24
P. ROEPSTORFF
new peaks upon phosphatase treatment (Jensen et ai. 1999, Kussmann et al.
1999). Phosphorylated pep tides can also be selectively detected by monitoring
precursor ions that generate a diagnostic fragment ion at m/z 79 due to loss of
the phosphate group in negative ion mode ESI-MS/MS (Carr et al. 1996). This
method has recently been applied successfully to identify phosphorylated peptides directly in the mixtures derived from gel-separated proteins (Neubauer and
Mann 1999). Purification of the phosphorylated peptides from the mixture by
affinity chromatography is an alternative to overcome the suppression phenomenon. The phosphopeptides can be isolated directly using a column containing
immobilized Fe 3 + or indirectly using avidin or streptavidin columns after conversion of the phosphopeptides to biotinylated pep tides by ~-elimination, followed
by ethanethiol addition to form S-ethylcystine and biotinylation of the free thiol
group (Oda and Chait 1998). Both methods have been demonstrated to enable
the identification of phosphopeptides in peptide mixtures generated by in-gel
digestion of proteins separated by 2D-PAGE (O.N. Jensen and M.R. Larsen personal communications).
4
When is de novo Protein Sequencing Relevant?
If a protein under identification in proteome studies comes out as 'unknown' in
the database search, then sufficient de novo sequence information must be generated to allow synthesis of nucleotide probes for subsequent isolation and
sequencing of the corresponding cDNA. This has been achieved from silverstained 2D-gels by nanoESI MS/MS (Wilm et al. 1996a, Wilm et al. 1996b, Shevchenko et ai. 1997). However, if no genomic or cDNA information is available for
the organism studied, then complete sequencing on the protein level might be
indicated as for example in our studies of insect and crustacean cuticle proteins
(Andersen et ai. 1995). A number of mass spectrometric approaches have been
suggested for de novo protein sequencing (listed in Fig. 2. 7). In our experience,
however, sequencing of proteins with mass spectrometry as the only sequencing
method nearly always fails to give the complete sequence. Some residues always
remain unidentified and frequently even longer stretches of the sequence. In
addition, the isomeric amino acid residues He and Leu cannot be distinguished.
At the 1992 MPSA meeting in Berlin we proposed a strategy for protein sequencing based on a combination of Edman degradation and mass spectrometry
(Roepstorff and H0jrup 1993). Although the different mass spectrometric
sequencing procedures play an increasing role in our present version of the strategy, we find that this combined approach is still the best strategy for complete de
novo protein sequencing when sufficient amounts of protein are available, i.e. in
the low to mid picomole range. Due to the inclusion of mass spectrometry this
combined approach can be used to sequence the individual proteins in mixtures
of proteins as demonstrated by the simultaneous sequencing of three isoforms of
a meal worm cuticle protein (Haebel et ai. 1995). Subsequent isolation of the proteins from single animals by 2D-PAGE and mass spectrometric determination of
their molecular mass after electroelution as well as mass spectrometric peptide
mapping demonstrated that the three forms represented allelic differences.
P. ROEPSTORFF
new peaks upon phosphatase treatment (Jensen et ai. 1999, Kussmann et al.
1999). Phosphorylated pep tides can also be selectively detected by monitoring
precursor ions that generate a diagnostic fragment ion at m/z 79 due to loss of
the phosphate group in negative ion mode ESI-MS/MS (Carr et al. 1996). This
method has recently been applied successfully to identify phosphorylated peptides directly in the mixtures derived from gel-separated proteins (Neubauer and
Mann 1999). Purification of the phosphorylated peptides from the mixture by
affinity chromatography is an alternative to overcome the suppression phenomenon. The phosphopeptides can be isolated directly using a column containing
immobilized Fe 3 + or indirectly using avidin or streptavidin columns after conversion of the phosphopeptides to biotinylated pep tides by ~-elimination, followed
by ethanethiol addition to form S-ethylcystine and biotinylation of the free thiol
group (Oda and Chait 1998). Both methods have been demonstrated to enable
the identification of phosphopeptides in peptide mixtures generated by in-gel
digestion of proteins separated by 2D-PAGE (O.N. Jensen and M.R. Larsen personal communications).
4
When is de novo Protein Sequencing Relevant?
If a protein under identification in proteome studies comes out as 'unknown' in
the database search, then sufficient de novo sequence information must be generated to allow synthesis of nucleotide probes for subsequent isolation and
sequencing of the corresponding cDNA. This has been achieved from silverstained 2D-gels by nanoESI MS/MS (Wilm et al. 1996a, Wilm et al. 1996b, Shevchenko et ai. 1997). However, if no genomic or cDNA information is available for
the organism studied, then complete sequencing on the protein level might be
indicated as for example in our studies of insect and crustacean cuticle proteins
(Andersen et ai. 1995). A number of mass spectrometric approaches have been
suggested for de novo protein sequencing (listed in Fig. 2. 7). In our experience,
however, sequencing of proteins with mass spectrometry as the only sequencing
method nearly always fails to give the complete sequence. Some residues always
remain unidentified and frequently even longer stretches of the sequence. In
addition, the isomeric amino acid residues He and Leu cannot be distinguished.
At the 1992 MPSA meeting in Berlin we proposed a strategy for protein sequencing based on a combination of Edman degradation and mass spectrometry
(Roepstorff and H0jrup 1993). Although the different mass spectrometric
sequencing procedures play an increasing role in our present version of the strategy, we find that this combined approach is still the best strategy for complete de
novo protein sequencing when sufficient amounts of protein are available, i.e. in
the low to mid picomole range. Due to the inclusion of mass spectrometry this
combined approach can be used to sequence the individual proteins in mixtures
of proteins as demonstrated by the simultaneous sequencing of three isoforms of
a meal worm cuticle protein (Haebel et ai. 1995). Subsequent isolation of the proteins from single animals by 2D-PAGE and mass spectrometric determination of
their molecular mass after electroelution as well as mass spectrometric peptide
mapping demonstrated that the three forms represented allelic differences.