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

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. Previous SRM analysis

48 proteins/group Working plan for MRM WP 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. Design of SRM mehods. Peptides and transitions. October 2013 May 2014 Nov 2013 Dec 2013 April2014 March 2014 Feb2014 Jan 2014 Excell with the proteins assigned to each group will be distributed Assays in biological matrices (at least 1 cell line) Preliminary assays based on experimental data Matching with SG data. Ranking by difficulty. Troubleshooting. Follow up meeting (SRM-psSRM) Consolidation of SRM methods (1-n cell lines) Follow up meeting (SRM-psSRM) Validation of assays in at least 2 labs Chr16 SRM Database MIAPE for SRM experiments Follow up meeting (SRM-psSRM) Topics for discussion MIDAS, Synthetic peptides Acceptance criteria. Reporting data. SRM database. Missing/difficult proteins. Gene expression data for cell line selection. Analysis of subcellular fractions. Analysis of plasma. PTMs by SRM. CRG

Searching for missing proteins 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.

neXtProt v ENSEMBL v73 UniProtKB v 2013_09 HPA v11 neXtProt v ENSEMBL v73 UniProtKB v 2013_09 HPA v genes 840 protein coding genes 143 missing proteins 2360 genes 840 protein coding genes 143 missing proteins 120 OMIM hits Obesity Neurodegenerative diseases Cancer 120 OMIM hits Obesity Neurodegenerative diseases Cancer Coverage of 73% gene products in Lymphoid cells Epitelial cells Fibroblasts Coverage of 73% gene products in Lymphoid cells Epitelial cells Fibroblasts Shotgun Proteomic analysis Shotgun Proteomic analysis Transcriptomic analysis Transcriptomic analysis 626 HPA antibodies for Chr16 proteins, 95 for missing Protein profile Gene expression profile Data integration Global and cell type specific outcomes Correlation proteome/transcriptome Proteome coverage and chromosome distribution Chr16 proteome coverage Global and cell type specific outcomes Correlation proteome/transcriptome Proteome coverage and chromosome distribution Chr16 proteome coverage Protein expression vectors for 64 Chr16 missing proteins

Tissue specific expression pattern of genes coding for missing proteins. HBM

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. B/D oriented strategy

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

Data management Data reporting, MIAPE. A common SRM database

Other topics for discussion. Analytical procedures Standardization of LC conditions. Reference peptides for retention time normalization. Acceptance criteria:  number of peptides and transitions  MIDAS  Synthetic peptides for validation Validation across labs (2-3). SOPs.

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. Proposal for extended working plan