la spettrometria di massa nello studio di proteine e …...2014/01/16 · 2 la spettrometria di...
TRANSCRIPT
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
1. Studio 1. Studio didi ProteineProteine
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
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
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)
i) Proteina in toto:
Determinazione della struttura: MSn
ECD = electron capture dissociation (FT-ICR)
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
16
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
17
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
36
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
Fragments identified with a average error of 0.58 ppm
[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
Fragments identified with a average error of 0.56 ppm
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
i) Proteina in toto:
Determinazione della struttura: MSn
ECD = electron capture dissociation (FT-ICR)
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