La Spettrometria di Massa nello Studio di Proteine e Peptidi

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1 La Spettrometria di Massa nello Studio di Proteine e Peptidi Gianluca Giorgi Università degli Studi di Siena Dipartimento di Biotecnologie, Chimica e Farmacia via Aldo Moro 531 Siena gianluca.giorgi@unisi.it Tel

2 La Spettrometria di Massa nello studio di Proteine e Peptidi 1. Studio di Proteine i) Estrazione ii) Separazione iii) Determinazione del PM i) MALDI-TF: spettro, risoluzione ii) ESI: ioni multicarica, risoluzione iii) Quale massa? iv) Ricerca in banca dati iv) Determinazione della struttura (Sequenza AA): MS n i) Proteina in toto: ECD, ETD ii) Digestione enzimatica Peptidi i) MALDI: peptide mass fingerprinting ii) HPLC-ESI-MS e MS n iii) Ricerca in banca dati

3 1. Studio di Proteine i) Estrazione ii) Separazione iii) Determinazione del peso molecolare MALDI ESI Proteine - Determinazione del PM MALDI-TF: spettro, risoluzione MALDI-TF linear mass spectrum of intact protein: Hg-Papain oligomerization

4 Proteine - Determinazione del PM MALDI-TF: spettro, risoluzione MALDI TF mass spectrum of a mixture of ubiquitin, cytochrome C and equine myoglobin using 2,5-dihydroxybenzoic acid (DHB) as the matrix. citocromoc #1-11 RT: AV: 11 SM: 9G NL: 7.4E5 F: + p Full ms [ 6.-2.] Proteine - Determinazione del PM ESI: ioni multicarica, risoluzione citocromoc #12-18 RT: AV:7 SM: 9G NL: 1.61E4 F: + Z ms [ ] R=

5 Assegnamento delle cariche Spettro deconvoluto in silico mass

6 ESI (+) of Ubiquitin 1,4, Resolution at 779.9u.9u Proteine & Peptidi- Determinazione del PM Spettrometria di massa.. Quale massa?

7 L accuratezza di massa diminuisce all aumentare della grandezza dello ione Poiché una proteina è una molecola grande, occorre elevatissima risoluzione e notevole accuratezza di massa (FT-ICR) per poter avere informazioni univoche sulla sua formula bruta La Spettrometria di Massa nello studio di Proteine e Peptidi 1. Studio di Proteine i) Estrazione ii) Separazione iii) Determinazione del PM i) MALDI-TF: spettro, risoluzione ii) ESI: ioni multicarica, risoluzione iii) Quale massa? iv) Ricerca in banca dati iv) Determinazione della struttura (Sequenza AA): MS n i) Proteina in toto: ECD, ETD ii) Digestione enzimatica Peptidi i) MALDI: peptide mass fingerprinting ii) HPLC-ESI-MS e MS n iii) Ricerca in banca dati

8 Determinazione della struttura: MS n i) Proteina in toto: CID non riesce a frammentare ioni prodotti da molecole grandi (PM > 3 ca) La decomposizione di ioni prodotti da molecole grandi può avvenire solo attraverso l interazione con elettroni: ECD = electron capture dissociation (FT-ICR) ETD = electron transfer dissociation (2D e 3D IT) Determinazione della struttura: MS n i) Proteina in toto: ECD = electron capture dissociation (FT-ICR) ETD = electron transfer dissociation (Linear IT) Approccio Top Down

9 Determinazione della struttura Proteina in toto ECD, EDT MS 2 Proteina [M+nH] n+ Frammenti Determinazione della struttura ii) Digestione enzimatica Peptidi Enzyme 1 Protein (in solution) Many Peptides (in solution)

10 Trypsin: Lys, Arg S S Approccio Bottom Up Database search

11 Identification of a Protein via Peptide Mass Fingerprinting Direct analysis of the entire peptide mixture Intensity Search Engine MQRVNMIMAESPSLITICLLGYLLSAECT VFLDHENANKILNRPKRYNSGKLEEFVQ GNLERECMEEKCSFEEAREVFENTEK TTEFWKQYVDGDQCESNPCLNGGSCK DDINSYECWCPFGFEGKNCELDVTCNI KNGRCEQFCKNSADNKVVCSCTEGYR LAENQKSCEPAVPFPCGRVSVSQTSK LTRAEAVFPDVDYVNPTEAETILDNITQ GTQSFNDFTRVVGGEDAKPGQFPWQ VVLNGKVDAFCGGSIVNEKWIVTAAHC VETGVKITVVAGEHNIEETEHTEQKRN VIRAIIPHHNYNAAINKYNHDIALLELDE PLVLNSYVTPICIADKEYTNIFLKFGSG YVSGWGRVFHKGRSALVLQYLRVPLV DRATCLRSTKFTIYNNMFCAGFHEGG RDSCQGDSGGPHVTEVEGTSFLTGII SWGEECAMKGKYGIYTKVSRYVNWIK EKTKLT Protein identification by peptide mapping 1 MALDI-TF-MS Relative Intensity # 16 Da ox Met Mass/Charge Database search ( Aldolase A 1 PYQYPALTPEQKKELSDIAHRIVAPGKGILAADESTGSIAKRLQSIGTEN 51 TEENRRFYRQLLLTADDRVNPCIGGVILFHETLYQKADDGRPFPQVIKSK 11 GGVVGIKVDKGVVPLAGTNGETTTQGLDGLSERCAQYKKDGADFAKWRCV 151 LKIGEHTPSALAIMENANVLARYASICQQNGIVPIVEPEILPDGDHDLKR 21 CQYVTEKVLAAVYKALSDHHIYLEGTLLKPNMVTPGHACTQKFSHEEIAM 251 ATVTALRRTVPPAVTGITFLSGGQSEEEASINLNAINKCPLLKPWALTFS 31 YGRALQASALKAWGGKKENLKAAQEEYVKRALANSLACQGKYTPSGQAGA 351 AASESLFVSNHAY

12 MS of a peptide mixture by MALDI-TF Peptide mass fingerprinting MALDI-TF

13 Identification of a spot based on peptide mapping Protein identifications were made by peptide mass mapping (peptide mass fingerprinting) using an unspecified search program and an unspecified database. Se il TF ha alta risoluzione, è possibile determinare la massa accurata e la formula bruta di ciascun peptide Se proteine diverse, digerite con lo stesso enzima nelle stesse condizioni sperimentali, producono lo stesso peptide mass fingerprint, le due proteine sono uguali

14 Proteine: Determinazione della struttura Digestione enzimatica Peptidi HPLC-ESI-MS e MS n Database search peptide MW HPLC separation ESI- MS Enzyme HPLC-MS Tempo di ritenzione di ciascun peptide MS

15 R=5 R=2 R= MS R=5 R=2 R=15. MS

16 Se si dispone di un analizzatore con alta risoluzione e massa accurata settori, ToF, rbitrap, FT-ICR è possibile misurare la massa accurata di ciascun peptide e quindi la formula bruta Poiché le tecniche di ionizzazione utilizzate sono soft non si ha frammentazione in sorgente e quindi non si hanno informazioni strutturali, ovvero non è possibile determinare la sequenza AA Proteine: Determinazione della struttura Digestione enzimatica Peptidi HPLC-ESI-MS e MS n Database search CID-MS n peptide MW HPLC separation ESI- MS

17 CID-MS n di peptidi x 3 y 3 z 3 x 2 y 2 z 2 x 1 y 1 z 1 a 1 b 1 c 1 a 2 b 2 c 2 a 3 b 3 c 3 Ioni C-terminali: y, x, z Ioni N-terminali: b, a, c

18 Why b ions are very stable species? They don t have an acylium ion structure R-C + but they have a cyclic oxazolonic structure Spectroscopic Evidence for an xazolone Structure of the b 2 Fragment Ion from Protonated Tri-Alanine omens J et al, J. Am. Soc. Mass. Spectrom. 2, 334 (29)

19 Whenever you identify a b ion look for an a ion at -28u. This gives some assurance that your assignment is correct. Also look for ammonia and water losses, -17 and -18u respectively.

20 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 #21-22 RT: AV: 2 NL: 2.41E6 F: + p Full ms [ 8.-8.] peptide99 #27-45 RT: AV: 19 NL: 6.73E5 F: + p Full ms2 556.@24. [ ] MS a b 4 peptide99 #46-79 RT: AV: 24 NL: 1.54E5 F: + p Full ms3 556.@ @19. [ ] 1 a Residui AA: Gly 57 Phe 147 Tyr 163 Leu/Ile MS b C:\CIADS\...\peptide-ms7\peptide99 21/2/ peptide99 #69-79 RT: AV: 1 NL: 6.2E3 F: + p Full ms4 556.@ @ @25. [ ] MS b a

21 556 peptide99 #8-16 RT: AV: 6 NL: 1.11E4 F: + p Full ms5 556.@ @ @ @22. [ ] MS 5 b 2 8 Residui AA: Gly 57 Phe 147 Tyr 163 Leu/Ile b peptide99 #85-16 RT: AV: 21 NL: 6.61E3 F: + p Full ms6 556.@ @ @ @ @21. [ ] MS 6 b 9 a a peptide99 # RT: AV: 38 NL: 7.87E2 F: + p Full ms7 556.@ @ @ @ @ @22. [ MS a b

22 H Tyr-Gly-Gly-Phe-Leu a 1 a 2 a 4 H 2 N NH NH NH NH H b 4 b 1 b 2 b 3 Attribuire la sequenza al peptide C:\CIADS\...\peptide-ms7\peptide99 peptide99 #21-22 RT: AV: 2 NL: 2.41E6 F: + p Full ms [ 8.-8.] peptide99 #27-45 RT: AV: 19 NL: 6.73E5 F: + p Full ms2 556.@24. [ ] MS y a b 4

23 Cyclic peptides: Naturally occurring in bacteria, fungi and plants Synthetic compounds They can have a lot of biological and pharmaceutical properties: enzyme inhibitors, antifungal and antibacterial agents, immunosuppressant, antibiotics and anticancer drugs Better bioavailability and higher resistance to proteolytic degradation than their linear analogues Highly selective metal ion chelators: carriers of metal ions inside the cells. Use in radiotherapy Brodbelt et al. J Am Soc Mass Spectrom 24, 15,

24 Characterization and sequencing of cyclic peptides represent an important challenge for mass spectrometry. No definite terminations, as it occurs in their linear analogues. The elimination of an aminoacidic residue must involve the fission of two bonds of the cycle. The ring-opening may occur at several backbone positions, meaning that all the fragment ions are not referenced to a single terminal position. peptidedk13_elisa_ #161 RT:1.5 AV:1 NL:1.6E9 F: FTMS + p ESI Full ms [ ] R= [M + 2H] [M + H] R= [M + 3H] R=

25 Resolving powers: rbitrap: at constant detection time: resolving power inversely proportional to Thus if R=1, at 1, at 1 it will be R=1,(1/1) 1/2 =31,646 RDB=double bond/ring equivalents For ions with odd charge: dd-electron ions = RDB integer Even-electron ions= RDB x.5 12 C 3 13 C 1 14 N H 6

26 peptidedk13_elisa_ # RT: AV:6 NL:1.9E9 F: FTMS + p ESI Full ms [ ] [M + 2H]

27 peptidedk13_elisa_ # RT: AV:6 NL:1.9E9 F: FTMS + p ESI Full ms [ ] peptidedk13_elisa_ #161 RT:1.5 AV:1 NL:8.24E7 F: FTMS + p ESI Full ms [ ] R= [M + 3H] R= R=

28 peptidedk13_elisa_ # RT: AV:54 NL:3.3E7 F: FTMS + p ESI Full ms2 474.@cid2. [13.-6.] (.49) (.7) 95 Residui AA: H 2 -Lys Lys 128 His 137 Ala/β-Ala (.43) -His -Lys β-ala NH 3 -His -Lys -His H 2 [MH (NH 3 CH 2 )] + NH [M+H]

29 Peptide solo2 #1 16 RT:.2.27AV:16 NL:2.1E5 F: + p Full ms3 474.@cid @cid22. [95.-1.] CID-MS 3 [MH-Lys] + Residui AA: Lys 128 His 137 Ala/β-Ala His H [MH-Lys] N NH NH HN NH NH NH 2 N NH c-(lys-d/l-his-β-ala-his)

30 N NH NH NH H + HN NH HN HN NH 2 N H 2 N H N +H + N N NH NH 2 N b + 3 K/H 346 NH HN - N H N H 2 N + H + N 29 N NH b 3 + K/H 346

31 N NH NH HN +H + NH NH HN - N H N H 2 N +H + HN N NH 2 HN N NH 2 N NH y 3+ K/H H 2 N NH H 2 N + H + N N NH 29 CID-MS 2 [M+2H] 2+ LysH LysH + [M H 2 +2H] LysH HisβAlaH + HisβAlaH HisH [M NH 3 H 2 +2H] [M+2H]

32 CID-MS 2 [M-H] His Lys IntensitàRelativa(%) β-ala His -His H β-ala His β-ala If you are working with a tryptic peptide, look for the arginine or lysine y 1 ion at the low end of the spectrum: [M+H] = penultimate b ion [M+H] = penultimate b ion Arg is the carboxy AA Lys is the carboxy AA This may give you a clue to the C-terminal residue of your tryptic peptide. In general: protonated peptide - AA residue mass - 18 = penultimate b ion or you could use the standard formula for calculating the corresponding b ion once the y ion is known. [M+H] + -y 1 +1 = penultimate b ion If you are using an ion trap you may not be able to observe the low end of the spectrum, and in this case, you will need to do both of these calculations

33 [M+H]+ - observed b ion + 1 = corresponding y ion. Digerito triptico [M+H] + = 1379 (14 AA) MS/MS 1379 (ion trap)

34 Digerito triptico [M+H] + = 1182 (1 AA) MS/MS 1182 (ion trap)

35 ETD, ECD High Collision CID Ions c, z

36

37 x 3 y 3 z 3 x 2 y 2 z 2 x 1 y 1 z 1 a 1 b 1 c 1 a 2 b 2 c 2 a 3 b 3 c 3 Loss of ne Phosphorylation Loss of Two Phosphorylations 74

38 Single Scan Ion Trap CID with ICR Cell Detection 1 R P K P Q Q F F G L M B B 8 2+ B B B 5 Y [M+2H] B B Ion Theoretical Measured ppm B B B B B [M+2H] B B B Y* B Y B Fragments identified with a average error of.3 ppm B B B Single Scan IRMPD with ICR Cell Detection R P K P Q Q F F G L M B B B [M+2H] B 2+ B B 6 + Ion Theoretical Measured ppm B B B B B [M-NH3] M B B B Y* Y Fragments identified with a average error of.58 ppm Y + 9 B

39 Single Scan ECD with ICR Cell Detection R P K P Q Q F F G L M C [M+2H] C + C Ion Theoretical Measured ppm C C [M+2H] C C C Z C C [M-NH3] [M+2H] Fragments identified with a average error of.56 ppm C C [M+2H] + [M-NH 3] Z 9 + C C Top down vs approaches Bottom up for studying large molecules

40 Determinazione della struttura: MS n i) Proteina in toto: ECD = electron capture dissociation (FT-ICR) ETD = electron transfer dissociation (Linear IT) Approccio Top Down

41 x 3 y 3 z 3 x 2 y 2 z 2 x 1 y 1 z 1 a 1 b 1 c 1 a 2 b 2 c 2 a 3 b 3 c 3

42 Top down

43 ETD Electron Transfer Dissociation Loss of ne Phosphorylation Loss of Two Phosphorylations Courtesy by G. Vago Thermo