Standards for proteomics: The HUPO Proteomics Standards Initiative (HUPO PSI) 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. Workgroups Mass Spectrometry Molecular Interactions Protein Modifications Protein Separation Proteomics Informatics Workflows/pipelines for proteomics. Many local. Some have broader appeal Seattle Proteome Center (SPC) - Proteomics Tools NHLBI Proteomics Center at the Institute for Systems Biology Peptide Atlas (uses the TTP) Trans Proteome Pipeline (TPP) Proteomics (19): Second Joint HUPO publication and Proteomics Standards Initiative workshop. Orchard SOrchard S, Binz PA, Hermjakob H.Binz PAHermjakob H Nucleic Acids Res (Database issue):D 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

Isolated BS and M chloroplast stroma and membranes 5,636,079 acquired MS/MS spectra Identified 2439 gene models; 1429 genes 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 ( 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 Friso et al (2010) Plant Physiology 152: Work flow for comparative proteomics – maize – organelles from two cell-types Submitted to PRIDE: 300 mgf files reformatted as PRIDE XML files

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%; 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.