La Spettrometria di Massa nello Studio di Proteine e …...2014/01/16 · 2 La Spettrometria di...

1

Gianluca GiorgiUniversità degli Studi di SienaDipartimento di Biotecnologie, Chimica e Farmaciavia Aldo Moro53100 Sienae-mail: [email protected]. 0577-234241

La Spettrometria di Massa nello Studio di Proteine e

Peptidi

2

La Spettrometria di Massa nello studio di Proteine e Peptidi

1. Studio 1. Studio didi ProteineProteine

i) Estrazione

ii) Separazione

iii) Determinazione del PMi) MALDI-TOF: spettro, risoluzioneii) ESI: ioni multicarica, risoluzioneiii) Quale massa?iv) Ricerca in banca dati

iv) Determinazione della struttura (Sequenza AA): MSn

i) Proteina in toto: ECD, ETD

ii) Digestione enzimatica Peptidii) MALDI: peptide mass fingerprintingii) HPLC-ESI-MS e MSn

iii) Ricerca in banca dati

3

MALDI ESI

iii) Determinazione del peso molecolare


i) Estrazione

ii) Separazione

MALDI-TOF linear mass spectrum of intact protein: Hg-Papain oligomerization

Proteine - Determinazione del PMMALDI-TOF: spettro, risoluzione

4

MALDI TOF mass spectrum of a mixture of ubiquitin, cytochrome C and equine myoglobin using 2,5-dihydroxybenzoic acid (DHB) as the matrix.

Proteine - Determinazione del PMMALDI-TOF: spettro, risoluzione

R=100.000

citocromoc #1-11 RT: 0.01-0.23 AV: 11 SM: 9G NL: 7.40E5F: + p Full ms [ 600.00-2000.00]

700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

942.2 1020.4

1112.9 1529.51359.81224.0875.1

816.91747.8

765.9721.1683.3

citocromoc #12-18 RT: 10.48-10.78 AV: 7 SM: 9G NL: 1.61E4

F: + Z ms [ 1480.00-1580.00]

1526 1528 1530 1532 1534 1536 1538 1540 1542

m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

1530.31530.0

1530.7

1529.8 1530.9

1531.0

1531.21529.6

1534.71531.7 1535.11529.5 1534.3 1539.51535.4 1539.81529.4

Proteine - Determinazione del PMESI: ioni multicarica, risoluzione

5

600 800 1000 1200 1400 1600 1800 2000m/z

0

10

20

30

40

50

60

70

80

90

100R

elat

ive

Abun

danc

e

1748

1529

13601224

1113

+121020

875

817

766

681

646

942

+7

+8

+9+10

+11

+14

+15

+16

+18

+19

+13

+17766

Assegnamento delle cariche

11500 12000 12500 13000

mass

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

12231.0

12326.0

12423.0

Spettro deconvolutoin silico

6

ESI (+) of Ubiquitin 1,040,000 Resolution at m/z 779

0.09u

0.09u

Spettrometria di massa ……..

Quale massa?

Proteine & Peptidi- Determinazione del PM

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


i) Estrazione

ii) Separazione

iii) Determinazione del PMi) MALDI-TOF: spettro, risoluzioneii) ESI: ioni multicarica, risoluzioneiii) Quale massa?iv) Ricerca in banca dati

iv) Determinazione della struttura (Sequenza AA): MSn

i) Proteina in toto: ECD, ETD

ii) Digestione enzimatica Peptidii) MALDI: peptide mass fingerprintingii) HPLC-ESI-MS e MSn

iii) Ricerca in banca dati

8

i) Proteina in toto:

Determinazione della struttura: MSn

CID non riesce a frammentare ioni prodotti da molecole grandi (PM > 3000 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)




ETD = electron transfer dissociation (Linear IT)

ApproccioTop Down

9

ECD, EDT

Proteina[M+nH]n+

Frammenti

Proteina in totoDeterminazione della struttura

MS2

Enzyme

1 Protein(in solution)

Many Peptides(in solution)

Determinazione della struttura

ii) Digestione enzimatica Peptidi

10

Approccio Bottom Up

Trypsin:Lys, Arg S

S

Databasesearch

11

Identification of a Protein via Peptide Mass Fingerprinting

m/z

Inte

nsity

MQRVNMIMAESPSLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTEKTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAEAVFPDVDYVNPTEAETILDNITQGTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRAIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT

SearchEngine

Direct analysis of the entireentire peptide peptide mixturemixture

Protein identification by peptide mapping

Database search (www.expasy.ch)

1 PYQYPALTPEQKKELSDIAHRIVAPGKGILAADESTGSIAKRLQSIGTEN51 TEENRRFYRQLLLTADDRVNPCIGGVILFHETLYQKADDGRPFPQVIKSK

101 GGVVGIKVDKGVVPLAGTNGETTTQGLDGLSERCAQYKKDGADFAKWRCV151 LKIGEHTPSALAIMENANVLARYASICQQNGIVPIVEPEILPDGDHDLKR201 CQYVTEKVLAAVYKALSDHHIYLEGTLLKPNMVTPGHACTQKFSHEEIAM251 ATVTALRRTVPPAVTGITFLSGGQSEEEASINLNAINKCPLLKPWALTFS301 YGRALQASALKAWGGKKENLKAAQEEYVKRALANSLACQGKYTPSGQAGA351 AASESLFVSNHAY

MALDI-TOF-MS

# 16 Da ⇒ ox Met

900 1200 1500 1800 2100 2400 2700 3000 33000

25

50

75

100

Rel

ativ

e In

tens

ity

Mass/Charge

Aldolase A

12

MS of a peptide mixture by MALDI-TOF

Peptide mass fingerprinting

MALDI-TOF

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 proteine diverse, digerite con lo stesso enzima nelle stesse condizioni sperimentali, producono lo

stesso peptide mass fingerprint, le due proteine sono uguali

Se il TOF ha alta risoluzione, è possibile determinare la massa accurata e la formula bruta di

ciascun peptide

14

Proteine: Determinazione della strutturaDigestione enzimatica Peptidi

Databasesearch

HPLC-ESI-MS e MSn

HPLCseparation

ESI- MS

peptide MW

Enzyme

HPLC-MS

Tempo di ritenzionedi ciascun peptide

MS

496

990

1012

15

R=500R=2000R=15.000

986 987 988 989 990 991 992 993 994 995 996 997 998m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

990

989.5561

990.5588

991.5592

992.5601

993.5613

990

991

992

993

MS

496

990

1012

R=500R=2000R=15.000

495.2817

495.7830

496.2832

496.7837497.2843

494.5 495.0 495.5 496.0 496.5 497.0 497.5 498.0m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

495.3

495.8

496.3

496.8497.3

MS

496

990

1012

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Se si dispone di un analizzatore con alta risoluzione e massa accurata

settori, ToF, Orbitrap, FT-ICR

è possibile misurare la massa accurata di ciascun peptide e quindi la formula bruta

Poiché le tecniche di ionizzazione utilizzate sono softnon si ha frammentazione in sorgente

e quindi non si hanno informazioni strutturali, ovvero non èpossibile determinare la sequenza AA

Proteine: Determinazione della strutturaDigestione enzimatica Peptidi

HPLC-ESI-MS e MSn

HPLCseparation

ESI- MS

peptide MW

CID-MSn

Databasesearch

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x3

a1 b1

y3

c1

z3 x2

a2 b2

y2

c2

z2

a3

x1

b3

y1

c3

z1

Ioni C-terminali:

y, x, z

Ioni N-terminali:

b, a, c

CID-MSn di peptidi

18

Why b ions are very stable species?

but they have a cyclic oxazolonic structure

They don’t have an acylium ion structure R-C≡O+

Spectroscopic Evidence for an Oxazolone Structure of the b2 Fragment Ion from Protonated Tri-Alanine

Oomens J et al, J. Am. Soc. Mass. Spectrom. 20, 334 (2009)

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

C:\CIADS\...\peptide-ms7\peptide99

peptide99 #21-22 RT: 0.31-4.05 AV: 2 NL: 2.41E6F: + p Full ms [ 80.00-800.00]

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

556.3

578.4

582.2

peptide99 #27-45 RT: 18.17-18.38 AV: 19 NL: 6.73E5F: + p Full ms2 [email protected] [ 150.00-650.00]

200 250 300 350 400 450 500 550m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

425.1

538.1397.2

556.1426.1 510.1336.2279.2 493.1205.1

m/z 556MS2

b4

a4

Attribuire la giusta sequenza al peptide

Gly Phe Tyr Gly Leu/IleResidui AA:Gly 57 Phe 147 Tyr 163 Leu/Ile 113

peptide99 #46-79 RT: 43.66-44.05 AV: 24 NL: 1.54E5F: + p Full ms3 [email protected] [email protected] [ 115.00-650.00]

200 250 300 350 400 450 500 550m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

397.0

424.9

380.1262.0

m/z 556MS3

m/z 425

m/z 556

MS4

m/z 425

m/z 397

C:\CIADS\...\peptide-ms7\peptide99 21/02/2007 12.23.21

peptide99 #69-79 RT: 48.00-48.20 AV: 10 NL: 6.20E3F: + p Full ms4 [email protected] [email protected] [email protected] [ 105.00-650.00]

150 200 250 300 350 400m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

380.0323.0

278.0

397.1

261.9234.1

352.0283.2238.1

a4

b3

Residui AA:Gly 57 Phe 147 Tyr 163 Leu/Ile 113

119a4

b4

21

m/z 556

MS5

m/z 425

m/z 397

m/z 278

peptide99 #80-106 RT: 57.64-57.75 AV: 6 NL: 1.11E4F: + p Full ms5 [email protected] [email protected] [email protected] [email protected] [ 75.00-650.00]

100 150 200 250 300m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

221.0

278.0

233.0

249.8 261.1

MS6

m/z 397

m/z 278

m/z 221

peptide99 #85-106 RT: 62.54-62.96 AV: 21 NL: 6.61E3F: + p Full ms6 [email protected] [email protected] [email protected] [email protected] [email protected] [ 60.00-650.00]

120 140 160 180 200 2200

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

193.0

220.9

136.0

203.8147.8

b2

a2

a1

Residui AA:Gly 57 Phe 147 Tyr 163 Leu/Ile 113

b3

b2

m/z 193

MS7m/z 397

m/z 278

m/z 221

peptide99 #106-144 RT: 72.78-73.65 AV: 38 NL: 7.87E2F: + p Full ms7 [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [ 50.00-6 ...

80 100 120 140 160 180 200m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

148.1

193.1

136.1164.8

b1

a2

22

H2NNH

NHNH

NHOH

O

O

O

O

O

HO

b1 b2 b3

b4

a4a2

Tyr-Gly-Gly-Phe-Leu

a1

C:\CIADS\...\peptide-ms7\peptide99

peptide99 #21-22 RT: 0.31-4.05 AV: 2 NL: 2.41E6F: + p Full ms [ 80.00-800.00]

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

556.3

578.4

582.2

peptide99 #27-45 RT: 18.17-18.38 AV: 19 NL: 6.73E5F: + p Full ms2 [email protected] [ 150.00-650.00]

200 250 300 350 400 450 500 550m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

425.1

538.1397.2

556.1426.1 510.1336.2279.2 493.1205.1

m/z 556MS2

b4

a4

Attribuire la sequenza al peptide

y3

23

CyclicCyclic peptidespeptides:

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

Highly selective metal ion chelators: carriers of metal ions inside the cells. Use in radiotherapy

Better bioavailability and higher resistance to proteolyticdegradation than their linear analogues

Brodbelt et al. J Am Soc Mass Spectrom 2004, 15, 1039–1054

24

Characterization and sequencing of cyclic peptides representan important challenge for mass spectrometry.

The elimination of an aminoacidic residue must involve the fission of two bonds of the cycle.

No definite terminations, as it occurs in their linear analogues.

The ring-opening may occur at several backbone positions,meaning that all the fragment ions are not referenced toa single “terminal” position.

peptideDK13_elisa_070919143425#161 RT:1.05 AV:1 NL:1.06E9F:FTMS + p ESI Full ms [125.00-1200.00]

150 200 250 300 350 400 450 500 550m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

237.6326R=43501

474.2574R=30701

158.7572R=53001

[M + H]+

[M + 2H]2+

[M + 3H]3+

25

Resolving powerS:OrbitrapOrbitrap:

at constant detection time:

resolving power inversely

proportional to m/z√

Thus if

R=100,000 at m/z 100,

at m/z 1000 it will be R=100,000(100/1000)1/2=31,646

RDB=double bond/ring equivalents

For ions with odd charge:Odd-electron ions = RDB integerEven-electron ions= RDB x.5

12C 0÷3013C 0÷114N 0÷1016O 0÷15

1H 0÷60

26

peptideDK13_elisa_070919143425#172 231RT:1.13 1.60AV:60 NL:1.09E9F:FTMS + p ESI Full ms [125.00-1200.00]

235 236 237 238 239 240 241m/z

05

1015

2025

3035

4045

50

5560

6570

7580

8590

95

100

Rel

ativ

e A

bund

ance

237.6325

238.1335

238.6358239.1395

[M + 2H]2+

27

peptideDK13_elisa_070919143425#172 231RT:1.13 1.60AV:60 NL:1.09E9F:FTMS + p ESI Full ms [125.00-1200.00]

235 236 237 238 239 240 241m/z

05

1015

2025

3035

4045

50

5560

6570

7580

8590

95

100

Rel

ativ

e A

bund

ance

237.6325

238.1335

238.6358239.1395

peptideDK13_elisa_070919143425#161 RT:1.05 AV:1 NL:8.24E7F:FTMS + p ESI Full ms [125.00-1200.00]

158.6 158.8 159.0 159.2 159.4 159.6 159.8m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

158.7572R=53001

159.0917R=53504

159.4287R=22804

[M + 3H]3+

28

— H2O

[M+H]+

— NH3

[MH—(NH3—CH2O)]+

-Lys(0.07)

-Lys

(0.49)

337.1986 (0.43)

-His

-His

peptideDK13_elisa_070919143425#538 591RT:4.41 7.95AV:54 NL:3.03E7F:FTMS + p ESI Full ms2 [email protected] [130.00-600.00]

150 200 250 300 350 400 450m/z

05

1015

2025

3035

4045

50

5560

6570

7580

8590

95

100

Rel

ativ

e A

bund

ance

346.1625328.1520

474.2571

456.2468

209.1035266.1615

237.0985

302.1615

439.2203180.0769 427.2204365.1935

-Lys

-His

-β-Ala

— H2O

318.1677

— NH3

Residui AA:

Lys 128His 137Ala/β-Ala 71

29

Peptide solo2#1 16 RT:0.02 0.27AV:16 NL:2.10E5F:+ p Full ms3 [email protected] [email protected] [95.00-1000.00]

100 120 140 160 180 200 220 240 260 280 300 320 340 360m/z

05

1015

2025

3035

4045

50

5560

6570

7580

8590

95

100

Rel

ativ

e A

bund

ance

328.2

346.1

237.0209.1318.1

[MH-Lys]+

— H2O

-His

CID-MS3 [MH-Lys]+

Residui AA:

Lys 128His 137Ala/β-Ala 71

c-(Lys-D/L-His-β-Ala-His)

NHN

NH

N

O

O

O

O

NH2

NH

HNNH

NH

30

NHN

NH

N

O

O

O

O

NH2

NH

HNNH

NH

H+

HN NH2

O

H2N

HN

NHN

O

NNH

N

O

O

m/z 346

+H+

H2N

O

NNH

N

O

m/z 209

+ H+

HN

NH

N

O

b3+ K/H

-

b3+ K/H m/z 346

31

NHN

NH

N

O

O

O

O

NH2

NH

HNNH

NH

HN

NH

N

O

NH2

HN

NHN

ON

O

O

H2N

+H+

+H+

y3+K/H m/z 337

H2N

O

NNH

N

O

+ H+

NHH2N

O

-

m/z 209

-

80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380m/z

0

20

40

60

80

100 129.1328.1

229.1

346.1

220.3209.1

237.7 337.1110.184.1 318.1

266.2180.1173.7 193.2 248.1 365.1159.7

—LysH+

[M+2H]2+

[M—H2O+2H]2+LysH+

—LysH+

[M—NH3—H2O+2H]2+

—HisβAlaHHisβAlaH+

—HisH+

CID-MS2 [M+2H]2+

32

150 200 250 300 350 400 450 500m/z

0

10

20

30

40

50

60

70

80

90

100

Intensità

Relativa (%

)246.3

335.3

281.3

472.3

454.3

383.3207.2

264.3401.3

150 200 250 300 350 400 450 500m/z

0

10

20

30

40

50

60

70

150 200 250 300 350 400 450 500m/z

0

10

20

30

40

50

60

70

80

90

100

Intensità

Relativa (%

)246.3

335.3

281.3

472.3

454.3

383.3207.2

264.3401.3

-His

- His

- His - β-Ala

- β-Ala

- β-Ala

— H2O

- Lys

CID-MS2 [M-H]–

-His

If you are working with a tryptic peptide, look for the arginine or lysine y1 ionat the low end of the spectrum:

[M+H] + -156 - 18 = penultimate b ion Arg is the carboxy AA

[M+H] + -128 -18 = penultimate b ion 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] + - y1+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 m/z 1379 (ion trap)

34

Digerito triptico

[M+H]+ = 1182 (10 AA)

MS/MS m/z 1182 (ion trap)

35

ETD, ECD

High Collision CID

Ions c, z

37

x3

a1 b1

y3

c1

z3 x2

a2 b2

y2

c2

z2

a3

x1

b3

y1

c3

z1

74

Loss of One Phosphorylation

Loss of Two Phosphorylations

38

75

Single Scan Ion Trap CID with ICR Cell DetectionSingle Scan Ion Trap CID with ICR Cell Detection

B2+

B3+

B102+

B6+B8

2+

B7+

B8+

Y9+

B9+

B5+

200 400 600 800 1000 1200 1400m/z

0

10

20

30

40

50

60

70

80

90

100

B10+

607.3675

600.3380

382.2561 1094.5815

254.1611

1077.5550

882.4948674.37101029.5631

735.42631199.6681

515.2853

1086.5840Rel

ativ

e Ab

unda

nce

R P K P Q Q F F G L MIon Theoretical Measured ppmB2

+ 254.1612 254.1611 0.19B3

+ 382.2561 382.2561 -0.04B8

2+ 515.2851 515.2853 0.64 B10

2+ 600.3378 600.3380 -0.06B5

+ 607.3675 607.3675 0.00[M+2H]2+ 674.3713 674.3710 -0.44 B6

+ 735.4260 735.4263 -0.31B7

+ 882.4944 882.4948 -0.00B8

+ 1029.5629 1029.5631 0.52Y*9

+ 1077.5550 1077.5550 -0.18B9

+ 1086.5843 1086.5840 -0.28Y9

+ 1094.5815 1094.5815 0.32B10

+ 1199.6684 1199.6681 1.17

Fragments identified with a average error of 0.03 ppm

[M+2H]2+

76

Single Scan IRMPD with ICR Cell DetectionSingle Scan IRMPD with ICR Cell Detection

200 400 600 800 1000 1200 1400m/z

0

2

4

6

8

1012

14

16

18

20

22

24

26

2830

Rel

ativ

e Ab

unda

nce

674.3708

254.1612

400.7012

1094.5833

600.3383

1194.9060946.5157

515.2853

382.2563

882.4956

735.4267

1029.5646

B2+

B3+

B102+

B6+

B82+ B7

+

B8+

Y9+

R P K P Q Q F F G L MIon Theoretical Measured ppmB2

+ 254.1612 254.1612 0.04B3

+ 382.2561 382.2563 0.38B8

2 515.2851 515.2853 0.51 B10

2+ 600.3378 600.3383 0.69B5

+ 607.3675 607.3679 0.68[M-NH3]2+ 665.8581 665.8581 -0.05M2+ 674.3713 674.3708 -0.87 B6

+ 735.4260 735.4267 0.85B7

+ 882.4944 882.4956 -1.30B8

+ 1029.5629 1029.5646 1.73Y*9

+ 1077.5550 1077.5547 -0.35Y9

+ 1094.5815 1094.5833 0.95


[M+2H]2+

39

77

Single Scan ECD with ICR Cell DetectionSingle Scan ECD with ICR Cell Detection

R P K P Q Q F F G L M


200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400m/z

0

2

4

6

8

10

12

14

16

18

20

22

24

26

28

30

Rel

ativ

e Ab

unda

nce

674.3715

624.3944

899.5217

752.4530496.3355 1216.6959

1046.5898

1348.74181331.7167

C4+ C6

+

C7+

C8+

C5+

C10+

[M+2H]+

[M+2H]2+

1078.5626Z9+

1103.6123C9

+

[M-NH3]+

Ion Theoretical Measured ppmC4

+ 496.3354 496.3355 0.20C5

+ 624.3940 624.3944 0.64[M+2H]2+ 674.3713 674.3715 0.29C6

+ 752.4526 752.4530 0.53C7

+ 899.5210 899.5217 0.78C8

+ 1046.5894 1046.5898 0.38Z9

+ 1078.5628 1078.5634 0.55C9

+ 1103.6109 1103.6112 0.27C10

+ 1216.6949 1216.6959 0.82[M-NH3]2+ 1331.7158 1331.7167 0.67[M+2H]+ 1348.7432 1348.7418 -1.00

Top downvs

Bottom up

approaches

for studying large molecules

40




ETD = electron transfer dissociation (Linear IT)

ApproccioTop Down

41

x3

a1 b1

y3

c1

z3 x2

a2 b2

y2

c2

z2

a3

x1

b3

y1

c3

z1

42

Top down

43

ETD

Electron Transfer Dissociation

Loss of One Phosphorylation

Loss of Two Phosphorylations

Courtesy by G. Vago Thermo

La Spettrometria di Massa nello Studio di Proteine e …...2014/01/16 · 2 La Spettrometria di...

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