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

2. Peptide synthesis for targeted proteomics  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 Stages:

3. Peptide selection Proteins of interest

4. KnownUnknown Protein Accession number NameProtein Accession number Name P02795MT2_HUMANMetallothionein-2Q96M29TEKT5_HUMANTektin-5 Q58EX7PKHG4_HUMANPuratrophin-1Q96MC5CP045_HUMANUncharacterized protein C16orf45 Q01726MSHR_HUMAN Melanocyte-stimulating hormone receptor Q14028CNGB1_HUMAN Cyclic nucleotide-gated cation channel beta-1 Q14764MVP_HUMANMajor vault proteinP23975SC6A2_HUMAN Sodium-dependent noradrenaline transporter Q0VD83APOBR_HUMANApolipoprotein B receptorQ8NC67NETO2_HUMANNeuropilin and tolloid-like protein 2 Q6EMK4VASN_HUMANVasorinQ8IZ96CKLF1_HUMAN CKLF-like MARVEL transmembrane domain-containing protein 1 O14983AT2A1_HUMAN Sarcoplasmic/endoplasmic reticulum calcium ATPase 1 P33527MRP1_HUMAN Multidrug resistance-associated protein 1 P22695QCR2_HUMAN Cytochrome b-c1 complex subunit 2, mitochondrial P60510PP4C_HUMAN Serine/threonine-protein phosphatase 4 catalytic subunit P55287CAD11_HUMANCadherin-11 Q49A26GLYR1_HUMANPutative oxidoreductase GLYR1 Q4KMP7TB10B_HUMANTBC1 domain family member 10B Q14019COTL1_HUMANCoactosin-like protein Q9NXV2KCTD5_HUMAN BTB/POZ domain-containing protein KCTD5 Q9Y221NIP7_HUMAN 60S ribosome subunit biogenesis protein NIP7 homolog P55259GP2_HUMAN Pancreatic secretory granule membrane major glycoprotein GP2 O75150BRE1B_HUMANE3 ubiquitin-protein ligase BRE1B Q2VPK5CTU2_HUMAN Cytoplasmic tRNA 2-thiolation protein 2 Q8N9N5BANP_HUMANProtein BANP Assigned proteins

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

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

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

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

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

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

19. 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.

20. 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

21. 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. PRACTICAL ISSUES