deep Coverage Proteome Analysis

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Highlights

• •Mechanistic insights into ionic liquids and proteins at molecular level.
• •Extractants prescreen for proteome analysis with MD simulation system.
• •A loss-less sample preparation method developed for in-depth proteome profiling.
• •Over 3,300 proteins were confidently identified from 1,000 HeLa cells in a 1 h run.
• •Label-free quantitative proteome analysis of human liver cancer tissues.

     

Stoichiometry of Nucleotide Binding to Proteasome AAA+ ATPase Hexamer Established by Native Mass Spectrometry

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Highlights

• •All six binding sites in PAN^WT are occupied by ADP- or ATP-type nucleotides.
• •PAN^KA Walker A mutant substoichiometrically binds ATP- but not ADP-type nucleotides.
• •PAN hexamer dissociation of the solution origin characteristics was observed in MS.
• •We posit that the PAN hexamer dissociation proceeds within the ESI droplets.

     

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.

     

High-speed Analysis of Large Sample Sets – How Can This Key Aspect of the Omics Be Achieved?

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Highlights

• •Future proteomic analyses for longitudinal studies and P4 medicine arguably require ≥1M samples/day.
• •Proteome depth/coverage is commonly the focus whereas analytical speed is typically neglected.
• •A compromise between analytical depth and speed is needed for future large-scale studies.
• •Ultrahigh-speed ‘omic’ analyses require tools that are intrinsically fast such as laser-based MS.

     

Proteomic Analysis Reveals that Topoisomerase 2A is Associated with Defective Sperm Head Morphology

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Highlights

• •Human spermatozoa possess cells of poor morphology that lack nuclear integrity.
• •These cells can be isolated by density separation.
• •Mass spectrometry reveals their nuclei contain excess protein.
• •TOP2A is a promising marker of this poor nuclear development.

     

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.

     

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.

     

Kir2.1 Interactome Mapping Uncovers PKP4 as a Modulator of the Kir2.1-Regulated Inward Rectifier Potassium Currents

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Highlights

• •Generation using BioID of a map of the Kir2.1 interactome with 218 interactions.
• •Identification of Kir2.1^WT- versus Kir2.1^Δ314-315-preferred interactors.
• •Identification of the desmosome protein PKP4 as a new modulator of I_Kir2.1 currents.