Transcript Slide 1

After collision with helium the peptide will break into characteristic fragments
The peptide bond is the weakest point,
so the protein will break into two pieces....
Bruch der Peptdibindung
R1
O
R
NH
NH2
R3
NH2
O
R3
O
R
NH
+
NH2
O
H
N
H2 N
R4
O
R2
B-Ion
Y-Ion
R1
R3
+
O
NH2
R4
O
R2
R1
R
H
N
+
OH
N
R2
B-Oxazalon-Ion
H
N
H2 N
O
R4
From the fragment spectra the peptide sequence can be generated
Example_1: Fragmentation of SLYK
Expected fragments: b-ions
SLY
SL
S
y-ions
K
YK
LYK
Total 7 peaks
I
SLYK
LYK
K
YK
L
Y
m/z
S
B-ions can help to identify the N-terminus of a peptide
Expected fragments: b-ions
SLY
SL
S
y-ions
K
YK
LYK
Total 7 peaks
I
SLYK
SLY
SL
Y
m/z
K
A completet spectra can be complex
Expected fragments: b-ions
SLY
SL
S
I
y-ions
K
YK
LYK
Total 7 peaks
SLY
SL
YK
K
m/z
LYK
SLYK
How to interprete MS-spectra?
Strategy #1
1. Try to find the C-terminal amino acid.
2. Try to find the next peak of the y-ion series in the spectra
3. Read the spectra from the tail to the head
Strategy #2
1. Find the peptide peak or the highest peak MW of one series.
2. Find the next peak in the series.
3. When the sequence is done, try to figure out in which way to read it.
Combined Strategy
1.
2.
3.
4.
Find the b2/a2 pair
Read the b-ion series as far as you can
Read the y-ion series.
Try to match them together
0
200
400
600
800
1000
1200
1400
1750.706
1536.440
1558.627
288.226
4
1576.901
1403.885
175.093
1192.473
1355.439
Example:
1445.772
1291.878
1220.151
1174.465
1267.254
955.985
831.417
1070.580
1016.653
939.622
793.346
744.401
643.397
514.391
401.309
2
600.240
195.116
45.831
Intens. [a.u.]
x10 4
8
6
0
1600
1800
m/z
0
200
400
600
800
1000
1200
1400
1536.440
1558.627
4
1576.901
1403.885
Δ233 Δ128
1445.772
1291.878
6
1220.151
1174.465
Δ101
Δ129 Δ87
1267.254
955.985
831.417
1070.580
1016.653
939.622
793.346
744.401
175.093
1192.473
C-terminal amino acid
1750.706
514.391
Δ113
Δ113
643.397
2
401.309
288.226
Δ113
600.240
195.116
45.831
1355.439
Intens. [a.u.]
x10 4
8
Peptide
Δ163
0
1600
1800
m/z
List of masses of amino acid and modifications
m/z
A=71,
P=97,
V=99,
I/L=113,
N=114,
D=115,
M=131,
H=137,
F=147,
K=129/147,
Q=128,
S=87
Y=163,
E= 129
T=101
R=175
phosphorylation = +80
ICPL= +86/92
0
200
400
600
800
1000
1200
1267.254
Q
K(12C)
1558.627
ICPL-K (13C)
4
1576.901
1403.885
1192.473
8
1400
1536.440
1445.772
1291.878
1220.151
1174.465
1070.580
1016.653
955.985
831.417
S
939.622
T
793.346
E
744.401
6
1750.706
514.391
I/L
643.397
I/L
600.240
2
401.309
I/L
288.226
175.093
R
195.116
45.831
1355.439
Intens. [a.u.]
x10 4
Sequence: YQKSTE(I/L)(I/L)(I/L)R
215
Y
173
0
1600
1800
m/z
A, Overexpression and enhanced activity of the epidermal growth factor receptor (EGFR)
is often observed in human cancers. EGFR is a tyrosine phosphotransferase (kinase) and
abnormal EGFR-kinase activity is related to poor prognosis. Upon binding of the ligand
EGFR undergoes autophosphorylation (self phosphorylation), an important step in EGF
signal transduction. Figure 1 shows the fragment spectra of the peptide (PDYQQDFFPK)
generated from isolated EGFR. Please confirm the predicted sequence and mark the
phosphorylation site.
1364
1267
147
I
244
391
538
58
198 210
378
422
653
1152
781
909
555
m/z
Figure 1: Fragment spectrum. (m/z A=71, P=97, V=99, I,=113, N=114, D=115, M=131,
H=137, F=147, K=129/147, Q=128, Y=163, phosphorylation = +80)
B, A pharmaceutical company wants to produce inhibitors against EGFR-kinase for cancer
therapy. Please explain briefly how mass spectrometry can help to identify an inhibitor compound.