Peptidi-1 Peptidi-2 1

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1 Peptidi-1 Peptidi-2 1

2 Peptidi-3 Peptidi-3 2

3 Peptidi-4 Peptidi-4 3

4 Peptidi-5 Peptidi-5 4

5 Peptidi-6 Peptidi-6 5

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9 Spettrometria di massa di biomolecole: esempi ed esercizi Margherita Ruoppolo Dipartimento di Medicina Molecolare e Biotecnologie Mediche Ceinge Biotecnologie Avanzate Università degli Studi di Napoli Federico II margherita.ruoppolo@unina.it Esercizio 1. Determinare la massa molecolare della proteina a partire dal suo spettro di massa ESI Spettro myoprova

10 Esercizio 2. Utilizzando il peso molecolare accurato determinato mediante ES/MS verificare la struttura primaria delle seguenti proteine. Laddove si osservano delle anomalie, formulare una ipotesi sulle possibili modifiche strutturali a) AcP (Acyl carrier protein) b) RNase A Acyl carrier Protein A B= /- 0.9 A= /- 1.1 B C C= /

11 AcP GLY-SER-MET-LYS-LYS-TRP-SER-ASP-THR-GLU-VAL-PHE-GLU-MET-LEU-LYS-ARG-MET-TYR-ALA ARG-VAL-TYR-GLY-LEU-VAL-GLN-GLY-VAL-GLY-PHE-ARG-LYS-PHE-VAL-GLN-ILE-HIS-ALA-ILE ARG-LEU-GLY-ILE-LYS-GLY-TYR-ALA-LYS-ASN-LEU-PRO-ASP-GLY-SER-VAL-GLU-VAL-VAL-ALA GLU-GLY-TYR-GLU-GLU-ALA-LEU-SER-LYS-LEU-LEU-GLU-ARG-ILE-LYS-GLN-GLY-PRO-PRO-ALA ALA-GLU-VAL-GLU-LYS-VAL-ASP-TYR-SER-PHE-SER-GLU-TYR-LYS-GLY-GLU-PHE-GLU-ASP-PHE- GLU-THR-TYR AVERAGE MOLECULAR WEIGHT Da SOLUZIONE Acyl carrier Protein B= A (5-103) B A= (1-103) C= C (7-103) 11

12 RNase A A= /- 0.8 A RNase A LYS GLU THR ALA ALA ALA LYS PHE GLU ARG GLN HIS MET ASP SER SER THR SER ALA ALA SER SER SER ASN TYR CYS ASN GLN MET MET LYS SER ARG ASN LEU THR LYS ASP ARG CYS LYS PRO VAL ASN THR PHE VAL HIS GJU SER LEU ALA ASP VAL GLN ALA VAL CYS SER GLN LYS ASN VAL ALA CYS LYS ASN GLY GLN THR ASN CYS TYR GLN SER TYR SER THR MET SER ILE THR ASP CYS ARG GLU THR GLY SER SER LYS TYR PRO ASN CYS ALA TYR LYS THR THR GLN ALA ASN LYS HIS ILE ILE VAL ALA CYS GLU GLY ASN PRO TYR VAL PRO VAL HIS PHE 125 ASP ALA SER VAL AVERAGE MOLECULAR WEIGHT Da 12

13 Esercizio n 3 Determinare la mappa peptidica della beta-globina umana sulla base dei dati ottenuti dall analisi MALDI/MS del digerito triptico, associando ciascun segnale di massa al corrispondente peptide nella sequenza della proteina: Beta-globina umana 13

14 Beta-globina umana VAL-HIS-LEU-THR-PRO-GLU-GLU-LYS-SER-ALA-VAL-THR-ALA-LEU-TRP-GLY-LYS-VAL-ASN-VAL ASP-GLU-VAL-GLY-GLY-GLU-ALA-LEU-GLY-ARG-LEU-LEU-VAL-VAL-TRP-PRO-TRP-THR-GLN-ARG PHE-PHE-SER-GLU-PHE-GLY-ASP-LEU-SER-THR-PRO-ASP-ALA-VAL-MET-GLY-ASN-PRO-LYS-VAL LYS-ALA-HIS-GLY-LYS-LYS-VAL-LEU-GLY-ALA-PHE-SER-ASP-GLY-LEU-ALA-HIS-LEU-ASP-ASN LEU-LYS-GLY-THR-PHE-ALA-THR-LEU-SER-GLU-LEU-HIS-CIS-ASP-LYS-LEU-HIS-VAL-ASP-PRO GLU-ASN-PHE-ARG-LEU-LEU-GLY-ASN-VAL-LEU-VAL-CYS-VAL-LEU-ALA-HIS-HIS-PHE-GLY-LYS GLU-PHE-THR-PRO-PRO-VAL-GLN-ALA-ALA-TYR-GLN-LYS-VAL-VAL-ALA-GLY-VAL-ALA-ASN-ALA- 145 LEU-ALA-HYS-LYS-TYR-HIS Beta-globina umana Peptide Average Molecular weight

15 Esercizio n 4 Assegnare i segnali non interpretati nello spettro dell idrolizzato triptico della b-globina umana, ipotizzando eventuali appaiamenti dei peptidi contenenti residui di cisteina. Usare la seguente tabella MH + peptide (Contiene Cys 93) (contiene Cys 112) (Contiene Cys 93) (96-104) (31-40) (18-30) ( ) (83-95) (67-82) (66-82) (41-59)

16 Beta-globina umana VAL-HIS-LEU-THR-PRO-GLU-GLU-LYS-SER-ALA-VAL-THR-ALA-LEU-TRP-GLY-LYS-VAL-ASN-VAL ASP-GLU-VAL-GLY-GLY-GLU-ALA-LEU-GLY-ARG-LEU-LEU-VAL-VAL-TRP-PRO-TRP-THR-GLN-ARG PHE-PHE-SER-GLU-PHE-GLY-ASP-LEU-SER-THR-PRO-ASP-ALA-VAL-MET-GLY-ASN-PRO-LYS-VAL LYS-ALA-HIS-GLY-LYS-LYS-VAL-LEU-GLY-ALA-PHE-SER-ASP-GLY-LEU-ALA-HIS-LEU-ASP-ASN LEU-LYS-GLY-THR-PHE-ALA-THR-LEU-SER-GLU-LEU-HIS-CIS-ASP-LYS-LEU-HIS-VAL-ASP-PRO GLU-ASN-PHE-ARG-LEU-LEU-GLY-ASN-VAL-LEU-VAL-CYS-VAL-LEU-ALA-HIS-HIS-PHE-GLY-LYS GLU-PHE-THR-PRO-PRO-VAL-GLN-ALA-ALA-TYR-GLN-LYS-VAL-VAL-ALA-GLY-VAL-ALA-ASN-ALA- 145 LEU-ALA-HYS-LYS-TYR-HIS 16

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19 Relative Abundance Relative Abundance Attribuire la giusta sequenza al peptide Gly Phe Tyr Gly Leu/Ile Residui AA: Gly 57 Phe 147 Tyr 163 Leu/Ile 113 C:\CIADS\...\peptide-ms7\peptide99 peptide99 # RT: AV: 2 NL: 2.41E6 F: + p Full ms [ ] m/z peptide99 # RT: AV: 19 NL: 6.73E5 F: + p Full ms @24.00 [ ] MS 2 m/z b a m/z 19

20 Relative Abundance Relative Abundance Relative Abundance Relative Abundance peptide99 # RT: AV: 24 NL: 1.54E5 F: + p Full ms @ @19.00 [ ] MS 3 m/z 556 m/z a Residui AA: Gly 57 Phe 147 Tyr 163 Leu/Ile m/z b 4 C:\CIADS\...\peptide-ms7\peptide99 21/02/ peptide99 # RT: AV: 10 NL: 6.20E3 F: + p Full ms @ @ @25.00 [ ] MS 4 m/z 556 m/z 425 m/z b m/z a 4 peptide99 # RT: AV: 6 NL: 1.11E4 F: + p Full ms @ @ @ @22.00 [ ] m/z b 2 80 MS 5 Residui AA: Gly 57 Phe 147 Tyr 163 Leu/Ile m/z 425 m/z m/z m/z peptide99 # RT: AV: 21 NL: 6.61E3 F: + p Full ms @ @ @ @ @21.00 [ ] MS 6 90 a m/z m/z 278 m/z

21 Relative Abundance Residui AA: Gly 57 Phe 147 Tyr 163 Leu/Ile 113 peptide99 # RT: AV: 38 NL: 7.87E2 F: + p Full ms @ @ @ @ @ @22.00 [ MS 7 m/z m/z m/z m/z 193 b m/z H O Tyr-Gly-Gly-Phe-Leu a 2 O a 4 O H 2 N N H N H N H N H O H O O b 4 O b 1 b 2 b 3 21

22 Relative Abundance Assegnare i vari ioni ESI-QIT (+) HO + H N m/z HF 100 HO H OH N N -HF -HF 80 OH N +H 2 O N N 60 F 3 C +H 2 O CF N N HF 40 H H 2 O H ESI-QqQ (+) ESI D:\Giacomo1&2\Peptide-solo 05/24/ :29:41 AM Peptide-solo #31-92 RT: AV: 62 SM: 9G NL: 5.74E4 F: - p ms [ ] m/z 22