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PEPTIDE SYNTHESIS in targeted proteomics

SpHPP. PEPTIDE SYNTHESIS in targeted proteomics. Manuel Lombardía Uría. Proteomics Facility – National Centre of Biotechnology. Peptide synthesis for targeted proteomics. Stages:. Selection and design of peptides for each protein of interest:

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PEPTIDE SYNTHESIS in targeted proteomics

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  1. SpHPP PEPTIDE SYNTHESIS in targeted proteomics Manuel Lombardía Uría Proteomics Facility – National Centre of Biotechnology

  2. Peptide synthesis for targeted proteomics Stages: • Selection and design of peptides for each protein of interest: • essential process for the success of SRM/MRM experiments • selection based on experimental and theoretical criteria • obtention of optimal proteotypic peptides • Chemical synthesis • Experimental validation of proteotypic peptides • Heavy isotope peptide synthesis

  3. Peptide selection Proteins of interest

  4. Assigned proteins

  5. Peptide selection Proteins of interest PTP prediction Candidate proteotypic peptides resulting from “in silico” digestion Experimental evidence Tryptic peptide observation by LC-MS/Ms analysis in biological samples

  6. Peptide selection Proteins of interest PTP prediction Candidate proteotypic peptides resulting from “in silico” digestion Experimental evidence Tryptic peptide observation by LC-MS/Ms analysis in biological samples Peptide selection

  7. Peptide selection criteria • Unique for each protein: Uniprot/Swiss prot searching (BLAST) • At least 3-4 peptides for protein • No missed cleavages • Containing 6-20 residues • Low hydrofobicity: values from 10 to 40 according to the SSRCalc algorithm • Avoid aa containing potential modification sites: • - Met and Trp undergo rapid oxidation • - Asn paired with G or P can be deamidated • - Gln or Glu at N-term undergo cyclization to pyroglutamic. Also C when carbamidomethylated • - Asn at N-term: protecting group can be difficult to remove during cleavage • - Asp paired with Gly, Pro or Ser can be hydrolised under acidic conditions • - Acidic aa at position P2 or P2’ promotes missed cleavage • - Asn in glycosilation consensus sequences (Asn-X-Ser/Thr) • - His residues close to either N or C terminus can undergo charge supression

  8. Peptide selection Proteins of interest PTP prediction Candidate proteotypic peptides resulting from “in silico” digestion Experimental evidence Tryptic peptide observation by LC-MS/Ms analysis in biological samples Peptide selection Búsqueda de evidencias en BD Not all the possible peptides can be experimentally observed. Select the most observed peptides in repositories such as GPMDB or SMS atlas.

  9. Peptide selection Proteins of interest PTP prediction Candidate proteotypic peptides resulting from “in silico” digestion Experimental evidence Tryptic peptide observation by LC-MS/Ms analysis in biological samples Peptide selection Búsqueda de evidencias en BD Not all the possible peptides can be experimentally observed. Select the most observed peptides in repositories such as GPMDB or SMS atlas. Peptide synthesis

  10. Peptide synthesis • Equipment: Authomatic synthesizer Multipep (Intavis AG) • Method: Solid phase peptide synthesis (SPPS) using polymeric resins proloaded and FMOC • Format: 96 minicolumn plates at a scale of 1-10 µmol • Peptide purification by semi-preparative HPLC • Resins preloaded with isotopically labeled Arg and Lys for the synthesis of peptides containing heavy Arg or Lys at the C-terminus • Heavy peptide quantification by amino acid analysis or fluorescence spectroscopy (commercial kit)

  11. Peptide validation Synthesized peptides crude or purified MALDI-TOF MS testing Validation by SRM/MRM experiments Optimal proteotypic peptides Heavy peptides synthesis

  12. Péptido 1 crudo Péptido 1 purificado

  13. Péptido 1 crudo Péptido 1 purificado

  14. Péptido 3 crudo Péptido 3 purificado

  15. Péptido 4 crudo Péptido 4 purificado

  16. Synthesized peptides

  17. Conclusion We can conclude from our results that our peptide synthesis method is suitable for the production of a high number of peptides with enough amount and quality for targeted proteomics applications.

  18. WE OFFER: • Regular Synthesis • Heavy Peptide Sinthesis • - Coupling to carrier protein for immunization • - N-term- Acetylation • - C-term Amidation • - Ser/Thr/Tyr Phosphorilation • - N-term Acylation • - Cys palmitoylatión • - N-term Biotinilation • - Peptide Arrays up to 620 spots per membrane

  19. PRACTICAL ISSUES Los precios 75 y 30€ (Arg y Lys) son sólo de la resina para cada péptido. Habría que añadir los 15 - 20€ por la síntesis. La cuantificación sólo se incluiría para los péptidos pesados precio? Tiempo?. Los tiempos para péptidos crudos, depende de cuántos sean, pero p.e. para media placa (48 péptidos) serían dos días para la síntesis y una semana para pasar por HPLC y al menos un día más para liofilizar, pesar y etiquetar. De 10-15 días si todo va bien.

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