Publications by authors named "Michael J MacCoss"

207 Publications

Slowed protein turnover in aging Drosophila reflects a shift in cellular priorities.

J Gerontol A Biol Sci Med Sci 2021 Jan 16. Epub 2021 Jan 16.

Department of Genome Sciences, University of Washington, Seattle, WA, USA.

The accumulation of protein aggregates and dysfunctional organelles as organisms age has led to the hypothesis that aging involves general breakdown of protein quality control. We tested this hypothesis using a proteomic and informatic approach in the fruit fly Drosophila melanogaster. Turnover of most proteins was markedly slower in old flies. However, ribosomal and proteasomal proteins maintained high turnover rates, suggesting that the observed slowdowns in protein turnover might not be due to a global failure of quality control. As protein turnover reflects the balance of protein synthesis and degradation, we investigated whether decreases in synthesis or decreases in degradation would best explain the observed slowdowns in protein turnover. We found that while many individual proteins in old flies showed slower turnover due to decreased degradation, an approximately equal number showed slower turnover due to decreased synthesis, and enrichment analyses revealed that translation machinery itself was less abundant. Mitochondrial complex I subunits and glycolytic enzymes were decreased in abundance as well, and proteins involved in glutamine-dependent anaplerosis were increased, suggesting that old flies modify energy production to limit oxidative damage. Together, our findings suggest that age-related proteostasis changes in Drosophila represent a coordinated adaptation rather than a systems collapse.
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http://dx.doi.org/10.1093/gerona/glab015DOI Listing
January 2021

Avant-garde: an automated data-driven DIA data curation tool.

Nat Methods 2020 12 16;17(12):1237-1244. Epub 2020 Nov 16.

Broad Institute of MIT and Harvard, Cambridge, MA, USA.

Several challenges remain in data-independent acquisition (DIA) data analysis, such as to confidently identify peptides, define integration boundaries, remove interferences, and control false discovery rates. In practice, a visual inspection of the signals is still required, which is impractical with large datasets. We present Avant-garde as a tool to refine DIA (and parallel reaction monitoring) data. Avant-garde uses a novel data-driven scoring strategy: signals are refined by learning from the dataset itself, using all measurements in all samples to achieve the best optimization. We evaluate the performance of Avant-garde using benchmark DIA datasets and show that it can determine the quantitative suitability of a peptide peak, and reach the same levels of selectivity, accuracy, and reproducibility as manual validation. Avant-garde is complementary to existing DIA analysis engines and aims to establish a strong foundation for subsequent analysis of quantitative mass spectrometry data.
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http://dx.doi.org/10.1038/s41592-020-00986-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7723322PMC
December 2020

A proteomic approach reveals possible molecular mechanisms and roles for endosymbiotic bacteria in begomovirus transmission by whiteflies.

Gigascience 2020 11;9(11)

Department of Entomology, The Volcani Center, HaMacabim Rd., Rishon LeZion, 50250, Israel.

Background: Many plant viruses are vector-borne and depend on arthropods for transmission between host plants. Begomoviruses, the largest, most damaging and emerging group of plant viruses, infect hundreds of plant species, and new virus species of the group are discovered each year. Begomoviruses are transmitted by members of the whitefly Bemisia tabaci species complex in a persistent-circulative manner. Tomato yellow leaf curl virus (TYLCV) is one of the most devastating begomoviruses worldwide and causes major losses in tomato crops, as well as in many agriculturally important plant species. Different B. tabaci populations vary in their virus transmission abilities; however, the causes for these variations are attributed among others to genetic differences among vector populations, as well as to differences in the bacterial symbionts housed within B. tabaci.

Results: Here, we performed discovery proteomic analyses in 9 whitefly populations from both Middle East Asia Minor I (MEAM1, formerly known as B biotype) and Mediterranean (MED, formerly known as Q biotype) species. We analysed our proteomic results on the basis of the different TYLCV transmission abilities of the various populations included in the study. The results provide the first comprehensive list of candidate insect and bacterial symbiont (mainly Rickettsia) proteins associated with virus transmission.

Conclusions: Our data demonstrate that the proteomic signatures of better vector populations differ considerably when compared with less efficient vector populations in the 2 whitefly species tested in this study. While MEAM1 efficient vector populations have a more lenient immune system, the Q efficient vector populations have higher abundance of proteins possibly implicated in virus passage through cells. Both species show a strong link of the facultative symbiont Rickettsia to virus transmission.
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http://dx.doi.org/10.1093/gigascience/giaa124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7662926PMC
November 2020

Development on Citrus medica infected with 'Candidatus Liberibacter asiaticus' has sex-specific and -nonspecific impacts on adult Diaphorina citri and its endosymbionts.

PLoS One 2020 6;15(10):e0239771. Epub 2020 Oct 6.

Department of Food Science and Technology, University of California, Davis, California, United States of America.

Huanglongbing (HLB) is a deadly, incurable citrus disease putatively caused by the unculturable bacterium, 'Candidatus Liberibacter asiaticus' (CLas), and transmitted by Diaphorina citri. Prior studies suggest D. citri transmits CLas in a circulative and propagative manner; however, the precise interactions necessary for CLas transmission remain unknown, and the impact of insect sex on D. citri-CLas interactions is poorly understood despite reports of sex-dependent susceptibilities to CLas. We analyzed the transcriptome, proteome, metabolome, and microbiome of male and female adult D. citri reared on healthy or CLas-infected Citrus medica to determine shared and sex-specific responses of D. citri and its endosymbionts to CLas exposure. More sex-specific than shared D. citri responses to CLas were observed, despite there being no difference between males and females in CLas density or relative abundance. CLas exposure altered the abundance of proteins involved in immunity and cellular and oxidative stress in a sex-dependent manner. CLas exposure impacted cuticular proteins and enzymes involved in chitin degradation, as well as energy metabolism and abundance of the endosymbiont 'Candidatus Profftella armatura' in both sexes similarly. Notably, diaphorin, a toxic Profftella-derived metabolite, was more abundant in both sexes with CLas exposure. The responses reported here resulted from a combination of CLas colonization of D. citri as well as the effect of CLas infection on C. medica. Elucidating these impacts on D. citri and their endosymbionts contributes to our understanding of the HLB pathosystem and identifies the responses potentially critical to limiting or promoting CLas acquisition and propagation in both sexes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0239771PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7537882PMC
November 2020

Acarbose has sex-dependent and -independent effects on age-related physical function, cardiac health, and lipid biology.

JCI Insight 2020 11 5;5(21). Epub 2020 Nov 5.

Department of Pathology, UM Medical School, Ann Arbor, Michigan, USA.

With an expanding aging population burdened with comorbidities, there is considerable interest in treatments that optimize health in later life. Acarbose (ACA), a drug used clinically to treat type 2 diabetes mellitus (T2DM), can extend mouse life span with greater effect in males than in females. Using a genetically heterogeneous mouse model, we tested the ability of ACA to ameliorate functional, pathological, and biochemical changes that occur during aging, and we determined which of the effects of age and drug were sex dependent. In both sexes, ACA prevented age-dependent loss of body mass, in addition to improving balance/coordination on an accelerating rotarod, rotarod endurance, and grip strength test. Age-related cardiac hypertrophy was seen only in male mice, and this male-specific aging effect was attenuated by ACA. ACA-sensitive cardiac changes were associated with reduced activation of cardiac growth-promoting pathways and increased abundance of peroxisomal proteins involved in lipid metabolism. ACA further ameliorated age-associated changes in cardiac lipid species, particularly lysophospholipids - changes that have previously been associated with aging, cardiac dysfunction, and cardiovascular disease in humans. In the liver, ACA had pronounced effects on lipid handling in both sexes, reducing hepatic lipidosis during aging and shifting the liver lipidome in adulthood, particularly favoring reduced triglyceride (TAG) accumulation. Our results demonstrate that ACA, already in clinical use for T2DM, has broad-ranging antiaging effects in multiple tissues, and it may have the potential to increase physical function and alter lipid biology to preserve or improve health at older ages.
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http://dx.doi.org/10.1172/jci.insight.137474DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710286PMC
November 2020

Highly Multiplex Targeted Proteomics Enabled by Real-Time Chromatographic Alignment.

Anal Chem 2020 09 12;92(17):11809-11817. Epub 2020 Aug 12.

Department of Genome Sciences, University of Washington, 3720 15th Street NE, Seattle, Washington 98195, United States.

Targeted mass spectrometry methods produce high-quality quantitative data in terms of limits of detection and dynamic range, at the cost of a substantial compromise in throughput compared to methods such as data independent and data dependent acquisition. The logistical and experimental issues inherent to maintaining assays of even several hundred targets are significant. Prominent among these issues is the drift in analyte retention time as liquid chromatography (LC) columns wear, forcing targeted scheduling windows to be much larger than LC peak widths. If these problems could be solved, proteomics assays would be capable of targeting thousands of peptides in an hour-long experiment, enabling large cohort studies to be performed without sacrificing sensitivity and specificity. We describe a solution in the form of a new method for real-time chromatographic alignment and demonstrate its application to a 56 min LC-gradient HeLa digest assay with 1489 targets. The method is based on the periodic acquisition of untargeted survey scans in a reference experiment and alignment to those scans during subsequent experiments. We describe how the method enables narrower scheduled retention time windows to be used. The narrower scheduling windows enables more targets to be included in the assay or proportionally more time to be allocated to each target, improving the sensitivity. Finally, we point out how the procedure could be improved and how much additional target multiplexing could be gained in the future.
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http://dx.doi.org/10.1021/acs.analchem.0c02075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7757911PMC
September 2020

Late-life restoration of mitochondrial function reverses cardiac dysfunction in old mice.

Elife 2020 07 10;9. Epub 2020 Jul 10.

Department of Pathology, University of Washington, Seattle, United States.

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.
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http://dx.doi.org/10.7554/eLife.55513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7377906PMC
July 2020

Composition of Caenorhabditis elegans extracellular vesicles suggests roles in metabolism, immunity, and aging.

Geroscience 2020 08 24;42(4):1133-1145. Epub 2020 Jun 24.

Department of Pathology, University of Washington, Seattle, WA, USA.

The nematode Caenorhabditis elegans has been instrumental in the identification of evolutionarily conserved mechanisms of aging. C. elegans also has recently been found to have evolutionarily conserved extracellular vesicle (EV) signaling pathways. We have been developing tools that allow for the detailed study of EV biology in C. elegans. Here we apply our recently published method for high specificity purification of EVs from C. elegans to carry out target-independent proteomic and RNA analysis of nematode EVs. We identify diverse coding and non-coding RNA and protein cargo types commonly found in human EVs. The EV cargo spectrum is distinct from whole worms, suggesting that protein and RNA cargos are actively recruited to EVs. Gene ontology analysis revealed C. elegans EVs are enriched for extracellular-associated and signaling proteins, and network analysis indicates enrichment for metabolic, immune, and basement membrane associated proteins. Tissue enrichment and gene expression analysis suggests the secreted EV proteins are likely to be derived from intestine, muscle, and excretory tissue. An unbiased comparison of the EV proteins with a large database of C. elegans genome-wide microarray data showed significant overlap with gene sets that are associated with aging and immunity. Taken together our data suggest C. elegans could be a promising in vivo model for studying the genetics and physiology of EVs in a variety of contexts including aging, metabolism, and immune response.
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http://dx.doi.org/10.1007/s11357-020-00204-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7394990PMC
August 2020

Audit logs to enforce document integrity in Skyline and Panorama.

Bioinformatics 2020 08;36(15):4366-4368

Department of Genome Sciences, University of Washington, Seattle, Washington 98195, USA.

Summary: Skyline is a Windows application for targeted mass spectrometry method creation and quantitative data analysis. Like most graphical user interface (GUI) tools, it has a complex user interface with many ways for users to edit their files which makes the task of logging user actions challenging and is the reason why audit logging of every change is not common in GUI tools. We present an object comparison-based approach to audit logging for Skyline that is extensible to other GUI tools. The new audit logging system keeps track of all document modifications made through the GUI or the command line and displays them in an interactive grid. The audit log can also be uploaded and viewed in Panorama, a web repository for Skyline documents that can be configured to only accept documents with a valid audit log, based on embedded hashes to protect log integrity. This makes workflows involving Skyline and Panorama more reproducible.

Availability And Implementation: Skyline is freely available at https://skyline.ms.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btaa547DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7520049PMC
August 2020

Acquiring and Analyzing Data Independent Acquisition Proteomics Experiments without Spectrum Libraries.

Mol Cell Proteomics 2020 07 20;19(7):1088-1103. Epub 2020 Apr 20.

Institute for Systems Biology, Seattle, Washington, USA. Electronic address:

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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http://dx.doi.org/10.1074/mcp.P119.001913DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7338082PMC
July 2020

Highly Parallel Quantification and Compartment Localization of Transcription Factors and Nuclear Proteins.

Cell Rep 2020 02;30(8):2463-2471.e5

Altius Institute for Biomedical Sciences, Seattle, WA 98121, USA; University of Washington, Department of Genome Sciences, Seattle, WA 98195, USA. Electronic address:

Transcription factors and other chromatin-associated proteins are difficult to quantify comprehensively. Here, we combine facile nuclear sub-fractionation with data-independent acquisition mass spectrometry to achieve rapid, sensitive, and highly parallel quantification of the nuclear proteome in human cells. We apply this approach to quantify the response to acute degradation of BET bromodomains, revealing unexpected chromatin regulatory dynamics. The method is simple and enables system-level study of previously inaccessible chromatin and genome regulators.
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http://dx.doi.org/10.1016/j.celrep.2020.01.096DOI Listing
February 2020

Matrix-Matched Calibration Curves for Assessing Analytical Figures of Merit in Quantitative Proteomics.

J Proteome Res 2020 03 24;19(3):1147-1153. Epub 2020 Feb 24.

Department of Genome Sciences, University of Washington, Seattle, Washington 98195, United States.

Mass spectrometry is a powerful tool for quantifying protein abundance in complex samples. Advances in sample preparation and the development of data-independent acquisition (DIA) mass spectrometry approaches have increased the number of peptides and proteins measured per sample. Here, we present a series of experiments demonstrating how to assess whether a peptide measurement is quantitative by mass spectrometry. Our results demonstrate that increasing the number of detected peptides in a proteomics experiment does not necessarily result in increased numbers of peptides that can be measured quantitatively.
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http://dx.doi.org/10.1021/acs.jproteome.9b00666DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7175947PMC
March 2020

Skyline for Small Molecules: A Unifying Software Package for Quantitative Metabolomics.

J Proteome Res 2020 04 26;19(4):1447-1458. Epub 2020 Mar 26.

Proteomics and Metabolomics Shared Resource, Duke University, Durham, North Carolina 27701, United States.

Vendor-independent software tools for quantification of small molecules and metabolites are lacking, especially for targeted analysis workflows. Skyline is a freely available, open-source software tool for targeted quantitative mass spectrometry method development and data processing with a 10 year history supporting six major instrument vendors. Designed initially for proteomics analysis, we describe the expansion of Skyline to data for small molecule analysis, including selected reaction monitoring, high-resolution mass spectrometry, and calibrated quantification. This fundamental expansion of Skyline from a peptide-sequence-centric tool to a molecule-centric tool makes it agnostic to the source of the molecule while retaining Skyline features critical for workflows in both peptide and more general biomolecular research. The data visualization and interrogation features already available in Skyline, such as peak picking, chromatographic alignment, and transition selection, have been adapted to support small molecule data, including metabolomics. Herein, we explain the conceptual workflow for small molecule analysis using Skyline, demonstrate Skyline performance benchmarked against a comparable instrument vendor software tool, and present additional real-world applications. Further, we include step-by-step instructions on using Skyline for small molecule quantitative method development and data analysis on data acquired with a variety of mass spectrometers from multiple instrument vendors.
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http://dx.doi.org/10.1021/acs.jproteome.9b00640DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7127945PMC
April 2020

Assessing Protein Sequence Database Suitability Using Sequencing.

Mol Cell Proteomics 2020 01 15;19(1):198-208. Epub 2019 Nov 15.

Department of Genome Sciences, University of Washington, Seattle, Washington.

The analysis of samples from unsequenced and/or understudied species as well as samples where the proteome is derived from multiple organisms poses two key questions. The first is whether the proteomic data obtained from an unusual sample type even contains peptide tandem mass spectra. The second question is whether an appropriate protein sequence database is available for proteomic searches. We describe the use of automated sequencing for evaluating both the quality of a collection of tandem mass spectra and the suitability of a given protein sequence database for searching that data. Applications of this method include the proteome analysis of closely related species, metaproteomics, and proteomics of extinct organisms.
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http://dx.doi.org/10.1074/mcp.TIR119.001752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6944239PMC
January 2020

The ProteomeXchange consortium in 2020: enabling 'big data' approaches in proteomics.

Nucleic Acids Res 2020 01;48(D1):D1145-D1152

European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-EBI), Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SD, UK.

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.
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http://dx.doi.org/10.1093/nar/gkz984DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7145525PMC
January 2020

Kinetochore-associated Stu2 promotes chromosome biorientation in vivo.

PLoS Genet 2019 10 4;15(10):e1008423. Epub 2019 Oct 4.

Howard Hughes Medical Institute, Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America.

Accurate segregation of chromosomes to daughter cells is a critical aspect of cell division. It requires the kinetochores on duplicated chromosomes to biorient, attaching to microtubules from opposite poles of the cell. Bioriented attachments come under tension, while incorrect attachments lack tension and must be released to allow proper attachments to form. A well-studied error correction pathway is mediated by the Aurora B kinase, which destabilizes low tension-bearing attachments. We recently discovered that in vitro, kinetochores display an additional intrinsic tension-sensing pathway that utilizes Stu2. The contribution of kinetochore-associated Stu2 to error correction in cells, however, was unknown. Here, we identify a Stu2 mutant that abolishes its kinetochore function and show that it causes biorientation defects in vivo. We also show that this Stu2-mediated pathway functions together with the Aurora B-mediated pathway. Altogether, our work indicates that cells employ multiple pathways to ensure biorientation and the accuracy of chromosome segregation.
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http://dx.doi.org/10.1371/journal.pgen.1008423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6795502PMC
October 2019

Thesaurus: quantifying phosphopeptide positional isomers.

Nat Methods 2019 08 29;16(8):703-706. Epub 2019 Jul 29.

Department of Genome Sciences, University of Washington, Seattle, WA, USA.

Proteins can be phosphorylated at neighboring sites resulting in different functional states, and studying the regulation of these sites has been challenging. Here we present Thesaurus, a search engine that detects and quantifies phosphopeptide positional isomers from parallel reaction monitoring and data-independent acquisition mass spectrometry experiments. We apply Thesaurus to analyze phosphorylation events in the PI3K/AKT signaling pathway and show neighboring sites with distinct regulation.
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http://dx.doi.org/10.1038/s41592-019-0498-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7012383PMC
August 2019

First Community-Wide, Comparative Cross-Linking Mass Spectrometry Study.

Anal Chem 2019 06 22;91(11):6953-6961. Epub 2019 May 22.

Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Charles Tanford Protein Center , Martin Luther University Halle-Wittenberg , Kurt-Mothes-Strasse 3a , 06120 Halle/Saale , Germany.

The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein-protein interactions has increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats. Such consensus formats should become accepted in the field and be shown to lead to reproducible results. This first, community-based harmonization study on XL-MS is based on the results of 32 groups participating worldwide. The aim of this paper is to summarize the status quo of XL-MS and to compare and evaluate existing cross-linking strategies. Our study therefore builds the framework for establishing best practice guidelines to conduct cross-linking experiments, perform data analysis, and define reporting formats with the ultimate goal of assisting scientists to generate accurate and reproducible XL-MS results.
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http://dx.doi.org/10.1021/acs.analchem.9b00658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6625963PMC
June 2019

Autophagy accounts for approximately one-third of mitochondrial protein turnover and is protein selective.

Autophagy 2019 09 21;15(9):1592-1605. Epub 2019 Mar 21.

a Department of Genome Sciences, University of Washington , Seattle , WA , USA.

The destruction of mitochondria through macroautophagy (autophagy) has been recognised as a major route of mitochondrial protein degradation since its discovery more than 50 years ago, but fundamental questions remain unanswered. First, how much mitochondrial protein turnover occurs through auto-phagy? Mitochondrial proteins are also degraded by nonautophagic mechanisms, and the proportion of mitochondrial protein turnover that occurs through autophagy is still unknown. Second, does auto-phagy degrade mitochondrial proteins uniformly or selectively? Autophagy was originally thought to degrade all mitochondrial proteins at the same rate, but recent work suggests that mitochondrial autophagy may be protein selective. To investigate these questions, we used a proteomics-based approach in the fruit fly , comparing mitochondrial protein turnover rates in autophagy-deficient mutants and controls. We found that ~35% of mitochondrial protein turnover occurred via autophagy. Similar analyses using mutants revealed that parkin-dependent mitophagy accounted for ~25% of mitochondrial protein turnover, suggesting that most mitochondrial autophagy specifically eliminates dysfunctional mitochondria. We also found that our results were incompatible with uniform autophagic turnover of mitochondrial proteins and consistent with protein-selective autophagy. In particular, the autophagic turnover rates of individual mitochondrial proteins varied widely, and only a small amount of the variation could be attributed to tissue differences in mitochondrial composition and autophagy rate. Furthermore, analyses comparing autophagy-deficient and control human fibroblasts revealed diverse autophagy-dependent turnover rates even in homogeneous cells. In summary, our work indicates that autophagy acts selectively on mitochondrial proteins, and that most mitochondrial protein turnover occurs through non-autophagic processes. ( (human); ( (human); DNA: deoxyribonucleic acid; ER: endoplasmic reticulum; GFP: green fluorescent protein; MS: mass spectrometry; ( ( (human); (human); RNA: ribonucleic acid; SD: standard deviation; Ub: ubiquitin/ubiquitinated; WT: wild-type; ( (human).
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http://dx.doi.org/10.1080/15548627.2019.1586258DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6693455PMC
September 2019

Improving Precursor Selectivity in Data-Independent Acquisition Using Overlapping Windows.

J Am Soc Mass Spectrom 2019 Apr 22;30(4):669-684. Epub 2019 Jan 22.

Department of Genome Sciences, University of Washington, 3720 15th Ave. NE, Seattle, WA, USA.

A major goal of proteomics research is the accurate and sensitive identification and quantification of a broad range of proteins within a sample. Data-independent acquisition (DIA) approaches that acquire MS/MS spectra independently of precursor information have been developed to overcome the reproducibility challenges of data-dependent acquisition and the limited breadth of targeted proteomics strategies. Typical DIA implementations use wide MS/MS isolation windows to acquire comprehensive fragment ion data. However, wide isolation windows produce highly chimeric spectra, limiting the achievable sensitivity and accuracy of quantification and identification. Here, we present a DIA strategy in which spectra are collected with overlapping (rather than adjacent or random) windows and then computationally demultiplexed. This approach improves precursor selectivity by nearly a factor of 2, without incurring any loss in mass range, mass resolution, chromatographic resolution, scan speed, or other key acquisition parameters. We demonstrate a 64% improvement in sensitivity and a 17% improvement in peptides detected in a 6-protein bovine mix spiked into a yeast background. To confirm the method's applicability to a realistic biological experiment, we also analyze the regulation of the proteasome in yeast grown in rapamycin and show that DIA experiments with overlapping windows can help elucidate its adaptation toward the degradation of oxidatively damaged proteins. Our integrated computational and experimental DIA strategy is compatible with any DIA-capable instrument. The computational demultiplexing algorithm required to analyze the data has been made available as part of the open-source proteomics software tools Skyline and msconvert (Proteowizard), making it easy to apply as part of standard proteomics workflows. Graphical Abstract.
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http://dx.doi.org/10.1007/s13361-018-2122-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6445824PMC
April 2019

Improving mitochondrial function with SS-31 reverses age-related redox stress and improves exercise tolerance in aged mice.

Free Radic Biol Med 2019 04 28;134:268-281. Epub 2018 Dec 28.

Department of Radiology, University of Washington, Seattle, WA, USA. Electronic address:

Sarcopenia and exercise intolerance are major contributors to reduced quality of life in the elderly for which there are few effective treatments. We tested whether enhancing mitochondrial function and reducing mitochondrial oxidant production with SS-31 (elamipretide) could restore redox balance and improve skeletal muscle function in aged mice. Young (5 mo) and aged (26 mo) female C57BL/6Nia mice were treated for 8-weeks with 3 mg/kg/day SS-31. Mitochondrial function was assessed in vivo using P and optical spectroscopy. SS-31 reversed age-related decline in maximum mitochondrial ATP production (ATPmax) and coupling of oxidative phosphorylation (P/O). Despite the increased in vivo mitochondrial capacity, mitochondrial protein expression was either unchanged or reduced in the treated aged mice and respiration in permeabilized gastrocnemius (GAS) fibers was not different between the aged and aged+SS-31 mice. Treatment with SS-31 also restored redox homeostasis in the aged skeletal muscle. The glutathione redox status was more reduced and thiol redox proteomics indicated a robust reversal of cysteine S-glutathionylation post-translational modifications across the skeletal muscle proteome. The gastrocnemius in the age+SS-31 mice was more fatigue resistant with significantly greater mass compared to aged controls. This contributed to a significant increase in treadmill endurance compared to both pretreatment and untreated control values. These results demonstrate that the shift of redox homeostasis due to mitochondrial oxidant production in aged muscle is a key factor in energetic defects and exercise intolerance. Treatment with SS-31 restores redox homeostasis, improves mitochondrial quality, and increases exercise tolerance without an increase in mitochondrial content. Since elamipretide is currently in clinical trials these results indicate it may have direct translational value for improving exercise tolerance and quality of life in the elderly.
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http://dx.doi.org/10.1016/j.freeradbiomed.2018.12.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6588449PMC
April 2019

Time-resolved interaction proteomics of the GIGANTEA protein under diurnal cycles in Arabidopsis.

FEBS Lett 2019 02 28;593(3):319-338. Epub 2018 Dec 28.

SynthSys and School of Biological Sciences, University of Edinburgh, UK.

The plant-specific protein GIGANTEA (GI) controls many developmental and physiological processes, mediating rhythmic post-translational regulation. GI physically binds several proteins implicated in the circadian clock, photoperiodic flowering, and abiotic stress responses. To understand GI's multifaceted function, we aimed to comprehensively and quantitatively identify potential interactors of GI in a time-specific manner, using proteomics on Arabidopsis plants expressing epitope-tagged GI. We detected previously identified (in)direct interactors of GI, as well as proteins implicated in protein folding, or degradation, and a previously uncharacterized transcription factor, CYCLING DOF FACTOR6 (CDF6). We verified CDF6's direct interaction with GI, and ZEITLUPE/FLAVIN-BINDING, KELCH REPEAT, F-BOX 1/LIGHT KELCH PROTEIN 2 proteins, and demonstrated its involvement in photoperiodic flowering. Extending interaction proteomics to time series provides a data resource of candidate protein targets for GI's post-translational control.
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http://dx.doi.org/10.1002/1873-3468.13311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6373471PMC
February 2019

Chromatogram libraries improve peptide detection and quantification by data independent acquisition mass spectrometry.

Nat Commun 2018 12 3;9(1):5128. Epub 2018 Dec 3.

Department of Genome Sciences, University of Washington, Seattle, WA, USA.

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.
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http://dx.doi.org/10.1038/s41467-018-07454-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6277451PMC
December 2018

Data-Independent Acquisition Mass Spectrometry To Quantify Protein Levels in FFPE Tumor Biopsies for Molecular Diagnostics.

J Proteome Res 2019 01 12;18(1):426-435. Epub 2018 Dec 12.

NantOmics , 9600 Medical Center Drive , Rockville , Maryland 20850 , United States.

Mass spectrometry-based protein quantitation is currently used to measure therapeutically relevant protein biomarkers in CAP/CLIA setting to predict likely responses of known therapies. Selected reaction monitoring (SRM) is the method of choice due to its outstanding analytical performance. However, data-independent acquisition (DIA) is now emerging as a proteome-scale clinical assay. We evaluated the ability of DIA to profile the patient-specific proteomes of sample-limited tumor biopsies and to quantify proteins of interest in a targeted fashion using formalin-fixed, paraffin-embedded (FFPE) tumor biopsies ( n = 12) selected from our clinical laboratory. DIA analysis on the tumor biopsies provided 3713 quantifiable proteins including actionable biomarkers currently in clinical use, successfully separated two gastric cancers from colorectal cancer specimen solely on the basis of global proteomic profiles, and identified subtype-specific proteins with prognostic or diagnostic value. We demonstrate the potential use of DIA-based quantitation to inform therapeutic decision-making using TUBB3, for which clinical cutoff expression levels have been established by SRM. Comparative analysis of DIA-based proteomic profiles and mRNA expression levels found positively and negatively correlated protein-gene pairs, a finding consistent with previously reported results from fresh-frozen tumor tissues.
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http://dx.doi.org/10.1021/acs.jproteome.8b00699DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6465117PMC
January 2019

The Interaction Dynamics of Two Potato Leafroll Virus Movement Proteins Affects Their Localization to the Outer Membranes of Mitochondria and Plastids.

Viruses 2018 10 26;10(11). Epub 2018 Oct 26.

United States Department of Agriculture, Biological Integrated Pest Management Research Unit, Robert W. Holley Center for Agriculture and Health, 538 Tower Road, Ithaca, NY 14853, USA.

The is an agriculturally important family of viruses whose replication and transport are restricted to plant phloem. Their genomes encode for four proteins that regulate viral movement. These include two structural proteins that make up the capsid and two non-structural proteins known as P3a and P17. Little is known about how these proteins interact with each other and the host to coordinate virus movement within and between cells. We used quantitative, affinity purification-mass spectrometry to show that the P3a protein of complexes with virus and that this interaction is partially dependent on P17. Bimolecular complementation assays (BiFC) were used to validate that P3a and P17 self-interact as well as directly interact with each other. Co-localization with fluorescent-based organelle markers demonstrates that P3a directs P17 to the mitochondrial outer membrane while P17 regulates the localization of the P3a-P17 heterodimer to plastids. Residues in the C-terminus of P3a were shown to regulate P3a association with host mitochondria by using mutational analysis and also varying BiFC tag orientation. Collectively, our work reveals that the PLRV movement proteins play a game of intracellular hopscotch along host organelles to transport the virus to the cell periphery.
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http://dx.doi.org/10.3390/v10110585DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6265731PMC
October 2018

Calibration Using a Single-Point External Reference Material Harmonizes Quantitative Mass Spectrometry Proteomics Data between Platforms and Laboratories.

Anal Chem 2018 11 23;90(21):13112-13117. Epub 2018 Oct 23.

Mass spectrometry (MS) measurements are not inherently calibrated. Researchers use various calibration methods to assign meaning to arbitrary signal intensities and improve precision. Internal calibration (IC) methods use internal standards (IS) such as synthesized or recombinant proteins or peptides to calibrate MS measurements by comparing endogenous analyte signal to the signal from known IS concentrations spiked into the same sample. However, recent work suggests that using IS as IC introduces quantitative biases that affect comparison across studies because of the inability of IS to capture all sources of variation present throughout an MS workflow. Here, we describe a single-point external calibration strategy to calibrate signal intensity measurements to a common reference material, placing MS measurements on the same scale and harmonizing signal intensities between instruments, acquisition methods, and sites. We demonstrate data harmonization between laboratories and methodologies using this generalizable approach.
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http://dx.doi.org/10.1021/acs.analchem.8b04581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6854904PMC
November 2018

Glucocerebrosidase deficiency promotes protein aggregation through dysregulation of extracellular vesicles.

PLoS Genet 2018 09 26;14(9):e1007694. Epub 2018 Sep 26.

Department of Genome Sciences, University of Washington, Seattle, WA, United States of America.

Mutations in the glucosylceramidase beta (GBA) gene are strongly associated with neurodegenerative diseases marked by protein aggregation. GBA encodes the lysosomal enzyme glucocerebrosidase, which breaks down glucosylceramide. A common explanation for the link between GBA mutations and protein aggregation is that lysosomal accumulation of glucosylceramide causes impaired autophagy. We tested this hypothesis directly by measuring protein turnover and abundance in Drosophila mutants with deletions in the GBA ortholog Gba1b. Proteomic analyses revealed that known autophagy substrates, which had severely impaired turnover in autophagy-deficient Atg7 mutants, showed little to no overall slowing of turnover or increase in abundance in Gba1b mutants. Likewise, Gba1b mutants did not have the marked impairment of mitochondrial protein turnover seen in mitophagy-deficient parkin mutants. Proteasome activity, microautophagy, and endocytic degradation also appeared unaffected in Gba1b mutants. However, we found striking changes in the turnover and abundance of proteins associated with extracellular vesicles (EVs), which have been proposed as vehicles for the spread of protein aggregates in neurodegenerative disease. These changes were specific to Gba1b mutants and did not represent an acceleration of normal aging. Western blotting of isolated EVs confirmed the increased abundance of EV proteins in Gba1b mutants, and nanoparticle tracking analysis revealed that Gba1b mutants had six times as many EVs as controls. Genetic perturbations of EV production in Gba1b mutants suppressed protein aggregation, demonstrating that the increase in EV abundance contributed to the accumulation of protein aggregates. Together, our findings indicate that glucocerebrosidase deficiency causes pathogenic changes in EV metabolism and may promote the spread of protein aggregates through extracellular vesicles.
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http://dx.doi.org/10.1371/journal.pgen.1007694DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6175534PMC
September 2018

Candidatus Liberibacter asiaticus Minimally Alters Expression of Immunity and Metabolism Proteins in Hemolymph of Diaphorina citri, the Insect Vector of Huanglongbing.

J Proteome Res 2018 09 27;17(9):2995-3011. Epub 2018 Aug 27.

Section of Plant Pathology and Plant-Microbe Biology, School of Integrated Plant Sciences, Cornell University , Ithaca , New York 14853 , United States.

Huanglongbing (HLB), also known as citrus greening disease, is the most serious disease of citrus plants. It is associated with the Gram-negative bacterium ' Candidatus Liberibacter asiaticus' ( CLas), which is transmitted between host plants by the hemipteran insect vector Diaphorina citri in a circulative, propagative manner involving specific interactions with various insect tissues including the hemolymph, fluid that occupies the body cavity akin to insect blood. High resolution quantitative mass spectrometry was performed to investigate the effect of CLas exposure on D. citri hemolymph at the proteome level. In contrast to the broad proteome effects on hundreds of proteins and a diverse array of metabolic pathways previously reported in gut and whole insect proteome analyses, the effect of CLas on the hemolymph was observed to be highly specific, restricted to key immunity and metabolism pathways, and lower in magnitude than that previously observed in the whole insect body and gut. Vitellogenins were abundantly expressed and CLas-responsive. Gene-specific RNA expression analysis suggests that these proteins are expressed in both male and female insects and may have roles outside of reproductive vitellogenesis. Proteins for fatty acid synthesis were found to be up-regulated, along with metabolic proteins associated with energy production, supported at the organismal level by the previously published observation that D. citri individuals experience a higher level of hunger when reared on CLas-infected plants. Prediction of post-translational modifications identified hemolymph proteins with phosphorylation and acetylation upon CLas exposure. Proteins derived from the three most prominent bacterial endosymbionts of the psyllid were also detected in the hemolymph, and several of these have predicted secretion signals. A DNAK protein, the bacterial HSP70, detected in the hemolymph expressed from Wolbachia pipientis was predicted to encode a eukaryotic nuclear localization signal. Taken together, these data show specific changes to immunity and metabolism in D. citri hemolymph involving host and endosymbiont proteins. These data provide a novel context for proteomic changes seen in other D. citri tissues in response to CLas and align with organismal data on the effects of CLas on D. citri metabolism and reproduction.
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http://dx.doi.org/10.1021/acs.jproteome.8b00183DOI Listing
September 2018

Using Skyline to Analyze Data-Containing Liquid Chromatography, Ion Mobility Spectrometry, and Mass Spectrometry Dimensions.

J Am Soc Mass Spectrom 2018 Nov 25;29(11):2182-2188. Epub 2018 Jul 25.

Biological Sciences Division, Pacific Northwest National Laboratory, 902 Battelle Blvd. MSIN K8-98, P.O. Box 999, Richland, WA, 99352, USA.

Recent advances in ion mobility spectrometry (IMS) have illustrated its power in determining the structural characteristics of a molecule, especially when coupled with other separations dimensions such as liquid chromatography (LC) and mass spectrometry (MS). However, these three separation techniques together greatly complicate data analyses, making better informatics tools essential for assessing the resulting data. In this manuscript, Skyline was adapted to analyze LC-IMS-CID-MS data from numerous instrument vendor datasets and determine the effect of adding the IMS dimension into the normal LC-MS molecular pipeline. For the initial evaluation, a tryptic digest of bovine serum albumin (BSA) was spiked into a yeast protein digest at seven different concentrations, and Skyline was able to rapidly analyze the MS and CID-MS data for 38 of the BSA peptides. Calibration curves for the precursor and fragment ions were assessed with and without the IMS dimension. In all cases, addition of the IMS dimension removed noise from co-eluting peptides with close m/z values, resulting in calibration curves with greater linearity and lower detection limits. This study presents an important informatics development since to date LC-IMS-CID-MS data from the different instrument vendors is often assessed manually and cannot be analyzed quickly. Because these evaluations require days for the analysis of only a few target molecules in a limited number of samples, it is unfeasible to evaluate hundreds of targets in numerous samples. Thus, this study showcases Skyline's ability to work with the multidimensional LC-IMS-CID-MS data and provide biological and environmental insights rapidly. Graphical Abstract ᅟ.
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http://dx.doi.org/10.1007/s13361-018-2028-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6191345PMC
November 2018