Skyline is a freely available, open‐source Windows client application for accelerating targeted proteomics experimentation, with an emphasis on the proteomics and mass spectrometry community as users ...and as contributors. This review covers the informatics encompassed by the Skyline ecosystem, from computationally assisted targeted mass spectrometry method development, to raw acquisition file data processing, and quantitative analysis and results sharing.
Data independent acquisition (DIA) mass spectrometry is a powerful technique that is improving the reproducibility and throughput of proteomics studies. Here, we introduce an experimental workflow ...that uses this technique to construct chromatogram libraries that capture fragment ion chromatographic peak shape and retention time for every detectable peptide in a proteomics experiment. These coordinates calibrate protein databases or spectrum libraries to a specific mass spectrometer and chromatography setup, facilitating DIA-only pipelines and the reuse of global resource libraries. We also present EncyclopeDIA, a software tool for generating and searching chromatogram libraries, and demonstrate the performance of our workflow by quantifying proteins in human and yeast cells. We find that by exploiting calibrated retention time and fragmentation specificity in chromatogram libraries, EncyclopeDIA can detect 20-25% more peptides from DIA experiments than with data dependent acquisition-based spectrum libraries alone.
Data independent acquisition (DIA) is an attractive method for quantitative proteomics. However, most DIA methods require collecting exhaustive, sample-specific spectrum libraries with data dependent ...acquisition (DDA) to detect and quantify peptides. Studies of non-human organisms, splice junctions, sequence variants, or simply working with small sample yields can make developing spectrum libraries impractical. Here we illustrate a method to efficiently generate DIA-only chromatogram libraries and demonstrate best practices for how to acquire, queue, and validate DIA data 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.
Data independent acquisition (DIA) is an attractive alternative to standard shotgun proteomics methods for quantitative experiments. However, most DIA methods require collecting exhaustive, sample-specific spectrum libraries with data dependent acquisition (DDA) to detect and quantify peptides. In addition to working with non-human samples, studies of splice junctions, sequence variants, or simply working with small sample yields can make developing DDA-based spectrum libraries impractical. Here we illustrate how to acquire, queue, and validate DIA data without spectrum libraries, and provide a workflow to efficiently generate DIA-only chromatogram libraries using gas-phase fractionation (GPF). We present best-practice methods for collecting DIA data using Orbitrap-based instruments and develop an understanding for why DIA using an Orbitrap mass spectrometer should be approached differently than when using time-of-flight instruments. Finally, we discuss several methods for analyzing DIA data without libraries.
Diastolic dysfunction is a prominent feature of cardiac aging in both mice and humans. We show here that 8-week treatment of old mice with the mitochondrial targeted peptide SS-31 (elamipretide) can ...substantially reverse this deficit. SS-31 normalized the increase in proton leak and reduced mitochondrial ROS in cardiomyocytes from old mice, accompanied by reduced protein oxidation and a shift towards a more reduced protein thiol redox state in old hearts. Improved diastolic function was concordant with increased phosphorylation of cMyBP-C Ser282 but was independent of titin isoform shift. Late-life viral expression of mitochondrial-targeted catalase (mCAT) produced similar functional benefits in old mice and SS-31 did not improve cardiac function of old mCAT mice, implicating normalizing mitochondrial oxidative stress as an overlapping mechanism. These results demonstrate that pre-existing cardiac aging phenotypes can be reversed by targeting mitochondrial dysfunction and implicate mitochondrial energetics and redox signaling as therapeutic targets for cardiac aging.
Research into the basic biology of human health and disease, as well as translational human research and clinical applications, all benefit from the growing accessibility and versatility of mass ...spectrometry (MS)-based proteomics. Although once limited in throughput and sensitivity, proteomic studies have quickly grown in scope and scale over the last decade due to significant advances in instrumentation, computational approaches, and bio-sample preparation. Here, we review these latest developments in MS and highlight how these techniques are used to study the mechanisms, diagnosis, and treatment of human diseases. We first describe recent groundbreaking technological advancements for MS-based proteomics, including novel data acquisition techniques and protein quantification approaches. Next, we describe innovations that enable the unprecedented depth of coverage in protein signaling and spatiotemporal protein distributions, including studies of post-translational modifications, protein turnover, and single-cell proteomics. Finally, we explore new workflows to investigate protein complexes and structures, and we present new approaches for protein-protein interaction studies and intact protein or top-down MS. While these approaches are only recently incipient, we anticipate that their use in biomedical MS proteomics research will offer actionable discoveries for the improvement of human health.
Post-transcriptional modifications of RNA strongly influence the RNA structure and function. Recent advances in RNA sequencing and mass spectrometry (MS) methods have identified over 140 of these ...modifications on a wide variety of RNA species. Most next-generation sequencing approaches can only map one RNA modification at a time, and while MS can assign multiple modifications simultaneously in an unbiased manner, MS cannot accurately catalog and assign RNA modifications in complex biological samples due to limitations in the fragment length and coverage depth. Thus, a facile method to identify novel RNA modifications while simultaneously locating them in the context of their RNA sequences is still lacking. We combined two orthogonal modes of RNA ion separation before MS identification: high-field asymmetric ion mobility separation (FAIMS) and electrochemically modulated liquid chromatography (EMLC). FAIMS RNA MS increases both coverage and throughput, while EMLC LC-MS orthogonally separates RNA molecules of different lengths and charges. The combination of the two methods offers a broadly applicable platform to improve the length and depth of MS-based RNA sequencing while providing contextual access to the analysis of RNA modifications.
Microproteins (MPs) are a potentially rich source of uncharacterized metabolic regulators. Here, we use ribosome profiling (Ribo-seq) to curate 3,877 unannotated MP-encoding small ORFs (smORFs) in ...primary brown, white, and beige mouse adipocytes. Of these, we validated 85 MPs by proteomics, including 33 circulating MPs in mouse plasma. Analyses of MP-encoding mRNAs under different physiological conditions (high-fat diet) revealed that numerous MPs are regulated in adipose tissue in vivo and are co-expressed with established metabolic genes. Furthermore, Ribo-seq provided evidence for the translation of Gm8773, which encodes a secreted MP that is homologous to human and chicken FAM237B. Gm8773 is highly expressed in the arcuate nucleus of the hypothalamus, and intracerebroventricular administration of recombinant mFAM237B showed orexigenic activity in obese mice. Together, these data highlight the value of this adipocyte MP database in identifying MPs with roles in fundamental metabolic and physiological processes such as feeding.
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•Thousands of unannotated smORFs encode microproteins in primary mouse adipocytes•Hundreds of microproteins are differentially expressed in obese versus lean mice•Dozens of microproteins are found to be secreted by mass spectrometry•Identified protein homolog of mouse FAM237B that is an orexigenic hormone in obese mice
Martinez et al. identify thousands of unannotated smORF-encoded microproteins in primary mouse adipocytes. Hundreds of microproteins are differentially expressed in tissues from obese versus lean mice. Dozens of secreted microproteins are also detected by mass spectrometry. A predicted FAM237B homolog is shown to be an orexigenic hormone in obese mice.
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