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 53100 Siena

e-mail: gianluca.giorgi@unisi.it Tel. 0577-234241

La Spettrometria di Massa nello Studio di Proteine e

Peptidi

La Spettrometria di Massa nello studio di Proteine e Peptidi

1. Studio

1. Studio didiProteineProteine i) Estrazione ii) Separazione

iii) Determinazione del PM

i) MALDI-TOF: spettro, risoluzione ii) ESI: ioni multicarica, risoluzione iii) Quale massa?

iv) Ricerca in banca dati

iv) Determinazione della struttura (Sequenza AA): MSⁿ i) Proteina in toto: ECD, ETD

ii) Digestione enzimatica ÆPeptidi i) MALDI: peptide mass fingerprinting ii) HPLC-ESI-MS e MSⁿ

iii) Ricerca in banca dati

MALDI ESI iii) Determinazione del peso molecolare

1. Studio

1. Studio didiProteineProteine i) Estrazione ii) Separazione

MALDI-TOF linear mass spectrum of intact protein: Hg-Papain oligomerization Proteine - Determinazione del PM

MALDI-TOF: spettro, risoluzione

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 PM MALDI-TOF: spettro, risoluzione

R=100.000

citocromoc#1-11RT:0.01-0.23 AV:11SM:9GNL:7.40E5 F:+ 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

Relative Abundance

942.2 1020.4

1112.9 1529.5

1359.8 1224.0 875.1

816.9

1747.8 765.9

721.1 683.3

citocromoc#12-18 RT:10.48-10.78 AV:7SM:9GNL: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

Relative Abundance

1530.3 1530.0

1530.7 1529.8 1530.9

1531.0 1531.2 1529.6

1534.7

1531.7 1535.1

1529.5 1534.3 1535.4 1539.5 1539.8

1529.4

Proteine - Determinazione del PM ESI: ioni multicarica, risoluzione

600 800 1000 1200 1400 1600 1800 2000 m/z

0 10 20 30 40 50 60 70 80 90 100

Relative Abundance

1748 1529

1224 1360 1113 +12

1020

875

817

766 681

646

942

+7 +8

+10 +9 +11

+14

+15

+16 +18

+19

+13

+17 766

Assegnamento delle cariche

11500 12000 12500 13000

mass

0 10 20 30 40 50 60 70 80 90 100

Relative Abundance

12231.0

12326.0

12423.0

Spettro deconvoluto

in silico

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

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

1. Studio didiProteineProteine i) Estrazione ii) Separazione

iii) Determinazione del PM

i) MALDI-TOF: spettro, risoluzione ii) ESI: ioni multicarica, risoluzione iii) Quale massa?

iv) Ricerca in banca dati

iv) Determinazione della struttura

ii) Digestione enzimaticaÆPeptidi i) MALDI: peptide mass fingerprinting ii) HPLC-ESI-MS e MSⁿ

iii) Ricerca in banca dati

i) Proteina in toto:

Determinazione della struttura: MS

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)

i) Proteina in toto:

Determinazione della struttura: MS

ECD = electron capture dissociation (FT-ICR) ETD = electron transfer dissociation (Linear IT)

Approccio Top Down

ECD, EDT

Proteina

[M+nH]

Frammenti

Proteina in toto

Determinazione della struttura

MS

Enzyme

1 Protein

(in solution)

Many Peptide s

(in solution) Determinazione della struttura

ii) Digestione enzimatica Æ Peptidi

Approccio Bottom Up

Trypsin:

Lys, Arg ^S

S

Database

search

Identification of a Protein via Peptide Mass Fingerprinting

m/z

Intensity

MQRVNMIMAESPSLITICLLGYLLSAECT VFLDHENANKILNRPKRYNSGKLEEFVQ GNLERECMEEKCSFEEAREVFENTEK TTEFWKQYVDGDQCESNPCLNGGSCK DDINSYECWCPFGFEGKNCELDVTCNI KNGRCEQFCKNSADNKVVCSCTEGYR LAENQKSCEPAVPFPCGRVSVSQTSK LTRAEAVFPDVDYVNPTEAETILDNITQ GTQSFNDFTRVVGGEDAKPGQFPWQ VVLNGKVDAFCGGSIVNEKWIVTAAHC VETGVKITVVAGEHNIEETEHTEQKRN VIRAIIPHHNYNAAINKYNHDIALLELDE PLVLNSYVTPICIADKEYTNIFLKFGSG YVSGWGRVFHKGRSALVLQYLRVPLV DRATCLRSTKFTIYNNMFCAGFHEGG RDSCQGDSGGPHVTEVEGTSFLTGII SWGEECAMKGKYGIYTKVSRYVNWIK EKTKLT

Search Engine

Direct analysis of the

entire peptide mixture entire peptide mixture

Protein identification by peptide mapping

Database search (www.expasy.ch)

1 PYQYPALTPEQKKELSDIAHRIVAPGKGILAADESTGSIAKRLQSIGTEN 51TEENRRFYRQLLLTADDRVNPCIGGVILFHETLYQKADDGRPFPQVIKSK 101 GGVVGIKVDKGVVPLAGTNGETTTQGLDGLSERCAQYKKDGADFAKWRCV 151 LKIGEHTPSALAIMENANVLARYASICQQNGIVPIVEPEILPDGDHDLKR 201 CQYVTEKVLAAVYKALSDHHIYLEGTLLKPNMVTPGHACTQKFSHEEIAM 251 ATVTALRRTVPPAVTGITFLSGGQSEEEASINLNAINKCPLLKPWALTFS 301 YGRALQASALKAWGGKKENLKAAQEEYVKRALANSLACQGKYTPSGQAGA 351 AASESLFVSNHAY

MALDI-TOF-MS

# 16 Da ⇒ ox Met

900 1200 1500 1800 2100 2400 2700 3000 3300 0

25 50 75 100

Relative Intensity

Mass/Charge

Aldolase A

MS of a peptide mixture by MALDI-TOF

Peptide mass fingerprinting

MALDI-TOF

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

Proteine: Determinazione della struttura Digestione enzimatica ÆPeptidi

Database search

HPLC-ESI-MS e MS

HPLC separation ESI-MS

peptide MW

Enzyme

HPLC-MS

Tempo di ritenzione di ciascun peptide

MS

496

990

1012

R=500 R=2000 R=15.000

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

0 10 20 30 40 50 60 70 80 90 100

Relative Abundance

990 989.5561

990.5588

991.5592

992.5601 993.5613 990

991

992 993

MS

496

990

1012

R=500 R=2000 R=15.000

495.2817

495.7830

496.2832

496.7837

497.2843

494.5 495.0 495.5 496.0 496.5 497.0 497.5 498.0 m/z

0 10 20 30 40 50 60 70 80 90 100

Relative Abundance

495.3

495.8

496.3

496.8

497.3

MS

496

990

1012

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 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 ÆPeptidi

HPLC-ESI-MS e MS

HPLC separation ESI-MS

peptide MW

CID-MS

Database search

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-MS

di peptidi

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 b

Fragment Ion from Protonated Tri-Alanine

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

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.

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

peptide99#21-22RT:0.31-4.05AV:2NL:2.41E6 F:+ p Full ms [ 80.00-800.00]

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

m/z 0

10 20 30 40 50 60 70 80 90 100

Relative Abundance

556.3

578.4 582.2

peptide99#27-45RT:18.17-18.38AV:19NL:6.73E5 F:+ p Full ms2 556.00@24.00 [ 150.00-650.00]

200 250 300 350 400 450 500 550

m/z 0

10 20 30 40 50 60 70 80 90 100

Relative Abundance

425.1

538.1

397.2 556.1

426.1 510.1

336.2

279.2 493.1

205.1 m/z 556

·

MS²

b₄

a₄ Attribuire la giusta sequenza al peptide

Gly Phe Tyr Gly Leu/Ile

Residui AA:

Gly 57 Phe 147 Tyr 163 Leu/Ile 113

556-425=131

peptide99#46-79RT:43.66-44.05AV:24NL:1.54E5 F:+ p Full ms3 556.00@24.00 425.00@19.00 [ 115.00-650.00]

200 250 300 350 400 450 500 550

m/z 0

10 20 30 40 50 60 70 80 90 100

Relative Abundance

397.0

424.9

380.1 262.0

m/z 556

·

MS³

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:6.20E3

F:+ p Full ms4 556.00@24.00 425.00@19.00 397.00@25.00 [ 105.00-650.00]

150 200 250 300 350 400

m/z 0

10 20 30 40 50 60 70 80 90 100

Relative Abundance

380.0 323.0

278.0

397.1 261.9

234.1

352.0 283.2

238.1

a₄

b₃

Residui AA:

Gly 57 Phe 147 Tyr 163 Leu/Ile 113

119 a₄

b₄

m/z 556

·

MS⁵

m/z 425

·

m/z 397

· m/z 278

· peptide99#80-106 RT:57.64-57.75 AV:6NL:1.11E4

F:+ p Full ms5 556.00@24.00 425.00@19.00 397.00@25.00 278.00@22.00 [ 75.00-650.00]

100 150 200 250 300

m/z 0

10 20 30 40 50 60 70 80 90 100

Relative Abundance

221.0

278.0 233.0

249.8 261.1

MS⁶ ·

m/z 397

· m/z 278

·

m/z 221

· peptide99#85-106RT:62.54-62.96AV:21NL:6.61E3

F:+ p Full ms6 556.00@24.00 425.00@19.00 397.00@25.00 278.00@22.00 221.00@21.00 [ 60.00-650.00]

120 140 160 180 200 220

0 10 20 30 40 50 60 70 80 90 100

Relative Abundance

193.0

220.9

136.0

203.8 147.8

b₂

a₂

a₁

Residui AA:

Gly 57 Phe 147 Tyr 163 Leu/Ile 113

b₃

b₂

278-221=57

m/z 193

·

MS^{7 ·}

m/z 397

· m/z 278

·

m/z 221

·

peptide99#106-144RT:72.78-73.65 AV:38NL:7.87E2

F:+ p Full ms7 556.00@24.00 425.00@19.00 397.00@25.00 278.00@22.00 221.00@21.00 193.00@22.00 [ 50.00-6 ...

80 100 120 140 160 180 200

m/z 0

10 20 30 40 50 60 70 80 90 100

Relative Abundance

148.1

193.1

136.1

164.8

b₁

a₂

H₂N NH

NH NH

NH OH

O

O

O

O

O HO

b

b

b

b

a

a

Tyr-Gly-Gly-Phe-Leu

a

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

peptide99#21-22RT:0.31-4.05AV:2NL:2.41E6 F:+ p Full ms [ 80.00-800.00]

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

m/z 0

10 20 30 40 50 60 70 80 90 100

Relative Abundance

556.3

578.4 582.2

peptide99#27-45RT:18.17-18.38AV:19NL:6.73E5 F:+ p Full ms2 556.00@24.00 [ 150.00-650.00]

200 250 300 350 400 450 500 550

m/z 0

10 20 30 40 50 60 70 80 90 100

Relative Abundance

425.1

538.1 397.2

556.1

426.1 510.1

336.2

279.2 493.1

205.1 m/z 556

·

MS²

b₄

a₄ Attribuire la sequenza al peptide

y₃

Cyclic peptides 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

Highly selective metal ion chelators:

carriers of metal ions inside the cells. Use in radiotherapy Better bioavailability and higher resistance to proteolytic degradation than their linear analogues

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

Characterization and sequencing of cyclic peptides represent an 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 to a single “terminal” position.

peptideDK13_elisa_070919143425#161RT:1.05AV:1NL:1.06E9 F:FTMS + p ESI Full ms [125.00-1200.00]

150 200 250 300 350 400 450 500 550

m/z 0

10 20 30 40 50 60 70 80 90 100

Relative Abundance

237.6326 R=43501

474.2574 R=30701

158.7572 R=53001

[M + H]⁺ [M + 2H]²⁺

[M + 3H]³⁺

Resolving power S:

Orbitrap Orbitrap:

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)

=31,646

RDB=double bond/ring equivalents

For ions with odd charge:

Odd-electron ions = RDB integer Even-electron ions= RDB x.5

12C 0÷30

13C 0÷1

14N 0÷10

16O 0÷15

1H 0÷60

peptideDK13_elisa_070919143425#172 231RT:1.13 1.60AV:60NL:1.09E9 F:FTMS + p ESI Full ms [125.00-1200.00]

235 236 237 238 239 240 241

m/z 0

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100

Relative Abundance

237.6325

238.1335

238.6358 239.1395

[M + 2H] ²⁺

peptideDK13_elisa_070919143425#172 231RT:1.13 1.60AV:60NL:1.09E9 F:FTMS + p ESI Full ms [125.00-1200.00]

235 236 237 238 239 240 241

m/z 0

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100

Relative Abundance

237.6325

238.1335

238.6358 239.1395

peptideDK13_elisa_070919143425#161RT:1.05AV:1NL:8.24E7 F:FTMS + p ESI Full ms [125.00-1200.00]

158.6 158.8 159.0 159.2 159.4 159.6 159.8

m/z 0

10 20 30 40 50 60 70 80 90 100

Relative Abundance

158.7572 R=53001

159.0917 R=53504

159.4287 R=22804

[M + 3H] ³⁺

—H₂O

[M+H]⁺

—NH₃ [MH—(NH₃—CH₂O)]⁺ (0.07)-Lys

-Lys (0.49)

337.1986(0.43)

-His -His peptideDK13_elisa_070919143425#538 591RT:4.41 7.95AV:54NL:3.03E7 F:FTMS + p ESI Full ms2 474.00@cid20.00 [130.00-600.00]

150 200 250 300 350 400 450

m/z 0

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100

Relative Abundance

346.1625 328.1520

474.2571 456.2468

209.1035

266.1615 237.0985

302.1615

439.2203

180.0769 365.1935 427.2204

-Lys -His

-β-Ala

—H₂O

318.1677

—NH₃

Residui AA:

Lys 128 His 137 Ala/β-Ala 71

Peptide solo2#1 16RT:0.02 0.27AV:16NL:2.10E5

F:+ p Full ms3 474.00@cid28.00 346.00@cid22.00 [95.00-1000.00]

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

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100

Relative Abundance

328.2

346.1

237.0 209.1

318.1

[MH-Lys]⁺

—H₂O

-His

CID-MS

[MH-Lys]

Residui AA:

Lys 128 His 137 Ala/β-Ala 71

c-(Lys- D/L -His-β-Ala-His)

NH N

NH N O

O

O O

NH₂

NH HN NH

NH

N NH

NH N O

O

O O

NH₂

NH HN NH

NH H⁺

HN NH₂

O

H₂N

HN N

HN

O N

NH N

O

O

m/z 346

+H⁺

H₂N

O

N NH

N

O

m/z 209

+ H⁺

HN

NH N O

b₃⁺ K/H

-

b₃⁺ K/H m/z 346

N NH

NH N O

O

O O

NH2

NH HN NH

NH

HN

NH N O

NH2

HN

HN N N O

O O H₂N

+H⁺

+H⁺

y₃⁺K/H m/z 337

H₂N

O

N NH

N

O + H⁺

H2N NH

O

-

m/z 209 -

80 100 120 140 160 180 200 220m/z240 260 280 300 320 340 360 380 0

20 40 60 80

100 129.1

328.1 229.1

346.1

220.3 209.1

237.7 337.1

110.1

84.1 318.1

266.2 180.1

173.7 193.2 248.1 365.1

159.7

—LysH⁺

[M+2H]²⁺

[M—H₂O+2H]²⁺

LysH⁺

—LysH⁺

[M—NH₃—H₂O+2H]²⁺

—HisβAlaH HisβAlaH⁺

—HisH⁺

CID-MS

[M+2H]

150 200 250 300 350 400 450 500 m/z

0 10 20 30 40 50 60 70 80 90 100

IntensitàRelativa (%)

246.3

335.3 281.3

472.3 454.3

383.3 207.2

264.3

401.3

150 200 250 300 350 400 450 500

m/z 0

10 20 30 40 50 60 70

150 200 250 300 350 400 450 500

m/z 0

10 20 30 40 50 60 70 80 90 100

IntensitàRelativa (%)

246.3

335.3 281.3

472.3 454.3

383.3 207.2

264.3

401.3

-His - His

- His - β-Ala

- β-Ala - β-Ala

—H₂O

- Lys

CID-MS

[M-H]

-His

If you are working with a

tryptic peptide

[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

[M+H]+ - observed b ion + 1 = corresponding y ion.

Digerito triptico [M+H]⁺ = 1379 (14 AA) MS/MS m/z 1379 (ion trap)

Digerito triptico [M+H]⁺ = 1182 (10 AA) MS/MS m/z 1182 (ion trap)

ETD, ECD

High Collision CID

Ions c, z

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

75

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

B2+ B3+

B102+

B6+ B82+

B7+ B8+

Y9+ B9+ B5+

200 400 600 800 1000 1200 1400

m/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 882.4948

674.3710

1029.5631 735.4263

1199.6681 515.2853

1086.5840

Relative Abundance

R P K P Q Q F F G L M

Ion Theoretical Measured ppm

B₂⁺ 254.1612 254.1611 0.19

B₃+ 382.2561 382.2561 -0.04 B₈²⁺ 515.2851 515.2853 0.64 B₁₀²⁺ 600.3378 600.3380 -0.06 B₅⁺ 607.3675 607.3675 0.00 [M+2H]²⁺ 674.3713 674.3710 -0.44 B₆⁺ 735.4260 735.4263 -0.31 B₇+ 882.4944 882.4948 -0.00 B₈⁺ 1029.5629 1029.5631 0.52 Y*₉⁺ 1077.5550 1077.5550 -0.18

B₉⁺ 1086.5843 1086.5840 -0.28

Y₉⁺ 1094.5815 1094.5815 0.32 B₁₀⁺ 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 Detection Single Scan IRMPD with ICR Cell Detection

200 400 600 800 1000 1200 1400

m/z 0

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Relative Abundance

674.3708

254.1612

400.7012

1094.5833 600.3383

1194.9060 946.5157

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 M

Ion Theoretical Measured ppm

B₂⁺ 254.1612 254.1612 0.04

B₃⁺ 382.2561 382.2563 0.38

B₈² 515.2851 515.2853 0.51

B₁₀²⁺ 600.3378 600.3383 0.69

B₅+ 607.3675 607.3679 0.68 [M-NH3]²⁺ 665.8581 665.8581 -0.05 M2+ 674.3713 674.3708 -0.87 B₆+ 735.4260 735.4267 0.85

B₇⁺ 882.4944 882.4956 -1.30

B₈⁺ 1029.5629 1029.5646 1.73

Y*₉+ 1077.5550 1077.5547 -0.35 Y₉+ 1094.5815 1094.5833 0.95 Fragments identified with a average error of 0.58 ppm [M+2H]²⁺

77

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

R P K P Q Q F F G L M

Fragments identified with a average error of 0.56 ppm

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

m/z 0

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Relative Abundance

674.3715

624.3944

899.5217 752.4530

496.3355 1216.6959

1046.5898

1348.7418 1331.7167

C₄⁺ C₆⁺

C₇⁺

C₈⁺ C₅⁺

C₁₀⁺

[M+2H]⁺ [M+2H]²⁺

1078.5626 Z₉+

1103.6123 C₉⁺

[M-NH_3]+

Ion Theoretical Measured ppm

C4+ 496.3354 496.3355 0.20

C5+ 624.3940 624.3944 0.64

[M+2H]²⁺ 674.3713 674.3715 0.29

C6+ 752.4526 752.4530 0.53

C7+ 899.5210 899.5217 0.78

C8+ 1046.5894 1046.5898 0.38

Z9+ 1078.5628 1078.5634 0.55

C9+ 1103.6109 1103.6112 0.27

C10+ 1216.6949 1216.6959 0.82

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

Top down

vs

Bottom up

approaches

for studying large molecules

i) Proteina in toto:

Determinazione della struttura: MS

ECD = electron capture dissociation (FT-ICR) ETD = electron transfer dissociation (Linear IT)

Approccio Top Down

x₃

a₁ b₁ y₃

c₁ z₃ x₂

a₂ b₂ y₂

c₂ z₂

a₃ x₁

b₃ y₁

c₃ z₁

Top

down

ETD

Electron Transfer Dissociation

Loss of One Phosphorylation Loss of Two Phosphorylations

Courtesy by G. Vago Thermo