Protein Sequencing or Genome Sequencing
21
PAGE digestion. The extract obtained by in situ digestion of one of the spots with
trypsin was micropurified, loaded direct in the nanospray needle and analyzed
with nanoESI-MS on a micromass Q-TOF mass spectrometer (Fig. 2.4A). A peak
derived from a doubly-charged ion was selected for MS/MS in the peptide map.
The Q-TOF instrument has sufficient resolution to determine the charged state of
an ion based on the mass differences between the isotope signals (Fig. 2.4B). In
the MS/MS spectrum of the selected ion (Fig. 2.4C) singly-charged ions are identified (Fig. 2.4D) and a peptide sequence tag is generated (Mann and Wilm 1994).
A search in the EBI non-redundant database for this sequence tag identified the
peptide as being a tryptic peptide derived from the constant region of a mouse Ig
kappa light chain. Peptide sequence tags derived by MS/MS of several of the other
peaks present in the spectrum (Fig. 2.4A) resulted in identification of the same
protein. Thus, the unknown spot represented the antibody used for immune precipitation.
3
Characterization of Secondary Modifications in Proteins
Once a protein is identified, the next obvious questions are: Is the identified protein post-translationally modified and if so, how? If the purified protein is available then the strategy is to compare its molecular mass determined by MS with
that calculated from its DNA sequence. If these masses are different, then the
modified sites and the types of modification are identified by mass spectrometric
peptide mapping and, when relevant, supplemented with MS/MS of selected peptide ions or degradation with appropriate enzymes, e.g. glycosidases or phosphatases (Burlingame 1996, Bean et al. 1995). If the proteins are available only as
spots or bands in electrophoretic gels, then it is often not possible to determine
the molecular mass of the intact protein, and characterization of posttranslational modifications must rely on peptide mapping before and after enzymatic treatments and, when appropriate, MS/MS. In such cases it is essential to
obtain complete or very high sequence coverage in the peptide maps (Moertz et
al. 1996, Wilm et al. 1996a).
The most frequent secondary modifications are: proteolytic processing, acylation of the N-terminus and Lys to-amino groups, phosphorylation of Ser, Thr and
Tyr, and N -glycosylation of Asn or O-glycosylation of Ser and Thr. The two first
types of modifications are normally easily recognized in the peptide maps whenever the corresponding signals are present. However, suppression of some of the
peptide signals in the spectra of the mixtures is a frequent phenomenon. This is
especially true for pep tides modified with acetic groups, e.g. phosphate groups or
sialic acid containing glycans. This can be overcome if sufficient protein is available for separation of the pep tides. Glycosylated peptides can easily be recognized by mass spectrometry due to the characteristic peak pattern caused by the
glycan heterogeneity (Fig. 2.5) and phosphorylated peptides can be identified by
the loss of the phosphate group either due to post source decay in MALDI (Fig.
2.6) or by collision-induced dissociation in ESI.
The peak pattern from the glycopeptides derived by enzymatic digestion of
glycoproteins can be used to generate a site specific glycoprofile for a protein
21
PAGE digestion. The extract obtained by in situ digestion of one of the spots with
trypsin was micropurified, loaded direct in the nanospray needle and analyzed
with nanoESI-MS on a micromass Q-TOF mass spectrometer (Fig. 2.4A). A peak
derived from a doubly-charged ion was selected for MS/MS in the peptide map.
The Q-TOF instrument has sufficient resolution to determine the charged state of
an ion based on the mass differences between the isotope signals (Fig. 2.4B). In
the MS/MS spectrum of the selected ion (Fig. 2.4C) singly-charged ions are identified (Fig. 2.4D) and a peptide sequence tag is generated (Mann and Wilm 1994).
A search in the EBI non-redundant database for this sequence tag identified the
peptide as being a tryptic peptide derived from the constant region of a mouse Ig
kappa light chain. Peptide sequence tags derived by MS/MS of several of the other
peaks present in the spectrum (Fig. 2.4A) resulted in identification of the same
protein. Thus, the unknown spot represented the antibody used for immune precipitation.
3
Characterization of Secondary Modifications in Proteins
Once a protein is identified, the next obvious questions are: Is the identified protein post-translationally modified and if so, how? If the purified protein is available then the strategy is to compare its molecular mass determined by MS with
that calculated from its DNA sequence. If these masses are different, then the
modified sites and the types of modification are identified by mass spectrometric
peptide mapping and, when relevant, supplemented with MS/MS of selected peptide ions or degradation with appropriate enzymes, e.g. glycosidases or phosphatases (Burlingame 1996, Bean et al. 1995). If the proteins are available only as
spots or bands in electrophoretic gels, then it is often not possible to determine
the molecular mass of the intact protein, and characterization of posttranslational modifications must rely on peptide mapping before and after enzymatic treatments and, when appropriate, MS/MS. In such cases it is essential to
obtain complete or very high sequence coverage in the peptide maps (Moertz et
al. 1996, Wilm et al. 1996a).
The most frequent secondary modifications are: proteolytic processing, acylation of the N-terminus and Lys to-amino groups, phosphorylation of Ser, Thr and
Tyr, and N -glycosylation of Asn or O-glycosylation of Ser and Thr. The two first
types of modifications are normally easily recognized in the peptide maps whenever the corresponding signals are present. However, suppression of some of the
peptide signals in the spectra of the mixtures is a frequent phenomenon. This is
especially true for pep tides modified with acetic groups, e.g. phosphate groups or
sialic acid containing glycans. This can be overcome if sufficient protein is available for separation of the pep tides. Glycosylated peptides can easily be recognized by mass spectrometry due to the characteristic peak pattern caused by the
glycan heterogeneity (Fig. 2.5) and phosphorylated peptides can be identified by
the loss of the phosphate group either due to post source decay in MALDI (Fig.
2.6) or by collision-induced dissociation in ESI.
The peak pattern from the glycopeptides derived by enzymatic digestion of
glycoproteins can be used to generate a site specific glycoprofile for a protein