Difference between revisions of "Widespreadaggregation.2013"
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Latest revision as of 16:11, 13 October 2014
This is supplementary website for the following paper:
Jeremy O’Connell, Mark Tsechansky, Ariel Royal, Dan Boutz, Andrew D. Ellington, Edward M. Marcotte, A proteomic survey of widespread protein aggregation in yeast, Molecular BioSystems, in press (2014).
Many normally cytosolic yeast proteins form insoluble intracellular bodies in response to nutrient depletion, suggesting the potential for widespread protein aggregation in stressed cells. Nearly 200 such bodies have been found in yeast by screening libraries of fluorescently tagged proteins. In order to more broadly characterize the formation of these bodies in response to stress, we employed a proteome-wide shotgun mass spectrometry assay in order to measure shifts in the intracellular solubilities of endogenous proteins following heat stress. As quantified by mass spectrometry, heat stress tended to shift the same proteins into insoluble form as did nutrient depletion; many of these proteins were also known to form foci in response to arsenic stress. Affinity purification of several foci-forming proteins showed enrichment for co-purifying chaperones, including Hsp90 chaperones. Tests of induction conditions and co-localization of metabolic enzymes participating in the same metabolic pathways suggested those foci did not correspond to multi-enzyme organizing centers. Thus, in yeast, the formation of stress bodies appears common across diverse, normally diffuse cytoplasmic proteins and is induced by multiple types of cell stress, including thermal, chemical, and nutrient stress.
Shotgun proteomics experimental design (reproduced from O'Connell et al.)
Insoluble fractions were resuspended in denaturing buffer consisting of 50% TFE in lysis buffer (50 mM Tris, 50 mM NaCl, 5 mM MgCl2). Soluble protein fractions were reduced to near-dry (<10 μl) by speedvac and resuspended in denaturing buffer (50% TFE in lysis buffer). All samples were then subjected to reduction, alkylation, and digestion with trypsin as previously described. Following digestion, trypsin activity was halted by the addition of 1% formic acid. Sample volume was reduced to [similar]100 μl by SpeedVac centrifugation and the volume adjusted to 150 μl with Buffer C (95% H2O, 5% acetonitrile (ACN), 0.1% formic acid). Tryptic peptides were bound and washed on Hypersep C-18 SpinTips (Thermo), eluted with 60% acetonitrile, 0.1% formic acid, reduced to near-dry by speedvac and resuspended in Buffer C. Soluble and insoluble fractions from heat-shock experiments were additionally filtered through Microcon 10[thin space (1/6-em)]000 NMWL Centrifugal Filters (Millipore) to remove larger contaminants and undigested proteins.
Soluble and insoluble fractions and Gln1-GFP immunoprecipitations were analyzed by nano LC-MS/MS using a Thermo Surveyor Plus HPLC coupled to an LTQ-Orbitrap Classic hybrid mass spectrometer (Thermo Scientific). Analyses the of remaining immunoprecipitations were carried out on a Dionex Ultimate 3000 nanoRSLC system coupled to an LTQ-Orbitrap Velos Pro hybrid mass spectrometer (Thermo Scientific). Data-dependent ion selection was activated, with parent ion scans (MS1) collected at high resolution (60[thin space (1/6-em)]000 for Classic, 100[thin space (1/6-em)]000 for Velos Pro). Ions with charge >+1 were selected for collision-induced dissociation fragmentation, with fragment spectra (MS2) collected by LTQ (12 MS2 per MS1 for Classic, 20 MS2 per MS1 for Velos Pro). Dynamic exclusion was activated, with an exclusion time of 45 seconds for ions selected more than twice in a 30 second window. Four injections (technical replicates) were performed for each biological replicate.
With a reference database of non-redundant yeast protein-coding sequences downloaded from SGD, mass spectra were interpreted using the MSBlender search algorithm, which employed an ensemble of Tide, MS-GFDB, and InsPecT search algorithms. Results were filtered to achieve a 1% false discovery rate for peptide spectrum matches (PSMs), using a reverse-sequence decoy database. As an additional confidence filter, peptides observed in only one injection were removed.
LC-MS/MS injections were analyzed independently for each biological replicate. Data from multiple injections per biological replicate were combined by adding the total count of peptide mass spectra for each protein across injections. Proteins observed only once in a biological replicate were subsequently omitted from further analyses. For heat-shock experiments, the data sets were curated to assign peptides to a single proteins or protein groups to account for the occurrence of degenerate peptides assigned to multiple proteins (e.g., paralogs). For IP samples, peptide spectral counts were divided evenly among all protein group members sharing that common peptide.
Shotgun proteomics datasets
- RAW files - http://data.marcottelab.org/MSdata/Widespreadaggregation.2013/RAW/
- mzXML files - http://data.marcottelab.org/MSdata/Widespreadaggregation.2013/mzXML/
- FASTA file - http://data.marcottelab.org/MSdata/Widespreadaggregation.2013/fasta
Database search results
- We used 3 search engines (Tide, InsPecT, MS-GFDB) before MSblender integration.
- We used the relative abundance version of the APEX quantification method for quantification.
To be added