CHAPTER 6
Characterization of Gel Separated Proteins
A.-CH. BERGMAN l M. OPPERMANN!, U. OPPERMANN l , H. JORNVALL l
and T. BERGMAN l
Recent developments in preparation and structure determination of proteins
have made analysis possible at a sensitivity and with a throughput that was
hardly anticipated only a few years ago. In particular, efficient tools for screening
and characterization of gene products are important. Protocols and combinations involving sequencer analysis and mass spectrometry have in this regard a
high potential (Bergman et aI., 1998).
Gel electrophoresis in two dimensions is well suited for preparation of protein
material for structural analysis (cf. Patterson 1994) and can replace long purification protocols with column approaches. In addition, SDS/polyacrylamide gel
electrophoresis is often the only choice for efficient purification of hydrophobic
polypeptides.
For characterization of gel separated proteins regarding amino acid sequence
and identity we use a strategy involving in-gel digestion and analysis of proteolytic pep tides or electroblotting of the intact protein to a polyvinylidene difluoride (PVDF) membrane for direct sequencer analysis (Fig. 6.1). Both routes are
efficient and the choice depends on the amount available but also on time and
likelihood of finding a blocked N-terminus (Fig. 6.1).
1
In-Gel Digestion and Further Analysis of Fragments
After electrophoresis, the one-dimensional (i-D) or two-dimensional (2-D) gel is
stained, preferrentiallywith Coomassie blue. Although silver-staining is compatible with the protocol for proteolytic cleavage, the recovery of fragments is low.
The protein band or spot is cut and applied to in-gel cleavage with trypsin followed by mass spectrometry (MS) of proteolytic fragments or Microblotter peptide isolation (Fig. 6.1). Peptide mass mapping is powerful for identification
(Henzel et aI., 1993) and in combination with 2-D gel electrophoresis, hundreds
of spots can be analyzed in just one separation (Schevchenko et aI., 1996). Mass
mapping is frequently carried out by matrix-assisted laser desorption ionization
(MALDI) MS but electrospray ionization (ESI) MS is also used to analyze the
unseparated peptide mixture. Computer algorithms applied to the mass data
obtained, are combined with the cleavage specificity of the enzyme and the estiI Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm,
Sweden.
Characterization of Gel Separated Proteins
A.-CH. BERGMAN l M. OPPERMANN!, U. OPPERMANN l , H. JORNVALL l
and T. BERGMAN l
Recent developments in preparation and structure determination of proteins
have made analysis possible at a sensitivity and with a throughput that was
hardly anticipated only a few years ago. In particular, efficient tools for screening
and characterization of gene products are important. Protocols and combinations involving sequencer analysis and mass spectrometry have in this regard a
high potential (Bergman et aI., 1998).
Gel electrophoresis in two dimensions is well suited for preparation of protein
material for structural analysis (cf. Patterson 1994) and can replace long purification protocols with column approaches. In addition, SDS/polyacrylamide gel
electrophoresis is often the only choice for efficient purification of hydrophobic
polypeptides.
For characterization of gel separated proteins regarding amino acid sequence
and identity we use a strategy involving in-gel digestion and analysis of proteolytic pep tides or electroblotting of the intact protein to a polyvinylidene difluoride (PVDF) membrane for direct sequencer analysis (Fig. 6.1). Both routes are
efficient and the choice depends on the amount available but also on time and
likelihood of finding a blocked N-terminus (Fig. 6.1).
1
In-Gel Digestion and Further Analysis of Fragments
After electrophoresis, the one-dimensional (i-D) or two-dimensional (2-D) gel is
stained, preferrentiallywith Coomassie blue. Although silver-staining is compatible with the protocol for proteolytic cleavage, the recovery of fragments is low.
The protein band or spot is cut and applied to in-gel cleavage with trypsin followed by mass spectrometry (MS) of proteolytic fragments or Microblotter peptide isolation (Fig. 6.1). Peptide mass mapping is powerful for identification
(Henzel et aI., 1993) and in combination with 2-D gel electrophoresis, hundreds
of spots can be analyzed in just one separation (Schevchenko et aI., 1996). Mass
mapping is frequently carried out by matrix-assisted laser desorption ionization
(MALDI) MS but electrospray ionization (ESI) MS is also used to analyze the
unseparated peptide mixture. Computer algorithms applied to the mass data
obtained, are combined with the cleavage specificity of the enzyme and the estiI Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm,
Sweden.