SRM working plan.

1. SRM working plan. La Cristalera, 5-6 November 2013.

2. Previous SRM analysis • To deliver reliable SRM methods, we proposed a stepwise strategy that involves data sharing and cross-validation across different laboratories and this process must be optimized under standard conditions • An initial set of 120 Chr-16 proteins was selected on the basis of their GPMDB scores, log e in the range -175 to -6000, belonging to the group defined as “known” Chr16 proteins. • Each laboratory explored the detectability by SRM of the assigned proteins in digests from at least three cell lines : MCF7, CCD18, and Ramos. • For 106 out of the 120 proteins selected (88.3%) a minimum of three co-eluting SRM transition signals were observed for a number of peptides ranging from 1-10 per protein, with an average of 4.16 peptides/protein. • After the initial round of SRM method development at the six laboratories, a second round of cross-validation was performed, assigning each of the final 51 proteins detected to two laboratories different from the laboratory that initially developed the SRM method. After this second round of analysis validation, a total of 149 peptides from 49 proteins.

5. Working plan for MRM WP 2013-14 • All proteins identified by SG analyses will be distributed in packages of 48 protein across 8 labs. • Analysis will be also performed by pseudoSRM in additional 8 labs. • A minimum of 25 proteins must be analysed. • Development of absolute quantitation methods. October 2013 Design of SRM mehods. Peptides and transitions. • Preliminary assays based on experimental data • Matching with SG data. • Ranking by difficulty. Troubleshooting. • Follow up meeting (SRM-psSRM) Nov 2013 Assays in biological matrices (at least 1 cell line) Dec 2013 Jan 2014 • Consolidation of SRM methods (1-n celllines) • Follow up meeting (SRM-psSRM) Feb2014 • Validation of assays in at least 2 labs • Chr16 SRM Database • MIAPE for SRM experiments • Follow up meeting (SRM-psSRM) March 2014 April2014 May 2014 • Topicsfordiscussion • MIDAS, Syntheticpeptides • Acceptancecriteria. • Reporting data. • SRM database. • Missing/difficultproteins. • Gene expression data forcell line selection. • Analysis of subcellularfractions. • Analysis of plasma. • PTMsby SRM. 48 proteins/group CRG Excellwiththeproteinsassignedtoeachgroupwill be distributed

6. Searchingformissingproteins • Definition of the missing protein group is already made (NextProt) but it may be worth to consider low abundance proteins which detection will be challenging. If so, define criterion. • Identify which tissues or cell lines should be explored to identify chr16 missing proteins based on transcriptomic profiles and HPA evidences (for 95 chr16 missing proteins). Primary cells should be considered, in particular PBL. • Study of secretome and body fluids. • In some cases, subcellular fractionation or any other enrichment procedure should be considered. The case of plasma. • Searching on cells upon stimulation. Cell lines, type of stimuli and conditions should be defined. • Revise the biological function of the missing proteins, if known, to infer potential localizations.

7. Chromosome 16 626HPA antibodiesfor Chr16 proteins, 95formissing neXtProtv2013-10-10 ENSEMBL v73 UniProtKBv2013_09 HPA v11 120OMIM hits Obesity Neurodegenerativediseases Cancer 2360genes 840proteincodinggenes 143missingproteins Proteinexpressionvectorsfor64Chr16 missingproteins Coverage of 73% gene products in Lymphoidcells Epitelial cells Fibroblasts Shotgun Proteomic analysis Transcriptomic analysis Data integration Gene expressionprofile Proteinprofile Global and celltypespecificoutcomes Correlationproteome/transcriptome Proteomecoverage and chromosomedistribution Chr16 proteomecoverage

8. Tissuespecificexpressionpattern of genes codingformissingproteins. HBM

9. B/D orientedstrategy • Targeting disease related proteins. • Identify proteins of chr16 involved in B/D conditions within the consortium. • Configuration of disease related protein lists within the different B/D areas of the SpHPP. • Targeted monitoring of proteins of cellular pathways of particular relevance (metabolic or signaling pathways, etc). PTMs? • Proposals from B/D related groups • Integration with transcriptomic, metabolomics, others.

10. Cardiovascular Diseases Chair: L. Rguez.-Padial Co-Chair: F. Vivanco Osteoarthritis Rheumatoidarthritis SLE Brain tumors Breast cancer Colon cancer Infectious Diseases Chair: J. Fortun Co-Chair: C. Gil Rheumatic Diseases Chair: FJ. Blanco Co-Chair: JP. Albar Neurologic Disorders Chair: A. López-Munain Co-chair: JM. Arizmendi Red RECAVA Cancer Chair: C. Belda Co-Chair: I. Casal Obesity Chair: J. Prieto Co-Chair:F. Corrales Obesity NAFLD Artherosclerosis Valvular diseases Muscular dystrophy Parkinson diseases Biology Biomarkers (D/P/P/T) 1ª Phase: Known Proteins 2ª Phase: Unknown Proteins B/D-SpHPP Coordinator: FJ. Blanco Candidiasis CAIBER-ISCIII (clinicalresearch) Biobanks-ISCIII

11. Data management • Data reporting, MIAPE. A common • SRM database

12. Othertopicsfordiscussion. Analyticalprocedures • Standardization of LC conditions. Reference peptidesforretention time normalization. • Acceptancecriteria: • number of peptides and transitions • MIDAS • Syntheticpeptidesforvalidation • Validationacrosslabs (2-3). • SOPs.

13. Proposalfor extended working plan • SRM for SG proteins. All, according with the proposed pipeline. Results warranted. Reduce the workload if we agree to tackle missing proteins and B/D. • SRM for missing proteins • Biological analysis of chr16 missing proteins (databases). Groups • Biophysical features of chr16 missing proteins (Pino’s work). • Identification of tissues and cell lines for missing proteins search. Transcriptomic, HPA, others. CIMA. • Development of SRM assays. • SRM for B/D • Specific proteins, drivers of disease, already described by B/D groups. • Configuration of disease related lists of proteins (form datasets of B/D groups), any of them from chr16? • Analysis of all lists and identification of cellular pathways and corresponding proteins of special interest (biological, belong to chr16, common across lists, etc.) • Development of SRM assays.