A strategy for isolating rare peptides: isolation and sequencing of a large peptide present in a single neuron of the nematode Ascaris suum.
Monoclonal antibody G15-6A was generated by immunizing mice with Ascaris head extracts. It recognizes an antigen present in a single neuron, with a cell body in the dorsal rectal ganglion, that projects along the ventral cord to the nerve ring. Ascaris extracts were fractionated by HPLC and ammonium sulfate precipitation, and fractions assayed by dot-blotting with antibody G15-6A. A single immunoreactive polypeptide was purified; mass spectrometry showed a molecular weight of 11,542 Da. Partial N-terminal sequencing, followed by cloning of the transcript encoding the peptide, revealed a predicted peptide product comprising 109 amino acids, and a molecular mass of 11,863 Da. The N-terminus of the predicted peptide includes four more amino acids than are found in the isolated product.
D000595:Amino Acid Sequence; D000645:Ammonium Sulfate / Q000737:chemistry; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology; D000947:Antigens, Helminth / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification; D017165:Ascaris suum / Q000033:anatomy & histology / Q000737:chemistry* / Q000502:physiology; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D030562:Databases, Protein; D015801:Helminth Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D015151:Immunoblotting; D007150:Immunohistochemistry; D008722:Methods; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009474:Neurons / Q000737:chemistry* / Q000276:immunology; D009479:Neuropeptides / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D011402:Pronase / Q000378:metabolism; D011480:Protease Inhibitors / Q000737:chemistry; D018510:RNA, Helminth / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000911:Antibodies, Monoclonal; D000947:Antigens, Helminth; D018076:DNA, Complementary; D015801:Helminth Proteins; D009479:Neuropeptides; D011480:Protease Inhibitors; D018510:RNA, Helminth; D011402:Pronase; D000645:Ammonium Sulfate