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WG2 - SRM Group

WG2 - SRM Group. 11-12- 2012. 16. Genes on Chromosome 16. 16. More than 85 % of protein coding genes of chromosome 16 are expressed in lymphoid cells , epitelial cells and fibroblasts . 16. Workplan proposal. 16.

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WG2 - SRM Group

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  1. WG2 - SRM Group 11-12- 2012

  2. 16 Genes onChromosome 16 16 More than85 % of proteincoding genes of chromosome 16 are expressed in lymphoidcells, epitelial cells and fibroblasts.

  3. 16 Workplanproposal 16 • Characterizethetranscriptometo define the actual chr16’s gen set expressed and mostimportantlywhich genes are notexpressed in thethreeselectedcelltypes. • Characterizetheproteome in detail. • Expression, purification, and characterization of 22 unknownproteinsavailable in NAPPA collection. • Developmentof SRM quantitativeassays. • Proteins (862) will be distributed in known (562) and unknowngroups (300) and thestudywill be performedalong 3 years, starting in 2012 (214 proteins). • Protein packs will be assignedtoeach of the 8 MRM groups (24), includingequalproportion of known (20, withdifferentrange of theoreticaldifficulty) and unknownproteins (4). • Unknownproteinswill be firstsearchedbytargeteddiscovery (TD) and relevantinformationresultingfromthisstudieswill be usedtodesign MRM assays. TD groups (9) willanalyze 54 proteinsthefirstyear. • Eachgroupwill use the master sample (thismightrequirethe use of syntheticpeptidesduringtherefinementsteps). TD will be done in allthreeselectedcelllines. Regarding MRM, a pool of thethreecelllineswill be usedforthe 20 knownproteins. Decisiononusing a pool or a particular cell line fortheunknownproteinswill be taken in light of theresultsfrom TD. • Once developed, theassayswill be validatedby 2 additionalgroups. Finally, thequantitativeassaywill be set up using heavy peptides. • Definition of Bioinformaticstandards and SOPs. • Definition of Biobankingstrategy, standards... in collaborationwiththeSpanishBiobankingPlatform.

  4. PROTEINS ASSIGNED - 2012 160 known Proteins, 8 groups → 20 proteins/group Sampletypes : MCF7/ CCD18/ Ramos/ HUH7/ Plasma/ TC28 Instruments: 4000 QTRAP, 5500 QTRAP Columns: PepMap100, C18, 3um 100A, 75umx15cm (3) PepMapRSLC, C18, 2um 100A, 75umx15cm EksigentChrome XP C18 3 µm,, 0,075 x 150 mm OnyxMonolithic C18, 150x0.1mm Gradients: 300 nL/min 35-85 min

  5. PROTEINS • Results for 90 proteins • 1775 assayed peptides • 413 observed peptides • 157 verified peptides

  6. Templateresults MRM_121127.xlsx Proteins

  7. TemplateresultsMRM_121127.xlsx Peptides

  8. TemplateresultsMRM_121127.xlsx Transitions

  9. PEPTIDES – OBSERVED /VERIFIED From 413 observed peptides *No Data forallthe 413 observedpeptides

  10. PEPTIDES/PROTEIN 86 Proteins 33 Proteins

  11. PEPTIDES/PROTEIN – Sample type 81 protevaluated 66 in RAMOS 48 in CCD18 63 in MCF7

  12. OtherSamples

  13. TRANSITIONS/ PEPTIDE 1360 Transitions

  14. DISCUSSION POINTS • Peptide /Transitionsverificationcriteria. IDA, MSMS # transitions • Proteotypicpeptides – Isoforms • Scoring “goodness” for SRM • Optimization of measurementconditions. Gradients – CE • Use of StandardsforrelativeRetention Time evaluation • Use of (Interna) Standard forrelativeintensitymeasurement • Selection of proteinsforrecombinantexpression • Selection of syntheticpeptidesfor SRM assaydevelopment • Selection of labelessyntheticpeptidesforquantitativeassays • Unknown- lowabundantprotein : detectionlimits? Fractionation? • Multi- laboratoryvalidation • NextProteinAssignment (when, number, unknown, samples…)

  15. THANK YOU !!!

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