sample preparation and sequencinglzidek/c9530/priprava_vzorku_a... · 2018-11-22 · + 2m nacl...

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