Mass spectrometry (MS) is by far the most used experimental approach in high-throughput proteomics. The ProteomeXchange (PX) consortium of proteomics resources (http://www.proteomexchange.org) was ...originally set up to standardize data submission and dissemination of public MS proteomics data. It is now 10 years since the initial data workflow was implemented. In this manuscript, we describe the main developments in PX since the previous update manuscript in Nucleic Acids Research was published in 2020. The six members of the Consortium are PRIDE, PeptideAtlas (including PASSEL), MassIVE, jPOST, iProX and Panorama Public. We report the current data submission statistics, showcasing that the number of datasets submitted to PX resources has continued to increase every year. As of June 2022, more than 34 233 datasets had been submitted to PX resources, and from those, 20 062 (58.6%) just in the last three years. We also report the development of the Universal Spectrum Identifiers and the improvements in capturing the experimental metadata annotations. In parallel, we highlight that data re-use activities of public datasets continue to increase, enabling connections between PX resources and other popular bioinformatics resources, novel research and also new data resources. Finally, we summarise the current state-of-the-art in data management practices for sensitive human (clinical) proteomics data.
To address the growing need for a centralized, community resource of published results processed with Skyline, and to provide reviewers and readers immediate visual access to the data behind ...published conclusions, we present Panorama Public (https://panoramaweb.org/public.url), a repository of Skyline documents supporting published results. Panorama Public is built on Panorama, an open source data management system for mass spectrometry data processed with the Skyline targeted mass spectrometry environment. The Panorama web application facilitates viewing, sharing, and disseminating results contained in Skyline documents via a web-browser. Skyline users can easily upload their documents to a Panorama server and allow other researchers to explore uploaded results in the Panorama web-interface through a variety of familiar summary graphs as well as annotated views of the chromatographic peaks processed with Skyline. This makes Panorama ideal for sharing targeted, quantitative results contained in Skyline documents with collaborators, reviewers, and the larger proteomics community. The Panorama Public repository employs the full data visualization capabilities of Panorama which facilitates sharing results with reviewers during manuscript review.
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.
Mammalian cells have about 30,000 times as many protein molecules as mRNA molecules, which has major implications in the development of proteomics technologies. We discuss strategies that have been ...helpful for counting billions of protein molecules by liquid chromatography–tandem mass spectrometry and suggest that these strategies can benefit single-molecule methods, especially in mitigating the challenges posed by the wide dynamic range of the proteome.
Percolator is a widely used software tool that increases yield in shotgun proteomics experiments and assigns reliable statistical confidence measures, such as
q
values and posterior error ...probabilities, to peptides and peptide-spectrum matches (PSMs) from such experiments. Percolator’s processing speed has been sufficient for typical data sets consisting of hundreds of thousands of PSMs. With our new scalable approach, we can now also analyze millions of PSMs in a matter of minutes on a commodity computer. Furthermore, with the increasing awareness for the need for reliable statistics on the protein level, we compared several easy-to-understand protein inference methods and implemented the best-performing method—grouping proteins by their corresponding sets of theoretical peptides and then considering only the best-scoring peptide for each protein—in the Percolator package. We used Percolator 3.0 to analyze the data from a recent study of the draft human proteome containing 25 million spectra (PM:24870542). The source code and Ubuntu, Windows, MacOS, and Fedora binary packages are available from
http://percolator.ms/
under an Apache 2.0 license.
Graphical Abstract
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Photoperiod is one of the most reliable environmental cues for plants to regulate flowering timing. In Arabidopsis thaliana, CONSTANS (CO) transcription factor plays a central role in regulating ...photoperiodic flowering. In contrast to posttranslational regulation of CO protein, still little was known about CO transcriptional regulation. Here we show that the CINCINNATA (CIN) clade of class II TEOSINTE BRANCHED 1/ CYCLOIDEA/ PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR (TCP) proteins act as CO activators. Our yeast one-hybrid analysis revealed that class II CIN-TCPs, including TCP4, bind to the CO promoter. TCP4 induces CO expression around dusk by directly associating with the CO promoter in vivo. In addition, TCP4 binds to another flowering regulator, GIGANTEA (GI), in the nucleus, and induces CO expression in a GI-dependent manner. The physical association of TCP4 with the CO promoter was reduced in the gi mutant, suggesting that GI may enhance the DNA-binding ability of TCP4. Our tandem affinity purification coupled with mass spectrometry (TAP-MS) analysis identified all class II CIN-TCPs as the components of the in vivo TCP4 complex, and the gi mutant did not alter the composition of the TCP4 complex. Taken together, our results demonstrate a novel function of CIN-TCPs as photoperiodic flowering regulators, which may contribute to coordinating plant development with flowering regulation.
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.
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.
Abstract
The ProteomeXchange (PX) consortium of proteomics resources (http://www.proteomexchange.org) has standardized data submission and dissemination of mass spectrometry proteomics data worldwide ...since 2012. In this paper, we describe the main developments since the previous update manuscript was published in Nucleic Acids Research in 2017. Since then, in addition to the four PX existing members at the time (PRIDE, PeptideAtlas including the PASSEL resource, MassIVE and jPOST), two new resources have joined PX: iProX (China) and Panorama Public (USA). We first describe the updated submission guidelines, now expanded to include six members. Next, with current data submission statistics, we demonstrate that the proteomics field is now actively embracing public open data policies. At the end of June 2019, more than 14 100 datasets had been submitted to PX resources since 2012, and from those, more than 9 500 in just the last three years. In parallel, an unprecedented increase of data re-use activities in the field, including ‘big data’ approaches, is enabling novel research and new data resources. At last, we also outline some of our future plans for the coming years.
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.
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