18
P. ROEPSTORFF
a. 2D gel
b. MALDIMa spectnlm
r - -
"Jl
Co
•
' "
{;
"5
1/
1:
)
"
~ ~ / ~
r.
f " J:
.1.
~
.... , ... II .. , ... no. " .. , ...
I
~hss ( Oa)
Index Pent. matched Acc. number
Mw(Da)
Protein name
1 23532
Q
swissIP08699 27070.44
LEG3 RAT GALECTIN-3
2 143771
9
T/J02962
27229.70
RNBPIGE 1 Rat Il!E binding protein
3 144409
9
TIM 13697
15670.16
RNIGEBP 1 Rat IgE binding protein
4 125590
5
TIX60200
80330.02 fECTNI000. 1 E . coli transposon
5 61878
4
Tnew1U33115 95593.73
SC33115 1 gene: "PSP 1 "; product
6 65552
4
Tnew1L77216 90710.31
BBLONNA 1 gene: "Ion"; product
7 67081
4
Tnew/Z49988
75189.10
SPMMSAGEN 2 gene:"rnrnsA"; produ ct
d. Galectin-3 Sequence
MADGFSLNDALAGSGNPNPQGWPGAWGNQPGAGGYPGASYPGAYPGQAPPGGYPGQAPPSAYPGPTGPSA
YPGPTAPGAYPGPTAPGAFPGQPGGPGAYPSAPGAYPSAPGAYPATGPFGAPTGPLTVPYDMPLPGGVMP
RMLITIIGTVKPNANSITLNFKKGNDIAFHFNPRFNENNRRVIVCNTKQDNNWGREERQSAFPFESGKPF
KIQVLVEADHFKVAVNDVHLLQYNHRMKNLREISQLGIIGDITLTSASHAMI
Fig. 2.3. Example of protein identification in proteome studies. (a) NEPHGE gel of proteins from ~cells from rat Langerhans Islets. (b) MALDI spectrum of the peptide mixture derived by in-gel tryptic
digestion of the spot indicated with an arrow in the gel. (c) top ranking candidates after a search in
the EBI non-redundant database using the Peptide Search program. The three top ranking sequences
are the same protein. (d) The sequence of the identified protein rat Galectin-3. The sequence covered
by the peptide map is underlined
ing 12 ng/ml trypsin. After digestion the pep tides were extracted from the gel,
vacuum dried and re-dissolved in 1O-20!-tl 5 % formic acid_ 0.5!-tl of this solution was applied to a mass spectrometric target precoated with a matrix (acyano-4-hydroxycinnamic acid) nitrocellulose mixture (Kussmann et al. 1997)
and dried. The surface was washed with 3 -1 0 !-t1 of 0.1 % TFA, dried and the mixture analyzed on a MALDI-TOF instrument in reflector and delayed extraction
mode. The resulting mass spectrum shown in Fig. 2.3B was first externally calibrated and then re-calibrated based on a few peaks identified as autodigestion
P. ROEPSTORFF
a. 2D gel
b. MALDIMa spectnlm
r - -
"Jl
Co
•
' "
{;
"5
1/
1:
)
"
~ ~ / ~
r.
f " J:
.1.
~
.... , ... II .. , ... no. " .. , ...
I
~hss ( Oa)
Index Pent. matched Acc. number
Mw(Da)
Protein name
1 23532
Q
swissIP08699 27070.44
LEG3 RAT GALECTIN-3
2 143771
9
T/J02962
27229.70
RNBPIGE 1 Rat Il!E binding protein
3 144409
9
TIM 13697
15670.16
RNIGEBP 1 Rat IgE binding protein
4 125590
5
TIX60200
80330.02 fECTNI000. 1 E . coli transposon
5 61878
4
Tnew1U33115 95593.73
SC33115 1 gene: "PSP 1 "; product
6 65552
4
Tnew1L77216 90710.31
BBLONNA 1 gene: "Ion"; product
7 67081
4
Tnew/Z49988
75189.10
SPMMSAGEN 2 gene:"rnrnsA"; produ ct
d. Galectin-3 Sequence
MADGFSLNDALAGSGNPNPQGWPGAWGNQPGAGGYPGASYPGAYPGQAPPGGYPGQAPPSAYPGPTGPSA
YPGPTAPGAYPGPTAPGAFPGQPGGPGAYPSAPGAYPSAPGAYPATGPFGAPTGPLTVPYDMPLPGGVMP
RMLITIIGTVKPNANSITLNFKKGNDIAFHFNPRFNENNRRVIVCNTKQDNNWGREERQSAFPFESGKPF
KIQVLVEADHFKVAVNDVHLLQYNHRMKNLREISQLGIIGDITLTSASHAMI
Fig. 2.3. Example of protein identification in proteome studies. (a) NEPHGE gel of proteins from ~cells from rat Langerhans Islets. (b) MALDI spectrum of the peptide mixture derived by in-gel tryptic
digestion of the spot indicated with an arrow in the gel. (c) top ranking candidates after a search in
the EBI non-redundant database using the Peptide Search program. The three top ranking sequences
are the same protein. (d) The sequence of the identified protein rat Galectin-3. The sequence covered
by the peptide map is underlined
ing 12 ng/ml trypsin. After digestion the pep tides were extracted from the gel,
vacuum dried and re-dissolved in 1O-20!-tl 5 % formic acid_ 0.5!-tl of this solution was applied to a mass spectrometric target precoated with a matrix (acyano-4-hydroxycinnamic acid) nitrocellulose mixture (Kussmann et al. 1997)
and dried. The surface was washed with 3 -1 0 !-t1 of 0.1 % TFA, dried and the mixture analyzed on a MALDI-TOF instrument in reflector and delayed extraction
mode. The resulting mass spectrum shown in Fig. 2.3B was first externally calibrated and then re-calibrated based on a few peaks identified as autodigestion