hEIDI: An Intuitive Application Tool To Organize and Treat Large-Scale Proteomics Data

Advances in high-throughput proteomics have led to a rapid increase in the number, size, and complexity of the associated data sets. Managing and extracting reliable information from such large series of data sets require the use of dedicated software organized in a consistent pipeline to reduce, validate, exploit, and ultimately export data. The compilation of multiple mass-spectrometry-based identification and quantification results obtained in the context of a large-scale project represents a real challenge for developers of bioinformatics solutions. In response to this challenge, we developed a dedicated software suite called hEIDI to manage and combine both identifications and semiquantitative data related to multiple LC–MS/MS analyses. This paper describes how, through a user-friendly interface, hEIDI can be used to compile analyses and retrieve lists of nonredundant protein groups. Moreover, hEIDI allows direct comparison of series of analyses, on the basis of protein groups, while ensuring consistent protein inference and also computing spectral counts. hEIDI ensures that validated results are compliant with MIAPE guidelines as all information related to samples and results is stored in appropriate databases. Thanks to the database structure, validated results generated within hEIDI can be easily exported in the PRIDE XML format for subsequent publication. hEIDI can be downloaded from http://biodev.extra.cea.fr/docs/heidi

MOESM4 of Systematic quantitative analysis of H2A and H2B variants by targeted proteomics

Additional file 4. Details of the SRM transitions for each signature peptide. SRM assay parameters including precursor and fragment ion type, charge state, elution time as well as raw data are provided in Suppl. data. (*) Indicates peptides monitored only in their endogenous form

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Additional file 5. Composition of the mixture of standard peptides

Main features of the identified chloroplastic methylproteins and methylation sites.

<p>(a) Curated subcellular/subplastidial location of methylproteins (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095512#pone-0095512-t002" target="_blank">Table 2</a>). (b) Functional categories of chloroplastic methylproteins. Annotated proteins from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095512#pone-0095512-t002" target="_blank">Table 2</a> were grouped to create categories ‘metabolism (other than photosynthesis)’ and ‘protein synthesis and targeting’. (c) Amino acid motif surrounding Lys methylation sites was created using WebLogo (<a href="http://weblogo.berkeley.edu/" target="_blank">http://weblogo.berkeley.edu/</a>). (d) Positioning of the Lys395 methylation site on the 3D-structure model of fructose bisphosphate aldolase (FBA1, At2g21330). The model was generated with the Phyre² server using the 3D structure of aldolase from rabbit muscle (PDB entry 1ZAI) and imaged with the PyMOL software.</p

Immunodetection of Lys- and Arg-methylated proteins in chloroplast stroma and membranes subfractions.

<p>Chloroplasts from Arabidopsis, spinach and pea leaves were purified using Percoll gradients and fractionated into soluble (stroma) and membrane (thylakoids and envelope) fractions. Fifty µg of proteins were analyzed by SDS-PAGE (Coomassie blue staining) and immunoblotting with antibodies against trimethyl-Lys (anti-K_me3), mono- and dimethyl-Lys (anti-K_me1/2), or mono- and dimethyl-Arg (anti-R_me1/2). The major polypeptides detected by the anti-K_me3 antibodies are RbcL (*) and fructose bisphosphate aldolases (**) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095512#pone.0095512-Mininno1" target="_blank">[30]</a>.</p

Biochemical validation of methylproteins through <i>in vitro</i> methylation assays using stroma from Arabidopsis chloroplasts.

<p>Methylation assays were done in the presence of 20 µg purified recombinant targets (FBA2, GAPA1, PRPL11, or ATP-B), 80 µg stroma from Arabidopsis Col-0 chloroplasts, 20 µM [methyl-³H]-AdoMet and 100 nM <i>S</i>-adenosylhomocysteine hydrolase. After incubation at 30°C for 1 to 2 hours, assays were split into two equals parts and radioactivity incorporated into proteins was counted by liquid scintillation (panel a) and analyzed by phosphorimaging (panels b–c). In panels (a–c), the symbol Ø means that no recombinant protein was added to the stromal extract. Purified recombinant substrates are indicated by asterisks: ATP-B, 54 kDa; FBA2, 40 kDa; GAPA1, 38 kDa; PRPL11, 18 kDa (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095512#pone.0095512.s003" target="_blank">Fig. S3</a>). Activities with the FBA2, PRPL11 and ATP-B substrates were strictly dependent on the addition of stroma. Values are mean ± SD of two to six independent determinations.</p

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Additional file 9. Rules used to select or reject peptides using their transition profiles. The validation of the best transitions was performed using a signal-to-noise ratio (> 5) and a perfect co-elution of the heavy standard peptide with the endogenous peptide. Three fragment ions (F1, F2, and F3) are represented for the heavy and the endogenous peptides. a All fragment ions can be integrated because the heavy and endogenous fragment ions co-elute in the same intensity order. b In that case, only F2 can be integrated because the ratio heavy/endogenous is different for F1 and F3. c The fragment F2 is contaminated by another analyte eluting at a slightly later time; it has to be excluded from the analysis. d Here, the signal-to-noise ratio is below five, no fragment ion can be integrated. e. The endogenous peptide traces do not co-elute with the heavy peptide traces

Main properties of Lys and Arg methylproteins and methylation sites from the AT_CHLORO_Me inventory.

<p>Protein accession, symbol and description were from TAIR, Uniprot and PPDB databases. Functional annotation was done according to MapMan bins and sub-bins <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095512#pone.0095512-Thimm1" target="_blank">[67]</a>. Subcellular and subplastidial location was curated using data from AT_CHLORO <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095512#pone.0095512-Ferro1" target="_blank">[36]</a>, PPDB <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095512#pone.0095512-Sun1" target="_blank">[62]</a> and dedicated studies. The trimethylated vs. acetylated status of Lys residues was confirmed (c) or remained ambiguous (a). Modeling of 3D-structures was done using the Phyre² server <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095512#pone.0095512-Kelley1" target="_blank">[52]</a>. NA, not available (the protein structure could not be modeled or the model does not cover the methylation site).</p

<i>In vitro</i> methylation of PRPL11 by PrmA-like.

<p>(a,b) Kinetic analysis of PRPL11 methylation by PrmA-like. Purified recombinant PRPL11 (2.5 µg) and PrmA-like (0.25 µg) were incubated at 30°C with 20 µM [methyl-³H]-AdoMet. Radioactivity incorporated into proteins was counted by liquid scintillation (a) or analyzed by phosphorimaging (b). A time-course analysis representative of three repeats is shown. The activity (2.0±0.2 nmol methyl incorporated. min⁻¹.mg⁻¹ protein) was strictly dependent on the presence of PrmA-like. (c) Screening of five chloroplastic PKMTs for their ability to methylate PRPL11. Purified recombinant PRPL11 (10 µg) and PKMTs (1 µg) were incubated at 30°C for 15 min with 20 µM [methyl-³H]-AdoMet and radioactivity incorporated into proteins was counted by liquid scintillation. Enzyme nomenclature is as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095512#pone-0095512-t001" target="_blank">Table 1</a>. Values are mean ± SD of three determinations.</p

Inventory of Arabidopsis PRMT and PKMTs known or predicted to be targeted to chloroplasts.

<p>Potential protein Lys/Arg methyltransferases found in the Arabidopsis genome were analyzed for their predicted subcellular localization by using SUBA3, AtSubP, and ChloroP 1.1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095512#pone.0095512-Tanz1" target="_blank">[44]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095512#pone.0095512-Emanuelsson1" target="_blank">[46]</a>. Proteins with a confident prediction for plastid targeting are shown. Data extracted from the predictors are: for SUBA3, consensus localization and number of predictors (out of 18) indicating a plastid targeting; for AtSubP, prediction using the best hybrid-based classifier (AA+PSSM+N-Center-C+PSI-BLAST); for ChloroP 1.1, presence of a predictable chloroplast transit peptide (cTP). Experimental localizations using GFP-tagging or mass spectrometry (MS) have been reported in the indicated references.</p