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Comprehensive Analysis of Chromosome 16 Protein Coding Genes

This project aims to characterize the transcriptome and proteome of chromosome 16, focusing on lymphoid, epithelial, and fibroblast cells. It includes the expression, purification, and characterization of unknown proteins, development of SRM quantitative assays, bioinformatic standards, and biobanking strategy definition.

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Comprehensive Analysis of Chromosome 16 Protein Coding Genes

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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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