Characterization of Gel Separated Proteins
83
sociation (cm) of proteolytic peptides, sequence stretches of 10-20 residues can
be obtained. Even low ng amounts of in-gel digested proteins have provided
sequence information helpful for cloning of the corresponding gene (cf. Wilm et
aI., 1996).
We have applied the in-gel approach to polypeptides of widely different properties and sizes. The technique was tested in studies regarding protein expression
in transformed versus normal cells (Bergman et aI., 1997; Bergman et aI., 1999a).
The amount of a 49 kDa protein separated by 2-D gel electrophoresis was
found to be three times higher in c-jun transformed rat fibroblasts (Bergman et
aI., 1997). To identify this polypeptide the gel spot was cut after staining with
Coomassie followed by thorough destaining and washing in 30 % methanol by
end-over-end mixing for two days. The latter step is important for removal of as
much as possible of the low-molecular-weight contaminants from the gel matrix
that will otherwise interfere with subsequent mass spectrometry. This treatment
was followed by further washing/equilibration in 0.1 M ammonium bicarbonate,
pH 8, mixed 1:1 with acetonitrile. The washed gel pieces were placed in an
Eppendorf tube and dried in a Speedvac centrifuge. The dry gel pieces were
reconstituted in buffer (0.1 M ammonium bicarbonate, pH 8) containing trypsin
(0.2 [!ghtl) in a volume just sufficient to cover the gel pieces. Digestion was
allowed to proceed for 20 h at 37°C and the liquid was recovered to a new tube.
The gel pieces were subsequently extracted with 60 % acetonitrile containing
0.1 % formic acid at 30°C for 30 mins after which the two peptide extracts were
combined and dried. The masses of the tryptic fragments were determined via
ESI MS. The masses of 13 tryptic fragments were screened in the SwissProt database and found to match the published sequence of rat a-enolase. To further verify the identity, cm MS of fragments was applied to determine parts of the
amino acid sequence (Bergman et aI., 1997).
A similar strategy was tested to identify genes affected by inhibition of the
protein kinase Raf-MEK-ERK signal pathway (Bergman et aI., 1999a) which is of
major importance for transformation by oncogenes (Cowley et al., 1994). An
inhibitor of MEK1 was applied to c-jun transformed rat fibroblasts followed by
cell lysis and 2-D gel electrophoresis. Gene products for which expression was
altered by MEK1 inhibition were identified both via MALDI MS and ESI MS/MS
of fragments from in-gel tryptic digestion. The protocol for in-gel digestion was
slightly modified from Bergman et aI. (1997). Conditions employed were ammonium bicarbonate at 20 mM for equilibration/digestion, 1 pic om ole trypsin per [!l
(or 0.02 [!g/[!l) for digestion and 0.1 % trifluoroacetic acid (with 60 % acetonitrile) for peptide extraction (Bergman et aI., 1999a). The combined data from
mass mapping and cm identified several proteins with altered expression in
response to MEK1 inhibition. Pirin, a nuclear factor I interacting protein, was
down-regulated which was also the case with many other proteins. Truncated
proteins were detected and up-regulation after MEK1 inhibition was established
for ornithine aminotransferase (Bergman et aI., 1999a). The results illustrate the
usefulness of the combination 2-D gel electrophoresis, in -gel digestion and MS in
studies of signal transduction pathways. The concept was further applied to
screening of potential disease-marker proteins in cells from solid tumors of the
breast, lung and ovary (Bergman et aI., 1999b).
83
sociation (cm) of proteolytic peptides, sequence stretches of 10-20 residues can
be obtained. Even low ng amounts of in-gel digested proteins have provided
sequence information helpful for cloning of the corresponding gene (cf. Wilm et
aI., 1996).
We have applied the in-gel approach to polypeptides of widely different properties and sizes. The technique was tested in studies regarding protein expression
in transformed versus normal cells (Bergman et aI., 1997; Bergman et aI., 1999a).
The amount of a 49 kDa protein separated by 2-D gel electrophoresis was
found to be three times higher in c-jun transformed rat fibroblasts (Bergman et
aI., 1997). To identify this polypeptide the gel spot was cut after staining with
Coomassie followed by thorough destaining and washing in 30 % methanol by
end-over-end mixing for two days. The latter step is important for removal of as
much as possible of the low-molecular-weight contaminants from the gel matrix
that will otherwise interfere with subsequent mass spectrometry. This treatment
was followed by further washing/equilibration in 0.1 M ammonium bicarbonate,
pH 8, mixed 1:1 with acetonitrile. The washed gel pieces were placed in an
Eppendorf tube and dried in a Speedvac centrifuge. The dry gel pieces were
reconstituted in buffer (0.1 M ammonium bicarbonate, pH 8) containing trypsin
(0.2 [!ghtl) in a volume just sufficient to cover the gel pieces. Digestion was
allowed to proceed for 20 h at 37°C and the liquid was recovered to a new tube.
The gel pieces were subsequently extracted with 60 % acetonitrile containing
0.1 % formic acid at 30°C for 30 mins after which the two peptide extracts were
combined and dried. The masses of the tryptic fragments were determined via
ESI MS. The masses of 13 tryptic fragments were screened in the SwissProt database and found to match the published sequence of rat a-enolase. To further verify the identity, cm MS of fragments was applied to determine parts of the
amino acid sequence (Bergman et aI., 1997).
A similar strategy was tested to identify genes affected by inhibition of the
protein kinase Raf-MEK-ERK signal pathway (Bergman et aI., 1999a) which is of
major importance for transformation by oncogenes (Cowley et al., 1994). An
inhibitor of MEK1 was applied to c-jun transformed rat fibroblasts followed by
cell lysis and 2-D gel electrophoresis. Gene products for which expression was
altered by MEK1 inhibition were identified both via MALDI MS and ESI MS/MS
of fragments from in-gel tryptic digestion. The protocol for in-gel digestion was
slightly modified from Bergman et aI. (1997). Conditions employed were ammonium bicarbonate at 20 mM for equilibration/digestion, 1 pic om ole trypsin per [!l
(or 0.02 [!g/[!l) for digestion and 0.1 % trifluoroacetic acid (with 60 % acetonitrile) for peptide extraction (Bergman et aI., 1999a). The combined data from
mass mapping and cm identified several proteins with altered expression in
response to MEK1 inhibition. Pirin, a nuclear factor I interacting protein, was
down-regulated which was also the case with many other proteins. Truncated
proteins were detected and up-regulation after MEK1 inhibition was established
for ornithine aminotransferase (Bergman et aI., 1999a). The results illustrate the
usefulness of the combination 2-D gel electrophoresis, in -gel digestion and MS in
studies of signal transduction pathways. The concept was further applied to
screening of potential disease-marker proteins in cells from solid tumors of the
breast, lung and ovary (Bergman et aI., 1999b).