Pl 0
()o ~
>-l
::l"""
()::r'
0..
S 0
(1)
()~gnlO'
g $
~ ~ ::::
S. ~ 0.. 0..
~
::r> (1)
'"0 ~
.....
dQ 0 (1)
0 ::l
(1)g~t"1CIQ
0..
..... ;:::>
'"0
..... >-l ~
_ .....
'-' '"Ij
;::!
0
::1.
> ~
o-q ()
1"'O..-...,.......(,;;)tiJ
::t>s . . . . -o..
'< oPl
0..;:;
..-...:::::s ~
..... (t)
~
_. r:.n ~
1
0 . . . . . . . . "':;;
V11-1~C.~
en ro ro 0 CI'.l
~o..",::lPl
~~ ~ 0
'"0
•
..... .....,'"0
I-rj
en
O· N
=-=
o '"0 ::l ::l (1)
I-tgN=o..
n -.
\0
~
§&,-,~
....
nll~nl&
go~;;l(1)
o' ;.... Pl
rp :::0
::ll=&~"d
Pl
t-'
~. Pl
~
;; a, ~ ~. ~
~&0..0..(J
.... (1) .....
-<
~ (1) 0
t;;.
~
0"~1::§'"O
~ ~ N
~ ~
(1) ().
N Pl
""""oro.-+........ /\ 0
0"
0..
O·
g,<'"O::l
tv 0 ......... 0
;....'"O~~.....,
S
::r: Pl
5' '"0
S'"O
~",..g
~~
0: ~ c.
~~e::r'~
'-" '-" ~
ro C/)
::ItIW
tD'
< ""'
tD
...
'" tD
9"1::1
::r
DI
'" tD
n
DI
~
iii"
-<
::c
"1::1
I"'"
n
3:
'"
>
::::I
DI
-<
'"
iii'
14000
12000
10000
'"
8000
1:
::I
0
u
6000
4000
2000
0
b
::.
~
~
~
.J~-L.~L_
2200
2400
2600
2800
3000
Mass
(mil)
R
~
iii
3200
3400
3600
~
~
' "
.;:
.,.
.,.
-
~
U
~
1
,
'IN
3800
40
Fig. 3.3. Streptokinase (0.5 ltg, 10
pmol) was separated on a 12 %
SDS-polyacrylamide gel, detected by Reverse-staining
and digested with trypsin in 50 mM ammonium bicarbonate during 4 h. The peptides were extracted by incubating under
vortexing for 10
mins the gel microparticles successively once in water, twice in 50 %
acetonitrile, 0.1 %
TFA and for 1
min
once in 80 %
ACN. These eluates were concentrated together up to about 50 It!. The gel pellet was then re-extracted with
100
Itl offormic acid: isopropanol: acetonitrile: water (20:15:25:40)
and the eluate concentrated to about 50
It!. These eluates (0.5
It!) were analyzed on DHB matrix in a PerSeptive Voyager Elite mass spectrometer equipped with a reflectron and
delayed extraction. Ion acceleration voltage was 20
kV and 200-256
scans were averaged. The peptide masses were matched with the cloned sequence using the program GPMA W (Lighthouse Data, Denmark). A: Analysis of pep tides eluted in
aqueous acetonitrile.
B: Analysis of pep tides eluted in formic acid: isopropanol: acetonitrile: water
""
' "
<
~
;;:
iD
z
' "
ICI
!!:.
:;r
' "
la
QI
;.
;.
ICI
Q
- ~
2'" ;.
Q
:I
m
iD
~
Cl
...
:r
Q
;;:
'"
;;;.
C'"I
' "
;;;
...
"