PˇR´IPRAVA VZORKU A SEKVENACE
mRNA
protein
A
CB
cDNA
B
B
B
B
B
B
C
72 C
65 C
95 CO
O
O
D
1
1 2
3
4
5
6
7
UUU Phe UCU Ser UAU Tyr UGU Cys
UUC Phe UCC Ser UAC Tyr UGC Cys
UUA Leu UCA Ser UAA UGA
UUG Leu UCG Ser UAG UGG Trp
CUU Leu CCU Pro CAU His CGU Arg
CUC Leu CCC Pro CAC His CGC Arg
CUA Leu CCA Pro CAA Gln CGA Arg
CUG Leu CCG Pro CAG Gln CGG Arg
AUU Ile ACU Thr AAU Asn AGU Ser
AUC Ile ACC Thr AAC Asn AGC Ser
AUA Ile ACA Thr AAA Lys AGA Arg
AUG Met ACG Thr AAG Lys AGG Arg
GUU Val GCU Ala GAU Asp GGU Gly
GUC Val GCC Ala GAC Asp GGC Gly
GUA Val GCA Ala GAA Glu GGA Gly
GUG Val GCG Ala GAG Glu GGG Gly
R
1
H
N
H O
CC
C SN
R
1
2
HO
H
N
N
S
OC=NH−CH(R2)−−
1
x3
b1
y3
c1
z3
a2
x2
b2
y2
c2
z2
a3
x1
b3
y1
c3
z1
NH
CH−CH
C=O
NH
CH−CH(OH)CH
C=O
NH
CH
C=O
NH
COOH
CH−CH SH
3
3
3
2
2
y1
z2 z3
y3
b3
a2 a3
c2 c3
x1 x2 x3
b1
a1
1c
50
NH
C=O
NH
CH(R1)
CH(R2)
a
x
b
y
z
c
−−NH=CH(R1)
−−NH−CH(R1)−C=O
H N−CH(R2)−−
−−NH−CH(R1)−CO−NH
CH(R2)−−
+
+
+
+
+
+
3
3
a
100 150 200 250 300 M/z
y2
2b
z1
Gly − H O (57.02)
Gly − H O (57.02)
Thr − H O (101.05)
Thr − H O (101.05)
2
2
2
2
2
2
2
2
2
2Ala − H O (71.04)
351.14
Cys + H (122.03)
Thr − H O (101.05)2
Gly − H O (57.02) Thr − H O (101.05)2 2
Thr − H O (101.05)
Thr − H O (101.05)
Gly − H O (57.02)
Gly − H O (57.02)
Gly − H O (57.02)
0
20
40
60
80
100
200 400 600 800 1000
Relativeintensity
Mass / charge
157.12
185.12
313.19
455.22
342.13
426.28
512.23
570.34
599.27
655.42
683.42
712.36
820.48
840.46
1024.63
a2 b2 b3
y3
y2
b4
y4
b6
y5
a7
b7
y6
b8
y7
[M+H]+
a
UG UA GA UU U AAU
C
U
C G
A U UCAC C
U
C
C
A
U
A
G
G
MCYEFHLKDAELRHVAKGF
LRHVAKGF
MCYE
FHLKDAE
LRHVAKGF
MCYE
FHLKDAE
MCYEFHLK
DAELR
HVAK
GF
GF
DAELR
HVAK
V8 (GluC) trypsin
MCYEFHLK
MCYEFHLK
b AUG
UUU Phe UCU Ser UAU Tyr UGU Cys
UUC Phe UCC Ser UAC Tyr UGC Cys
UUA Leu UCA Ser UAA UGA
UUG Leu UCG Ser UAG UGG Trp
CUU Leu CCU Pro CAU His CGU Arg
CUC Leu CCC Pro CAC His CGC Arg
CUA Leu CCA Pro CAA Gln CGA Arg
CUG Leu CCG Pro CAG Gln CGG Arg
AUU Ile ACU Thr AAU Asn AGU Ser
AUC Ile ACC Thr AAC Asn AGC Ser
AUA Ile ACA Thr AAA Lys AGA Arg
AUG Met ACG Thr AAG Lys AGG Arg
GUU Val GCU Ala GAU Asp GGU Gly
GUC Val GCC Ala GAC Asp GGC Gly
GUA Val GCA Ala GAA Glu GGA Gly
GUG Val GCG Ala GAG Glu GGG Gly
T
o
90 C, pH>7o
P−32
P−32
P−32
P−32
P−32
P−32
P−32
P−32
P−32
P−32
P−32
P−32
P−32
P−32
P−32
P−32
P−32
P−32
P−32
piperidin
+ 2M NaCl
hydrazinhydrazin
piperidin
pH < 7
CAGGTACGCGAT
pH > 7
CAGGTACGC
CAGGTAC
CAGGT
C
CAGGTACGC
CAGGTAC
C
CAGGTACGCGA
CAGGTACGCG
CAGGTACG
CAGGTA
CAGG
CAG
CA
CAGGTACGCG
CAGGTACG
CAGG
CAG
dimethylsulfat dimethylsulfat
G
A
G
C
G
C
A
T
90 C, pH>7
A
dideoxy
PCR PCR
dideoxy dideoxy
PCR PCR
CAGGTACGCGAT
dideoxy A T
GTCC
GTCCATGCGCT
G
GTCCA
GTCCATGCGC
GT
GTC
GTCCATG
GTCCATGCG
GTCCAT
GTCCATGC
A
A T
T
T
C
C
C
C G
G
G
C G
3’ 5’
C
A
T
G
C
G
C
T
GT
GTC
GTCCATG
GTCCATGCG
GTCCAT
GTCCATGC
T
C
C G
C
3’ 5’
GTCCATGCGCT
G
PCR
GTCCATGCGCT
T
C
G
G
C
A
A
dideoxy dideoxy dideoxy
CAGGTACGCGAT
dideoxyA T
GTCC G
GTCCA