Sequence determination of three cuticular proteins and isoforms from the migratory locust, Locusta migratoria, using a combination of Edman degradation and mass spectrometric techniques.
The cuticle (exoskeleton) is a characteristic structure of insects and other arthropods. It is an extracellular layer which surrounds and protects the insect, and it is composed of proteins, lipids, water molecules, phenolic materials and chitin. Four proteins isolated from the thorax and femur cuticle of pharate adult migratory locust, Locusta migratoria, have been purified by ion-exchange chromatography and reversed-phase high performance liquid chromatography (RP-HPLC). Their amino acid sequences were determined by combined use of mass spectrometry and automated Edman degradation. The cuticular extract was also separated by two-dimensional gel electrophoresis. In order to localize and identify the position of the proteins in the gel, a number of gel spots were excised and the proteins electroeluted. The molecular mass of some of the electroeluted proteins was determined by means of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) as well as by electrospray mass spectrometry (ESI-MS). Two of the sequenced proteins exist as pairs of closely related isoforms; one of the pairs contains the conserved 68-residue RR-2 motif, common for proteins from solid cuticles, and the other proteins contain the short motif Ala-Ala-Pro-Ala/Val repeatedly throughout the sequence.
Kalume
Dário Eluan
DE
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej, 55 DK-5230 Odense M, Denmark.
Kieffer
Sylvie
S
Rafn
Kate
K
Skou
Lene
L
Andersen
Svend Olav
SO
Roepstorff
Peter
P
D000595:Amino Acid Sequence; D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional; D005269:Femur; D006110:Grasshoppers / Q000235:genetics* / Q000378:metabolism; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D020033:Protein Isoforms / Q000235:genetics; D016415:Sequence Alignment; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013909:Thorax; D014357:Trypsin
D019476:Insect Proteins; D010446:Peptide Fragments; D020033:Protein Isoforms; C047663:cuticle proteins, insects; D014357:Trypsin