sample preparation and sequencinglzidek/c9530/priprava_vzorku_a... · 2018-11-22 · + 2m nacl...
TRANSCRIPT
SAMPLE PREPARATION AND SEQUENCING
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
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
AGCG
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
CGCT
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
UUU Phe UCU Ser UAU Tyr UGU CysUUC Phe UCC Ser UAC Tyr UGC CysUUA Leu UCA Ser UAA UGAUUG Leu UCG Ser UAG UGG Trp
CUU Leu CCU Pro CAU His CGU ArgCUC Leu CCC Pro CAC His CGC ArgCUA Leu CCA Pro CAA Gln CGA ArgCUG Leu CCG Pro CAG Gln CGG Arg
AUU Ile ACU Thr AAU Asn AGU SerAUC Ile ACC Thr AAC Asn AGC SerAUA Ile ACA Thr AAA Lys AGA ArgAUG Met ACG Thr AAG Lys AGG Arg
GUU Val GCU Ala GAU Asp GGU GlyGUC Val GCC Ala GAC Asp GGC GlyGUA Val GCA Ala GAA Glu GGA GlyGUG Val GCG Ala GAG Glu GGG Gly
R1
H
N
H O
CC
C SN
R1
2
HO
H
N
N
S
NH
C=O
NH
CH(R1)
CH(R2)
a
x
b
y
z
c
−−NH=CH(R1)
OC=NH−CO−NH−CH(R2)−−
−−NH−CH(R1)−C=O
H N−CH(R2)−−
−−NH−CH(R1)−CO−NH
CH(R2)−−
+
+
+
+
+
+3
3
a1x3b1y3c1z3a2x2b2y2c2z2a3x1b3y1c3z1
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
100 150 200 250 300 M/z
y2
2b
z1
b1
a1
1c
50
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)
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
Thr − H O (101.05)2
Cys + H (122.03)
0
20
40
60
80
100
200 400 600 800 1000
Rel
ativ
e in
tens
ity
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
.63a2 b2 b3
y3
y2
b4
y4b6
y5
a7b7
y6
b8
y7
[M+H]+
a
UG UA GA UU U AA UC
UC G
A U UCA C CUC
CA
UA
GG
MCYEFHLKDAELRHVAKGF
LRHVAKGFMCYEFHLKDAE
LRHVAKGF
MCYE
FHLKDAEMCYEFHLK
DAELR
HVAKGF
GFDAELRHVAK
V8 (GluC) trypsin
MCYEFHLK
MCYEFHLKb
AUG
UUU Phe UCU Ser UAU Tyr UGU CysUUC Phe UCC Ser UAC Tyr UGC CysUUA Leu UCA Ser UAA UGAUUG Leu UCG Ser UAG UGG Trp
CUU Leu CCU Pro CAU His CGU ArgCUC Leu CCC Pro CAC His CGC ArgCUA Leu CCA Pro CAA Gln CGA ArgCUG Leu CCG Pro CAG Gln CGG Arg
AUU Ile ACU Thr AAU Asn AGU SerAUC Ile ACC Thr AAC Asn AGC SerAUA Ile ACA Thr AAA Lys AGA ArgAUG Met ACG Thr AAG Lys AGG Arg
GUU Val GCU Ala GAU Asp GGU GlyGUC Val GCC Ala GAC Asp GGC GlyGUA Val GCA Ala GAA Glu GGA GlyGUG Val GCG Ala GAG Glu GGG Gly