571 S.O. Andersen Insect Biochemistry and Molecular Biology 30 2000 569–577
Elution was performed with 0.1 TFA in ultrahigh- quality UHQ water A-buffer in combination with
90 acetonitrile in 0.08 TFA B-buffer. To separate cuticular proteins the column was equilibrated with 20
B, during the first 2 min after application of the sample the concentration of B was increased from 20 to 30,
it was increased from 30 to 50 during the next 40 min and then kept constant at 50 for 3 min before
being readjusted to 20.
To separate peptides derived by proteolytic digestion of the proteins the same eluants were used with the elu-
tion scheme: 0–10 B for 2 min, 10–50 for 40 min, constant at 50 for 3 min, and thereafter returned to 0
for reequilibration.
2.3. Enzymatic digestion To obtain peptides of a reasonable size for sequence
studies the proteins were digested with one or more of the following proteolytic enzymes: trypsin and endoprot-
einase Glu-C from Promega Madison, WI, chymotryp- sin
and endoproteinase
Asp-N from
Boehringer Mannheim, Germany. The digestions were performed
as described previously Andersen, 1998. 2.4. Mass spectrometry
Matrix-assisted laser ionisation mass spectrometry MALDI-MS was performed at the Institute of Molecu-
lar Biology, Odense University, as described previously Jensen et al., 1998.
Plasma desorption mass spectrometry PDMS was performed on a BioIon 20 time-of-flight mass spec-
trometer BioIon, Uppsala, Sweden. Peptides or small proteins were dissolved in 0.1 TFA to a concentration
of approximately 50 pmol µ
l, and 5 µ
l applied to a nitro- cellulose covered target Roepstorff, 1993. Spectra were
acquired for 0.5–1 ×
10
6
fission events at 15 kV acceler- ation voltage and calibrated based on the H
+
and Na
+
ions. 2.5. Sequence determination
The intact proteins and selected peptides were sequenced by Edman degradation with an Applied Bios-
ystems 476A Protein Sequencer. Degradation, conver- sion and identification of the liberated phenylisothiohyd-
antoin amino acids were performed as described by the supplier.
2.6. Nomenclature The Locusta and Blaberus proteins will be given a
prefix, Lm and Bc, respectively, followed by the letters NCP for nymphal cuticular protein, and a number denot-
ing the molecular weight in kDa as calculated from the amino acid sequence.
3. Results
Urea-soluble proteins were extracted from fifth instar nymphs of B. craniifer and L. migratoria. Some of the
extracted proteins were purified, and their amino acid sequences were determined by the combined use of mass
spectrometry and Edman degradation. The proteins were chosen among the more abundant components in the two
extracts and both small and medium size proteins were represented.
The first step in the procedure for sequence determi- nation was to obtain the mass of the individual proteins;
plasma desorption mass spectrometry PDMS was used for the low-molecular weight proteins, and MALDI-MS
for proteins with masses above 10 kDa. Table 1 com- pares the mass values obtained by mass spectrometry
with the values obtained by sequence determination. The N-terminal sequences of the proteins, ranging from 35
to 50 residues, were obtained for most of the proteins by Edman degradation, but two of the Blaberus proteins
BcNCP14.9 and BcNCP21.1 appeared to be N-ter- minally blocked. Treatment of the proteins with pyroglu-
tamine aminopeptidase removed the blocking group, and Edman degradation could be carried out, indicating that
pyroglutamine is the N-terminal residue in these two proteins.
Samples of the proteins were digested by trypsin, and the tryptic peptides separated by RP-HPLC and sub-
jected to mass determination by PDMS and Edman degradation. The sum of the masses of the tryptic pep-
tides could in all cases account for the mass of the indi- vidual proteins, and good agreement was observed
between the peptide masses determined by mass spec- trometry and those calculated from the sequences, indi-
cating that no glycosylations or other secondary modifi- cations were present. A few tryptic peptides were too
long to be sequenced completely, and it was necessary to subject them to further digestion by other proteolytic
enzymes chymotrypsin or endoproteinase Glu-C, and the new set of peptides could be sequenced completely.
The intact proteins were also digested by chymotryp- sin or endoproteinase Glu-C to obtain peptides to estab-
lish the sequential order of the tryptic peptides. The masses of the chymotryptic and Glu-C peptides were
determined, and compared with the masses expected for peptides obtained by digestion of the tryptic peptides.
By means of the sequences of the peptides, which according to their masses could represent overlaps
between tryptic peptides, the complete amino acid sequences of the proteins were established.
The above procedure was sufficient to obtain all the sequences except one: the sequence of LmNCP21.3
572 S.O. Andersen Insect Biochemistry and Molecular Biology 30 2000 569–577
Table 1 The molecular masses of nymphal, post-ecdysial cuticular proteins from B. craniifer and L. migratoria determined by mass spectrometry and
calculated from the amino acid sequences together with the calculated isoelectric points Protein
Molecular mass according to: pI
Mass spectrometry Sequence
BcNCP3.8 3754.2
3753.3 9.3
BcNCP14.6 14592.9
14596.7 5.4
BcNCP14.9 14932.8
14930.5 5.3
BcNCP15.0 15043.8
15049.8 6.2
BcNCP21.1 21093.6
21085.5 8.7
LmNCP4.9 4921.3
4922.5 9.2
LmNCP5.1 5136.4
5142.7 9.2
LmNCP6.4 6446.0
6438.0 8.8
LmNCP9.5 9498.3
9505.6 9.1
LmNCP18.7 18733.0
18721.3 6.2
LmNCP19.8 19816.3
19815.3 6.1
LmNCP21.3 21329.8
21323.1 9.8
could only be determined unambiguously from residue 1 to residue 97, the remaining part of the sequence gave
six closely related tryptic peptides, which contained neither glutamic acid nor aspartic acid, preventing the
use of endoproteases Glu-C and Asp-N for establishing overlaps, and the content of tyrosine was so high that
insufficient information could be obtained from chymo- tryptic digests. The problem was solved by incubating
the protein with trypsin for 5 min at 0
° C, resulting in
partial digestion of the protein. The peptides from the partial digest were separated by RP-HPLC and their
masses were determined by PDMS. Several of the pep- tides had masses corresponding to the sum of the masses
of two or three of the peptides from the complete tryptic digest, and N-terminal sequence determination was used
for deciding the order of tryptic peptides in the products from the partial digest.
The complete amino acid sequences were thus obtained for five proteins from post-ecdysial nymphal
cuticle of B. craniifer Fig. 1 and seven proteins from post-ecdysial nymphal cuticle of L. migratoria Fig. 2.
The cockroach proteins range in mass from 3.8 kDa to 21.1 kDa, and the locust proteins range from 4.9 kDa to
21.3 kDa. The masses and isoelectric points of the pro- teins as calculated by use of the GPMAW program
Lighthouse Data, Odense, Denmark are shown in Table 1.
4. Discussion
