180
L. J. GONZALEZ et al.
on the column were eluted. Both retained and the non-retained fractions were
analyzed by rp-HPLC (Fig. 12.2B and 12.2C).
By comparing the chromatograms of the tryptic map of the native protein
(Fig. 12.2A) with the one obtained for the retained fraction (Fig. 12.2B), it is evident that the reaction has been completed, since all the N-terminal blocked peptides (Fig. 12.2B) have increased their retention time. Also, in the chromatogram
of the non-retained fraction (Fig. 12.2C), two peaks appeared with higher retention time than that corresponding to the C-terminal peptide.
The results of this methodology were not affected by adding different quantities of acetic acid anhydride, at least within a reasonable range of concentrations
(600-1500 [.lM), because the rp-HPLC profiles of the non-retained fractions were
very similar (data not shown). Amino acid analysis of peak 1 and 2 (Fig. 12.2C)
showed ten fold higher amounts of peak # 2 as well as the absence of lysine residues (data not shown). Therefore, non-specific acetylation at the E amino group
of the lysine residues had not occurred.
In the FAB mass spectra of peak # 1 (Fig. 12.3A) a signal appeared, matching
very well with peptides corresponding to five different regions in the whole protein. The sequencing by CID-linked scan (Fig. 12.3B) revealed that this is an
internal peptide (Ac-Gln 69 - TyrBO), originated by a non-specific cleavage of the
trypsin. It should be noticed that the non-specific cleavages of the trypsin should
be minimized, since those peptides will be also transformed as neutral species
after acetylation, and will co-elute with the C-terminal peptide in the nonretained fraction. Therefore, trypsin sequencing grade and the gentlest digestions should be used to avoid ambiguous results when an unknown protein is
analyzed.
In the FAB mass spectrum of peak # 2 (Fig. 12.3C), a signal at m/z 592.2 was
observed which matches very well with the theoretical value expected for the Cterminal pentapeptide of TAB-9 (AFVTI). The sequencing by CID-linked scan
confirmed this assignment (Fig. 12.3D).
However, it is clear that the presence of a basic amino acid at the C-terminus
of the protein could be a limitation for the methodology, since the C-terminal
peptide could also be retained on the column in the cation exchange chromatography. This disadvantage can be overcome if the basic residues (lysine or arginine), are blocked before the tryptic digestion. By this procedure, the C-terminal
peptide is also transformed into a neutral species and will also elute in the nonretained fraction.
The modified methodology was evaluated with the streptokinase (SKr). It is a
47 kDa recombinant protein with 32 lysine residues, one of them located just at
their C-terminal end.
Fig. 12.3. The FAB mass spectra A and C were obtained from the peaks 1 and 2 shown in the Fig.
12.2C. The CID-linked scan spectra shown in Band D correspond to the main molecular ions (1289.5
and 592.2 Da) observed in the FAB mass spectra shown in A and C, respectively. The peaks labeled
with asterisks in the spectra A and C are signals corresponding to the matrix (glycerol). Ac- represents a blocking acetyl group at the N -terminal end of the peptides
L. J. GONZALEZ et al.
on the column were eluted. Both retained and the non-retained fractions were
analyzed by rp-HPLC (Fig. 12.2B and 12.2C).
By comparing the chromatograms of the tryptic map of the native protein
(Fig. 12.2A) with the one obtained for the retained fraction (Fig. 12.2B), it is evident that the reaction has been completed, since all the N-terminal blocked peptides (Fig. 12.2B) have increased their retention time. Also, in the chromatogram
of the non-retained fraction (Fig. 12.2C), two peaks appeared with higher retention time than that corresponding to the C-terminal peptide.
The results of this methodology were not affected by adding different quantities of acetic acid anhydride, at least within a reasonable range of concentrations
(600-1500 [.lM), because the rp-HPLC profiles of the non-retained fractions were
very similar (data not shown). Amino acid analysis of peak 1 and 2 (Fig. 12.2C)
showed ten fold higher amounts of peak # 2 as well as the absence of lysine residues (data not shown). Therefore, non-specific acetylation at the E amino group
of the lysine residues had not occurred.
In the FAB mass spectra of peak # 1 (Fig. 12.3A) a signal appeared, matching
very well with peptides corresponding to five different regions in the whole protein. The sequencing by CID-linked scan (Fig. 12.3B) revealed that this is an
internal peptide (Ac-Gln 69 - TyrBO), originated by a non-specific cleavage of the
trypsin. It should be noticed that the non-specific cleavages of the trypsin should
be minimized, since those peptides will be also transformed as neutral species
after acetylation, and will co-elute with the C-terminal peptide in the nonretained fraction. Therefore, trypsin sequencing grade and the gentlest digestions should be used to avoid ambiguous results when an unknown protein is
analyzed.
In the FAB mass spectrum of peak # 2 (Fig. 12.3C), a signal at m/z 592.2 was
observed which matches very well with the theoretical value expected for the Cterminal pentapeptide of TAB-9 (AFVTI). The sequencing by CID-linked scan
confirmed this assignment (Fig. 12.3D).
However, it is clear that the presence of a basic amino acid at the C-terminus
of the protein could be a limitation for the methodology, since the C-terminal
peptide could also be retained on the column in the cation exchange chromatography. This disadvantage can be overcome if the basic residues (lysine or arginine), are blocked before the tryptic digestion. By this procedure, the C-terminal
peptide is also transformed into a neutral species and will also elute in the nonretained fraction.
The modified methodology was evaluated with the streptokinase (SKr). It is a
47 kDa recombinant protein with 32 lysine residues, one of them located just at
their C-terminal end.
Fig. 12.3. The FAB mass spectra A and C were obtained from the peaks 1 and 2 shown in the Fig.
12.2C. The CID-linked scan spectra shown in Band D correspond to the main molecular ions (1289.5
and 592.2 Da) observed in the FAB mass spectra shown in A and C, respectively. The peaks labeled
with asterisks in the spectra A and C are signals corresponding to the matrix (glycerol). Ac- represents a blocking acetyl group at the N -terminal end of the peptides