1. 10. Standards in Proteomics MS bioinformatics analysis for proteomics Salvador Martínez de Bartolomé smartinez@proteored.org Bioinformatics support – ProteoRed Proteomics Facility, National Center for Biotechnology, Madrid

2. Index Need of standards in Proteomics HUPO-PSI –Organization –Standard data formats –MIAPEs PEFF: A Common Sequence Database Format in Proteomics PRIDE Standard data format converters

3. Index Need of standards in Proteomics HUPO-PSI –Organization –Standard data formats –MIAPEs PEFF: A Common Sequence Database Format in Proteomics PRIDE Standard data format converters

4. Proteomics data is often only made available as arbitrarily formatted PDF tables, carrying important limitations: Source data (mass spectra) are not made available No peer review validation possible Very little raw materials for testing innovative in silico techniques are available Automated (re-)processing of the identifications is impossible (eliminating objective technique comparison) Need of standards in Proteomics

5. Thoughts in Standards Bradshaw RA, Burlingame AL, Carr S, Aebersold R. Reporting protein identification data: the next generation of guidelines. Mol Cell Proteomics. 2006 May;5(5):787-8. Wilkins et al. Guidelines for the next 10 years of proteomics. Proteomics. 2006 Jan;6(1):4-8. Nature Biotechnology 2006, Nov: Editorial: Standards Operating Procedures Burgoon LD. The need for standards, not guidelines, in biological data reporting and sharing. Ball C. Are we stuck in standards? Nature Biotechnology: Planned focus issue and Community Consultation on Standards: http://www.nature.com/nbt/consult/index.html

6. Need of standards in Proteomics Proteomics: No standardized reporting, not standard database submission Proteomics data is generated at a high rate, and lost at a high rate Experiments are repeated unnecessarily, the field advances slower than necessary

7. Need of standards in Proteomics Standards for: Exchange data Compare data Review data Reproduce results Store data

8. Index Need of standards in Proteomics HUPO-PSI –Organization –Standard data formats –CVs –MIAPEs PEFF: A Common Sequence Database Format in Proteomics PRIDE Standard data format converters

9. Index Need of standards in Proteomics HUPO-PSI –Organization –Standard data formats –CVs –MIAPEs PEFF: A Common Sequence Database Format in Proteomics PRIDE Standard data format converters

10. HUPO PSI Protein Standard Initiative http://www.psidev.info

11. HUPO PSI Protein Standard Initiative Meetingshttp://www.psidev.info

12. HUPO PSI Open community initiative Develop data format standards Data representation and annotation standards Involve data producers, database providers, software producers, publishers Protein Standard Initiative http://psidev.info The Proteomics Standards Initiative (PSI) aims to define community standards for data representation in proteomics and to facilitate data comparison, exchange and verification Proteomics 2003, 3 (7): The proteomics standards initiative. Orchard,S., Hermjakob,H., Apweiler,R.

13. HUPO PSI structure Main unit is workgroup Gel Electrophoresis Molecular Interactions Sample Processing Mass spectrometry Proteomic Informatics (MS oriented) Protein Modifications Transversal activities One Steering Group Controlled vocabulary MIAPE guidelines

14. HUPO PSI structure Annual workshop, reporting activity at annual HUPO, conference calls, dedicated workshops No permanent funding, active members work on their “spare time” Website (http://psidev.info) and mailing-lists PSI Document process Vizcaino, J.A., Martens, L., Hermjakob, H., Julian, R.K. and Paton, N.W. (2007) The PSI formal document process and its implementation on the PSI website. Proteomics 7: 2355-2357.

15. HUPO PSI document process Community consultation at: http://www.nature.com/nbt/consult/

16. HUPO PSI structure

17. HUPO-PSI Project status

18. HUPO-PSI PSI deliverables Data formats MIML mzML AnalysisXML gelML giML spML MIAPE minimal reporting requirements One parent document - The minimum information about a proteomics experiment (MIAPE), Nature Biotechnology 25, 887-893 (2007) MIAPE MI, MS, MSI, GE, GI, CC, CE, SP Formats (XML schema, instance docs, specification docs) Controlled Vocabularies MIAPE docs (representation and annotation standards)

19. Index Need of standards in Proteomics HUPO-PSI –Organization –Standard data formats –CVs –MIAPEs PEFF: A Common Sequence Database Format in Proteomics PRIDE Standard data format converters

20. Standard data formats for Experimental data: spectra, acquisition parameters, acquisition equipment,... Analyzed data: identifications, quantitations, data analysis software...

21. Standard data formats Experimental data: spectra, acquisition parameters, acquisition equipment,... mzXML 2.0 mzXML 1.05 mzXML 3.0 mzXML 4.0 mzXML 2.0 mzML 1.0 mzML: Released on June 1st, 2008 Seattle Proteome Center at the Institute for Systems Biology HUPO-PSI data format capturing peak list information. Its aim is to unite the large number of current formats (pkl's, dta's, mgf's,.....) into one It is NOT a substitute for the rawfile formats of the instrument vendors. Some vendors, if not all, will provide software transforming their raw files to that standards

22. Sample instance document mzML 1.0

23. Standard data formats for Experimental data: spectra, acquisition parameters, acquisition equipment,... Analyzed data: identifications, quantitations, data analysis software...

24. Standard data formats Analyzed data: identifications, quantitations, data analysis software... describes the results of identification and quantitation processes for proteins, peptides and protein modifications from mass spectrometry protXML pepXML AnalysisXML AnalysisXML: v1.0 – candidate (Dic 08) Seattle Proteome Center at the Institute for Systems Biology HUPO-PSI

25. Sample instance document AnalysisXML (beta)

26. Standard data formats Other data: XML data formatMIAPE GelMLMIAPE GE GelInfoMLMIAPE GI miXMLMIAPE MIMIX spMLMIAPE SP

27. proprie- tary format mass spectrometer B mass spectrometer A converter mzML search engine A search engine B analysisXML Public repository Standard data formats

28. Index Need of standards in Proteomics HUPO-PSI –Organization –Standard data formats –CVs –MIAPEs PEFF: A Common Sequence Database Format in Proteomics PRIDE Standard data format converters

29. Controlled Vocabularies The Controlled Vocabularies (CVs) of the Proteomic Standard Initiative (PSI) provide a consensus annotation system to standardize the meaning, syntax and formalism of terms used across proteomics, as required by the PSI Working Groups. Each PSI working group develop the CVs required by the technology or data type it aims to standardize, following common recommendations for development and maintenance. At the PSI meeting in Washington (Sept 06), it was decided that all PSI working groups should adopt the same CVs standardizing some overlapping concepts (units and resources).

30. 100173 TOFT.O.F. time of flight time-of-flight What is a CV? Term Synonyms Controlled Vocabularies

31. PSI CVs are composed of two documents: a design principle description the implementation of the CVs in OBO (Open Biomedical Ontologies) Developing CVs is a process of collecting, and if necessary defining terms. Every effort must be made to adopt and re-use existing ontologies or CVs where they exist, to avoid “re-inventing the wheel”.

32. Ontology Lookup Service http://www.ebi.ac.uk/ontology-lookup/ The OLS provides a web service interface to query multiple ontologies from a single location with a unified output format.

33. Ontology Lookup Service http://www.ebi.ac.uk/ontology-lookup/

35. Index Need of standards in Proteomics HUPO-PSI –Organization –Standard data formats –CVs –MIAPEs PEFF: A Common Sequence Database Format in Proteomics PRIDE Standard data format converters

36. Sufficiency and practicability Unambiguous description of the experimental context Allow understanding of the results and their interpretation Sufficient to permit a critical evaluation In principle allow recreation of the work MIAPE: Minimum Information About a Proteomics Experiment Taylor, C.F., Paton, N.W., Lilley, K.S., Binz, P.A., Julian, R.K., Jr., Jones, A.R., Zhu, W., Apweiler, R., Aebersold, R., Deutsch, E.W., Dunn, M.J., Heck, A.J., Leitner, A., Macht, M., Mann, M., Martens, L., Neubert, T.A., Patterson, S.D., Ping, P., Seymour, S.L., Souda, P., Tsugita, A., Vandekerckhove, J., Vondriska, T.M., Whitelegge, J.P., Wilkins, M.R., Xenarios, I., Yates, J.R., 3rd and Hermjakob, H. (2007) The minimum information about a proteomics experiment (MIAPE). Nat Biotechnol 25: 887-893.

37. It is: – Describing a list of information and data to provide when an experiment is reported (it is a content descriptor) Peptide sequence, scores, modifications, mass errors, etc. – Helping to assess quality control Number of replicates, expected error rate MIAPE guidelines

38. It is not: – Describing the way to run an experiment does not specify the use of a search engine in particular does not force the use of one protocol – Describing the data representation Use excel to create a table with these five following columns:… – Including any quality judgment need 30% sequence coverage to identify a protein “The absence of thorough validation of both analytical and biological results, including error analysis should result in rejection” “Authors should justify the use of a very small database or database that excludes common contaminants, since this may generate misleading assignments” MIAPE guidelines

39. MIAPE Gel Electrophoresis (GE) v1.4 MIAPE Gel Informatics (GI) v0.5 MIAPE Mass Spectrometry (MS) v2.22 MIAPE Mass Spectrometry Informatics (MSI) v0.8 MIAPE Column Chromatography (CC) v1.0 MIAPE Capillary Electrophoresis (CE) v0.7 MIAPE Sample Preparation and handling (SP) v0.2 MIAPE Molecular Interactions (MI) v1.1.2 MIAPE guidelines

40. Online tool to generate and store MIAPE documents http://www.proteored.org

41. A MIAPE generator tool Fill all minimal information by hand Fill only some changes or new items by hand, and add automatically static information from previous MIAPE documents ProteoRed server

42. A MIAPE generator tool http://www.proteored.org

43. A MIAPE generator tool

47. HUPO-PSI: MIAPE Gel Electrophoresis v1.2

49. Generate XML Generate report Delete documentEdit document

50. MIAPE Reports Generate report

51. MIAPE Reports

58. Index Need of standards in Proteomics HUPO-PSI –Organization –Standard data formats –MIAPEs PEFF: A Common Sequence Database Format in Proteomics PRIDE Standard data format converters

59. A Common Sequence Database Format in Proteomics P-A Binz, S Seymour, J Shofsthal, D Creasy, E Kapp Problem: interpretation of current fasta format by search engines: Protein identifiers Description Taxonomy Other annotation (PTMs, sequence variants, etc) Propose an alternative to the heterogeneous fasta format, ideally generated by the database providers, or alternatively via an accepted converter, to submit one single source sequence database to various search engines SwissProt and EBI already agreed on the principle Format proposal reached (not only for MS, flexible, extensible) PEFF: PSI Extended FASTA Format

60. A unified format for protein and nucleotide sequence databases to be used by sequence search engines and other associated tools (spectra library search tools, sequence alignment software, data repositories, etc). PEFF: PSI Extended FASTA Format Enables consistent extraction, display and processing of information such as protein/nucleotide sequence database entry identifier, description, taxonomy, etc. across software platforms. Allows the representation of structural annotations such as post-translational modifications, mutations and other processing events. Flat file that includes a header of meta data to describe relevant information about the database(s) from which the sequence has been obtained (i.e., name, version, etc). Sequence database providers are encouraged to generate this format as part of their release policy or to provide appropriate converters that can be incorporated into processing tools.

62. Index Need of standards in Proteomics HUPO-PSI –Organization –Standard data formats –MIAPEs PEFF: A Common Sequence Database Format in Proteomics PRIDE Standard data format converters

63. PRIDE – Protein Identification Database Turns publicly available data into publicly accessible data Protein identifications Experimental detail Peak lists Linkout to raw data Fully open source Fully open data Implementation of PSI standards as they are released

64. proprie- tary format mass spectrometer B mass spectrometer A converter mzML search engine A search engine B analysisXML Public repository PRIDE

65. PRIDE – Protein Identification Database...Tomorrow with Alberto Medina

66. Index Need of standards in Proteomics HUPO-PSI –Organization –Standard data formats –MIAPEs PEFF: A Common Sequence Database Format in Proteomics PRIDE Standard data format converters

67. msconvert (ProteoWizard): –From: mzML, mzXML, Thermo RAW, MGF –To: mzML, mzXML –Vendor format reading restrictions: Thermo RAW: Windows with XCalibur XDK installed

68. Standard data format converters ReAdW version 4.0.2: –From: Thermo RAW –Exports to: mzXML mzML (not yet updated to final mzML 1.0 standard; try msconvert) –Requires a valid installation of the Thermo XCalibur software system, as it relies on the XCalibur libraries.

69. Standard data format converters CompassXport 1.3.6 : –From: analysis.baf (instrument families: APEX, micrOTOF, micOTOF-Q,...) analysis.yep (esquire/HCT instrument family) AutoXecute run for LCMaldi (instrument family: autoFlex, ultraFlex,...) fid files (flex instrument family) –Exports to: mzXML version 2.1 mzData, version 1.05 mzML in progress –Do not requires to install Bruker propietary software –Replace to mzBruker

70. Standard data format converters massWolf 4.0.2 (1st july 08): –From: MassLynx native acquisition files –Exports to: mzXML –Requires installation of MassLynx software on the same computer –You must select the appropriate massWolf download to match the version of your MassLynx software (4.0 or 4.1).

71. Standard data format converters mzWiff 4.0.2 (1st July 08): –From: Analyst native acquisition (.wiff) files –Exports to: mzXML –Requires installation of Analyst software

72. Standard data format converters T2DExtractor (Dec 07): –From: data from a SCIEX/ABI 4000 series MALDI TOFTOF instruments –Exports to: mzXML

73. Standard data format converters Trapper 4.1.0 (17 th july 08): –From: Agilent MassHunter format (.d directories) –Exports to: mzXML –Requires Agilent's MHDAC software installed –This software will be included in the upcoming 4.1.0 TPP distribution