Reversible Negative Staining of Protein on Electrophoresis Gels
45
The efficiency in digestion and peptide elution has been evaluated by measuring the recovery of radioiodinated tryptic peptides generated from BSA. Yields
after two 10 min incubation of the gel microparticles with either the digestion
buffer or in combination with aqueous acetonitrile-0.1 % TFA solutions are
90-96 %. Slightly higher elution yields are obtained if an additional extraction is
accomplished with an aqueous solution of formic acid-isopropanol-acetonitrile
(98%).
3.1
MALDI-TOF MS Peptide Mass Fingerprint
Analysis of sequence coverage by MALDI-TOF MS after in-gel digestion. Streptokinase (SK), a 47 kDa protein presenting 32 lysine and 18 arginine residues, was
selected for evaluating the efficiency of this protocol by analyzing its tryptic map
by MALDI-TOF MS. The peptide fraction eluted in 50 % ACN, 0.1 % TFA showed
41 major signals, all identified in the sequence (Fig. 3.3A), and several minor signals corresponding to sodium adducts of the peptides. In addition, two signals
corresponded to peptides containing oxidized methionine residues, that accompanied the predominant reduced form and two signals corresponded to trypsin
autoproteolysis. The identified peptides on the acetonitrile/TFA eluate covered
90 % of the sequence. A further extraction of the gel pellet under more drastic
conditions by using a mixture of aqueous formic acid, isopropanol and acetonitrile showed 12 signals, 11 of them already present in the previously analyzed
fraction, and one corresponding to the N-terminal peptide (37 residues) (Fig.
3.3B), for a global coverage of 98.8 % of the sequence. The missing signals corresponded to the tripeptide LLK (185-187) (monoisotopic mass 372.51) and a
dipeptide AK (334-335) (monoisotopic mass 217.27), their presence could not be
established as they fell below the lower limit for data acquisition (400 Da). The
efficiency of the digestion was also evidenced by an analysis of the missed cleavage sites. From the total 42 major signals, 17 arose from total cleavage. The
remaining 25 presented one or two missing cleavage sites, 15 of them corresponded to the basic residue followed by proline, and 9 to basic residues linked
to aspartic or glutamic acid. These results are equivalent to those we have found
for an in-solution tryptic digestion of streptokinase for 4 h (not shown) indicating that the efficiency of gel digestion following the present protocol is very close
to that achieved for digestion in solution.
A second example of the application of these procedures is the in-gel deglycosylation of human erythropoietin, followed by in-gel digestion with trypsin.
Erythropoietin (EPO, MW 18.3 kDa) is a relatively highly glycosylated protein,
presenting both N- and O-glycosylation, migrating in gel electrophoresis as a 36
kDa protein. It contains 16 tryptic cleavage sites. It was deglycosylated in-gel for
2 h, separated by SDS-PAGE and the deglycosylated protein was digested in-gel
for 4 h with trypsin. According to MALDI-TOF MS analysis, the sequence coverage resulting from a single in-gel digestion experiment and considering all possible peptides was 91 % (148 from 165 residues) while coverage considering only
the peptides in the range of data acquisition was 95 %. The missing signals corresponded to peptide Al_R4 (439.25), and peptide Alll_K116 (586.34). The dipeptides
45
The efficiency in digestion and peptide elution has been evaluated by measuring the recovery of radioiodinated tryptic peptides generated from BSA. Yields
after two 10 min incubation of the gel microparticles with either the digestion
buffer or in combination with aqueous acetonitrile-0.1 % TFA solutions are
90-96 %. Slightly higher elution yields are obtained if an additional extraction is
accomplished with an aqueous solution of formic acid-isopropanol-acetonitrile
(98%).
3.1
MALDI-TOF MS Peptide Mass Fingerprint
Analysis of sequence coverage by MALDI-TOF MS after in-gel digestion. Streptokinase (SK), a 47 kDa protein presenting 32 lysine and 18 arginine residues, was
selected for evaluating the efficiency of this protocol by analyzing its tryptic map
by MALDI-TOF MS. The peptide fraction eluted in 50 % ACN, 0.1 % TFA showed
41 major signals, all identified in the sequence (Fig. 3.3A), and several minor signals corresponding to sodium adducts of the peptides. In addition, two signals
corresponded to peptides containing oxidized methionine residues, that accompanied the predominant reduced form and two signals corresponded to trypsin
autoproteolysis. The identified peptides on the acetonitrile/TFA eluate covered
90 % of the sequence. A further extraction of the gel pellet under more drastic
conditions by using a mixture of aqueous formic acid, isopropanol and acetonitrile showed 12 signals, 11 of them already present in the previously analyzed
fraction, and one corresponding to the N-terminal peptide (37 residues) (Fig.
3.3B), for a global coverage of 98.8 % of the sequence. The missing signals corresponded to the tripeptide LLK (185-187) (monoisotopic mass 372.51) and a
dipeptide AK (334-335) (monoisotopic mass 217.27), their presence could not be
established as they fell below the lower limit for data acquisition (400 Da). The
efficiency of the digestion was also evidenced by an analysis of the missed cleavage sites. From the total 42 major signals, 17 arose from total cleavage. The
remaining 25 presented one or two missing cleavage sites, 15 of them corresponded to the basic residue followed by proline, and 9 to basic residues linked
to aspartic or glutamic acid. These results are equivalent to those we have found
for an in-solution tryptic digestion of streptokinase for 4 h (not shown) indicating that the efficiency of gel digestion following the present protocol is very close
to that achieved for digestion in solution.
A second example of the application of these procedures is the in-gel deglycosylation of human erythropoietin, followed by in-gel digestion with trypsin.
Erythropoietin (EPO, MW 18.3 kDa) is a relatively highly glycosylated protein,
presenting both N- and O-glycosylation, migrating in gel electrophoresis as a 36
kDa protein. It contains 16 tryptic cleavage sites. It was deglycosylated in-gel for
2 h, separated by SDS-PAGE and the deglycosylated protein was digested in-gel
for 4 h with trypsin. According to MALDI-TOF MS analysis, the sequence coverage resulting from a single in-gel digestion experiment and considering all possible peptides was 91 % (148 from 165 residues) while coverage considering only
the peptides in the range of data acquisition was 95 %. The missing signals corresponded to peptide Al_R4 (439.25), and peptide Alll_K116 (586.34). The dipeptides