84
A.-CH. BERGMAN et al.
A different approach to identification and sequence analysis of gel separated
proteins is to carry out a traditional peptide mapping via reverse-phase HPLC
and collect the components for Edman degradation (Fig. 6.1). However, the
amounts of individual proteins separated in 2-D gels are seldom above a few
picomoles, thus requiring sensitive systems for peptide preparation and sequence
analysis. We use an instrument combination involving a capillary HPLC system,
the Microblotter, and the Procise cLC sequencer (both from PE-Applied Biosysterns) for peptide extracts at the low picomole to sub-picomole level recovered
after in-gel tryptic digestion (Fig. 6.1). The C18 chromatography is carried out
using a column with inner diameter 0.5 mm and flow rate 5 Ill/min. The low flow
rate necessitates small-scale fractionation of peptide components for microsequence analysis. The capillary outlet intermittently touches a membrane strip of
PVDF while moving. After sample collection, the strip is aligned with the chromatogram for excision of spots corresponding to peptide candidates for structural analysis. The PVDF membrane pieces are mounted in the cartridge of the
Procise cLC sequencer which has a matching sensitivity (Fig. 6.1). Peptide
amounts in the sub-picomole range can be analyzed for extended degradations
(15 cycles or more). The high sensitivity is achieved mainly via miniaturization
of vital parts of the sequencer instrument such as the cartridge and the column
for phenylthiohydantoin (PTH) identification. The chromatography system can
detect below 100 femtomole PTH amino acid.
Trypsin in-gel digestion followed by Microblotter peptide isolation was
applied to a 110 kDa insect protein separated by SDS/polyacrylamide gel electrophoresis in the low picomole range. The chromatogram revealed about 20 well
separated fragments suitable for sequence analysis. A PVDF-bound peptide fraction corresponding to approximately 1.5 picomole was applied to Procise cLC
analysis. The tyrosine residue detected in the first cycle corresponded to 900 femtomole and was followed by a phenylalanine at 870 femtomole in the second
cycle. Interpretation of each of the twelve cycles analyzed was straightforward. In
the last cycle, a serine residue corresponding to 150 femtomole was clearly
assigned which illustrates the sub-picomole capability of the sequencer system.
In the PTH analysis, each residue generated a signal that was on average more
than 5-fold above the level of the amino acid background. In addition, the carryover from cycle to cycle was less than 10 %. A database search using the BLAST
algorithm (Altschul and Gish, 1996) revealed a strong sequence homology
between the protein under investigation and a known Drosophila protein. Of the
twelve residues sequenced, ten were identical with the Drosophila sequence and
one of the two non-identical residues represented a conservative replacement. In
the Drosophila sequence, the first residue is preceded by a lysine residue and the
residue at position 13 is arginine. Since these residues both fit trypsin specificity,
the analyzed fragment is likely to be thirteen residues in total. Serine detected in
position 12 is then penultimate to the C-terminal residue. Still the yield is good,
150 femtomole, showing that wash-out losses are acceptable during degradation.
A.-CH. BERGMAN et al.
A different approach to identification and sequence analysis of gel separated
proteins is to carry out a traditional peptide mapping via reverse-phase HPLC
and collect the components for Edman degradation (Fig. 6.1). However, the
amounts of individual proteins separated in 2-D gels are seldom above a few
picomoles, thus requiring sensitive systems for peptide preparation and sequence
analysis. We use an instrument combination involving a capillary HPLC system,
the Microblotter, and the Procise cLC sequencer (both from PE-Applied Biosysterns) for peptide extracts at the low picomole to sub-picomole level recovered
after in-gel tryptic digestion (Fig. 6.1). The C18 chromatography is carried out
using a column with inner diameter 0.5 mm and flow rate 5 Ill/min. The low flow
rate necessitates small-scale fractionation of peptide components for microsequence analysis. The capillary outlet intermittently touches a membrane strip of
PVDF while moving. After sample collection, the strip is aligned with the chromatogram for excision of spots corresponding to peptide candidates for structural analysis. The PVDF membrane pieces are mounted in the cartridge of the
Procise cLC sequencer which has a matching sensitivity (Fig. 6.1). Peptide
amounts in the sub-picomole range can be analyzed for extended degradations
(15 cycles or more). The high sensitivity is achieved mainly via miniaturization
of vital parts of the sequencer instrument such as the cartridge and the column
for phenylthiohydantoin (PTH) identification. The chromatography system can
detect below 100 femtomole PTH amino acid.
Trypsin in-gel digestion followed by Microblotter peptide isolation was
applied to a 110 kDa insect protein separated by SDS/polyacrylamide gel electrophoresis in the low picomole range. The chromatogram revealed about 20 well
separated fragments suitable for sequence analysis. A PVDF-bound peptide fraction corresponding to approximately 1.5 picomole was applied to Procise cLC
analysis. The tyrosine residue detected in the first cycle corresponded to 900 femtomole and was followed by a phenylalanine at 870 femtomole in the second
cycle. Interpretation of each of the twelve cycles analyzed was straightforward. In
the last cycle, a serine residue corresponding to 150 femtomole was clearly
assigned which illustrates the sub-picomole capability of the sequencer system.
In the PTH analysis, each residue generated a signal that was on average more
than 5-fold above the level of the amino acid background. In addition, the carryover from cycle to cycle was less than 10 %. A database search using the BLAST
algorithm (Altschul and Gish, 1996) revealed a strong sequence homology
between the protein under investigation and a known Drosophila protein. Of the
twelve residues sequenced, ten were identical with the Drosophila sequence and
one of the two non-identical residues represented a conservative replacement. In
the Drosophila sequence, the first residue is preceded by a lysine residue and the
residue at position 13 is arginine. Since these residues both fit trypsin specificity,
the analyzed fragment is likely to be thirteen residues in total. Serine detected in
position 12 is then penultimate to the C-terminal residue. Still the yield is good,
150 femtomole, showing that wash-out losses are acceptable during degradation.