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.

     

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.

     

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.

     

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.

     

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.

     

MaxQuant Software for Ion Mobility Enhanced Shotgun Proteomics

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Highlights

• •Highly parallelizable 4D feature detection in ion mobility enhanced shotgun proteomics.
• •Multidimensional non-linear mass, retention time and ion mobility recalibration.
• •Collision cross section aware matching between runs.
• •Label-free quantification of ion mobility MS data.

     

Selection of Features with Consistent Profiles Improves Relative Protein Quantification in Mass Spectrometry Experiments

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Highlights

• •Automated statistical approach for detecting uninformative features and outliers.
• •Improved performance on relative protein quantification.
• •An option in the open-source R-based software MSstats.

     

Peptidomic Analysis of Urine from Youths with Early Type 1 Diabetes Reveals Novel Bioactivity of Uromodulin Peptides In Vitro

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Highlights

• •Urinary peptide profiling of youths with type 1 diabetes before clinical injury.
• •Internal validation of uromodulin peptides by parallel reaction monitoring.
• •Discovery of novel bioactivity of uromodulin peptides in vitro.
• •In silico prediction of proteases involved in uromodulin processing.

     

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.

     

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.

     

Acquiring and Analyzing Data Independent Acquisition Proteomics Experiments without Spectrum Libraries

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Highlights

• •Rapid DIA-only library building with gas-phase fractionation.
• •Recommended DIA acquisition strategies with staggered windows and forbidden zones.
• •Optimized DIA instrument settings for several Thermo Orbitrap instruments.
• •Data analysis tutorial using open source DIA software.

     

Identification of Microorganisms by Liquid Chromatography-Mass Spectrometry (LC-MS1) and in Silico Peptide Mass Libraries

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Highlights

• •Rapid identification of bacteria by LC-MS¹ and in silico databases.
• •Pattern analysis-based strategy for identifying bacteria by experimental LC-MS¹ data.
• •Compilation of an in silico database of taxon-specific tryptic peptide mass profiles.
• •Tests of the identification pipeline with LC-MS¹ data from bacteria.

     

Tandem Mass Tag Approach Utilizing Pervanadate BOOST Channels Delivers Deeper Quantitative Characterization of the Tyrosine Phosphoproteome

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Highlights

• •Detection of low-abundance phosphotyrosine-containing peptides is challenging.
• •Multiplexed TMT allows inclusion of modification(pTyr)-saturated boost channels.
• •Boost channels facilitate selection of pTyr precursor ions for fragmentation.
• •Quantitation depth is increased while maintaining accuracy and precision.

     

An in-depth Comparison of the Pediatric and Adult Urinary N-glycomes

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Highlights

• •N-glycan patterns are distinct in pediatric and adult urine.
• •Sex differences of N-glycans are much larger in adults.
• •Pediatric urine has almost no sex differences in N-glycan levels.
• •In adults, the majority of N-glycans were more abundant in males.

     

Spatially Resolved Activity-based Proteomic Profiles of the Murine Small Intestinal Lipases

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Highlights

• •Activity based serine hydrolase profiles in 11 fractions along the murine small intestine.
• •AADAC and Ces2e activity profiles correlate with chylomicron secretion.
• •Ces2e shows the highest activity based enrichment in the entire small intestine.

     

Fast Quantitative Analysis of timsTOF PASEF Data with MSFragger and IonQuant

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Highlights

• •MSFragger now supports raw timsTOF PASEF data.
• •IonQuant performs fast and accurate feature detection and quantification.
• •MSFragger and IonQuant provide excellent performance for timsTOF PASEF data.
• •Flexibility allows for complex analyses, such as semi-enzymatic and open search.

     

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.

     

Asparagine Hydroxylation is a Reversible Post-translational Modification

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Highlights

• •Quantitative mass spectrometric method to monitor PTM stability.
• •Pulse labeling reveals dehydroxylation of several asparagine hydroxylation sites.
• •Reversal of TNKS2, TRPV3 and HIF1a asparagine hydroxylation sites.
• •Protein dehydroxylation is an additional level of control for cellular signaling networks.

     

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.