Protein Sequencing or Genome Sequencing
19
products of trypsin. The spectra are isotopically resolved and with this calibration mode a mass accuracy better than 50 ppm is obtained for the monoisotopic
mass. The mass list, in this case containing 20 peptide masses, is then exported
into the peptide search program (Mann et al. 1994) and the entire EBI nonredundant database searched for matching proteins. The top ranking candidate
was Rat Galectin-3 with 9 matching peptides (Fig. 2.3C). Since the species (rat)
was correct and the calculated protein mass (27 kDa) was consistent with the
molecular mass observed in the gel (32 kDa), the protein is considered to be
unambiguously identified. Upon closer examination of the spectrum two additional peaks could be identified as a result of oxidized Methionine residues in the
corresponding peptides. The sequence coverage was 39 %. This is rather low.
However, examination of the sequence (Fig. 2.3D) revealed that the first 140
N-terminal amino acid residues did not contain any Lys or Arg residues at which
tryptic cleavage could take place. The peak corresponding to this large peptide
was not observed. The sequence coverage for the remaining part of the protein
was 84 %, which is a typical sequence coverage obtained from good quality spectra. Some of the remaining peaks were assigned to keratin-related peptides, keratin being a frequent contaminant when working at low protein levels, and a few
to the tryptic autodigestion products used for calibration. It is a precondition for
positive identification based on MALDI peptide mapping that the sequence of the
protein or the sequence of a highly homologous protein from another species is
present in the database and that a sufficient number of peptide signals (> 4-5)
are present. Sometimes when working on very low protein levels too few peaks
are present or the peptide signals may be suppressed by contaminant-derived signals. In such cases a concentration/purification step might improve the spectra
(see micro-purification below).
If the protein cannot be identified based on the peptide map then partial
sequence information is derived for selected peaks by performing nanoESI MS/
MS on an aliquot of the remaining part of the peptide mixture extracted from the
gel. This may be relevant when very few peaks are present, when identification is
made based on search in EST databases, or by homology to proteins from other
species. Recording of spectra by nanoESI always requires micro-purification to
remove salts and other contaminants.
Micro-purification is performed by applying the protein extract to a custommade miniature column with a bed volume of a few hundred nanoliters made in
an Eppendorph gel loader tip (Gobom et al. 1999). The extracted peptide mixture
dissolved in 5 % formic acid is loaded on the column, washed with 5 -1 0 f.ll of 5 %
formic acid and eluted directly into the nanospray needle with 50 % aqueous
methanol, 2 % formic acid. As mentioned above, micro-purification can also be
applied with advantage prior to MALDI peptide mapping when the spectra are of
poor quality. Inclusion of the micro-purification procedure usually leads to
improved sequence coverage. For MALDI the elution is performed with 1 f.ll of
matrix solution containing 70 % acetonitrile directly onto the mass spectrometric
target (Gobom et al. 1999).
Identification based on peptide sequence tags is illustrated in Fig. 2.4. A human
protein complex was isolated by immune precipitation with a mouse antibody
directed against one of the components and the components separated by 2D
19
products of trypsin. The spectra are isotopically resolved and with this calibration mode a mass accuracy better than 50 ppm is obtained for the monoisotopic
mass. The mass list, in this case containing 20 peptide masses, is then exported
into the peptide search program (Mann et al. 1994) and the entire EBI nonredundant database searched for matching proteins. The top ranking candidate
was Rat Galectin-3 with 9 matching peptides (Fig. 2.3C). Since the species (rat)
was correct and the calculated protein mass (27 kDa) was consistent with the
molecular mass observed in the gel (32 kDa), the protein is considered to be
unambiguously identified. Upon closer examination of the spectrum two additional peaks could be identified as a result of oxidized Methionine residues in the
corresponding peptides. The sequence coverage was 39 %. This is rather low.
However, examination of the sequence (Fig. 2.3D) revealed that the first 140
N-terminal amino acid residues did not contain any Lys or Arg residues at which
tryptic cleavage could take place. The peak corresponding to this large peptide
was not observed. The sequence coverage for the remaining part of the protein
was 84 %, which is a typical sequence coverage obtained from good quality spectra. Some of the remaining peaks were assigned to keratin-related peptides, keratin being a frequent contaminant when working at low protein levels, and a few
to the tryptic autodigestion products used for calibration. It is a precondition for
positive identification based on MALDI peptide mapping that the sequence of the
protein or the sequence of a highly homologous protein from another species is
present in the database and that a sufficient number of peptide signals (> 4-5)
are present. Sometimes when working on very low protein levels too few peaks
are present or the peptide signals may be suppressed by contaminant-derived signals. In such cases a concentration/purification step might improve the spectra
(see micro-purification below).
If the protein cannot be identified based on the peptide map then partial
sequence information is derived for selected peaks by performing nanoESI MS/
MS on an aliquot of the remaining part of the peptide mixture extracted from the
gel. This may be relevant when very few peaks are present, when identification is
made based on search in EST databases, or by homology to proteins from other
species. Recording of spectra by nanoESI always requires micro-purification to
remove salts and other contaminants.
Micro-purification is performed by applying the protein extract to a custommade miniature column with a bed volume of a few hundred nanoliters made in
an Eppendorph gel loader tip (Gobom et al. 1999). The extracted peptide mixture
dissolved in 5 % formic acid is loaded on the column, washed with 5 -1 0 f.ll of 5 %
formic acid and eluted directly into the nanospray needle with 50 % aqueous
methanol, 2 % formic acid. As mentioned above, micro-purification can also be
applied with advantage prior to MALDI peptide mapping when the spectra are of
poor quality. Inclusion of the micro-purification procedure usually leads to
improved sequence coverage. For MALDI the elution is performed with 1 f.ll of
matrix solution containing 70 % acetonitrile directly onto the mass spectrometric
target (Gobom et al. 1999).
Identification based on peptide sequence tags is illustrated in Fig. 2.4. A human
protein complex was isolated by immune precipitation with a mouse antibody
directed against one of the components and the components separated by 2D