1. Standards for proteomics: The HUPO Proteomics Standards Initiative (HUPO PSI) http://www.ebi.ac.uk/pride/ Public Repository for Mass spectrometry spectral data and metadata The PRIDE PRoteomics IDEntifications database is a centralized, standards compliant, public data repository for proteomics data. It has been developed to provide the proteomics community with a public repository for protein and peptide identifications together with the evidence supporting these identifications. PRIDE is also able to capture details of post-translational modifications coordinated relative to the peptides in which they have been found. http://www.psidev.info/ Workgroups Mass Spectrometry Molecular Interactions Protein Modifications Protein Separation Proteomics Informatics Workflows/pipelines for proteomics. Many local. Some have broader appeal http://tools.proteomecenter.org/software.php Seattle Proteome Center (SPC) - Proteomics Tools NHLBI Proteomics Center at the Institute for Systems Biology Peptide Atlas (uses the TTP) http://www.peptideatlas.org/ Trans Proteome Pipeline (TPP) Proteomics. 2009 9(19):4426-8.Second Joint HUPO publication and Proteomics Standards Initiative workshop. Orchard SOrchard S, Binz PA, Hermjakob H.Binz PAHermjakob H Nucleic Acids Res. 2010. 38(Database issue):D736-42. Proteomics Identifications database: 2010 update. Vizcaíno JAVizcaíno JA, Côté R, Reisinger F, Barsnes H, Foster JM, Rameseder J, Hermjakob H, Martens L.Côté RReisinger FBarsnes HFoster JMRameseder JHermjakob HMartens L The Arabidopsis proteomics community http://www.masc-proteomics.org/mascp/index.php/Main_Page

3. Isolated BS and M chloroplast stroma and membranes 5,636,079 acquired MS/MS spectra Identified 2439 gene models; 1429 genes 357955 matched spectra (adjSPC) to 1429 best gene models [250.5 (average) or 21.5 (median) adjSPC/protein] Proteins annotated for: Subcellular localization Function (MapMan) Remove 149 proteins with 1 SPC and/or 1 aa seq (<10 aa*) with I/L (<0.07% total mass (% adjSPC)) Remove 175 contaminant proteins (<0.6% of total mass (% adjSPC)) Final protein set for quantification (923 proteins or groups) 366 proteins and protein groups (≥ 100 adjSPC) (high confidence) 179 proteins and protein groups (40-100 adjSPC) (medium confidence) 378 protein and protein groups (< 40 adjSPC) (low confidence) 144 LC-MS/MS LTQ-Orbitrap runs (stroma) 156 LC-MS/MS LTQ-Orbitrap runs (thylakoids & envelopes) MASCOT search 4a53 genome + chlpst & mito genomes Post-MASCOT cleanup (FPR peptides < 1%) Calculate SPC, unique SPC and adjSPC for each gene model Select best scoring gene models for each gene Annotation by best BLAST Arabidopsis & rice Literature & manual evaluation 1105 proteins Grouping for highly related & duplicated proteins by similarity matrix and manual evaluation (313 proteins in 131 groups); refine annotations # of proteins # of adjSPC 1 10 100 1000 10000 100000 02004006008001000 378 179 366 40 Friso et al (2010) Plant Physiology 152:1219-1250 Work flow for comparative proteomics – maize – organelles from two cell-types Submitted to PRIDE: 300 mgf files reformatted as PRIDE XML files

4. Isolated Chloroplasts Stroma (supernatant) SEC & SDS-PAGE Membrane (pellet) discard In-gel tryptic digestion Peptide extraction NanoLCRP-LTQ-Orbitrap analysis MASCOT search ATH v8 Post-MASCOT cleanup (FPR peptides <1%; 700-3500 Da) Calculate NAdjSPC for each gene model Select best scoring gene models for each protein Profiling macromolecular assemblies from NadjSPC lyse in native conditions spin (100K xg) Hierachical clustering (adjSPC ≥ 20) Heat maps by function (adjSPC ≥ 2) Remove proteins with only one aa sequence Remove proteins with <2 adjSPC Remove non-chloroplast proteins Remove thylakoid & envelope proteins Work flow for oligomeric state analysis - ATH Olinares et al (2010) Molec. Cell Prot.