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Reliable sequence determination of ribosome‐ inactivating proteins by combining electrospray mass spectrometry and Edman degradation
Author(s) -
Maro Antimo Di,
Ferranti Pasquale,
Mastronicola Mariarosaria,
Polito Letizia,
Bolognesi Andrea,
Stirpe Fiorenzo,
Malorni Antonio,
Parente Augusto
Publication year - 2001
Publication title -
journal of mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 121
eISSN - 1096-9888
pISSN - 1076-5174
DOI - 10.1002/jms.102
Subject(s) - edman degradation , chemistry , saporin , electrospray ionization , amino acid , mass spectrometry , protein primary structure , peptide sequence , sequence (biology) , ribosome inactivating protein , molecular mass , chromatography , ribosome , biochemistry , rna , enzyme , immunotoxin , cytotoxicity , in vitro , gene
The primary structure of saporin‐S9 and MAP‐S, two type‐1 ribosome‐inactivating proteins isolated from the seeds of Saponaria officinalis L. and Mirabilis jalapa , respectively, was determined using a combined approach based on Edman degradation and electrospray ionization mass spectrometry (ESMS). Saporin‐S9 has 253 amino acids with a calculated molecular mass of 28 492.99, which is in good agreement with that determined by ESMS (28 495 ± 2 Da). Unlike other saporins with known primary structure, saporin‐S9 contains four histidinyl residues (positions 111, 121, 216 and 248). By comparing the amino acid sequence of saporin‐S9 with that of saporin‐S6, we found 22 amino acid substitutions (8.7%), 13 of which are conservative and nine non‐conservative. The residues known to be involved in the definition of the active site and with RNA base recognition are conserved. The four histidinyl residues and especially Lys for Gln203 contribute to the higher calculated p I value (10.17) of saporin‐S9 compared with saporin‐S6 (9.98). MAP‐S contains 250 amino acid residues with a calculated molecular mass of 27 789.49, in good agreement with that determined by ESMS (27 789 ± 2). Cys36 and Cys220 form a disulphide bridge and only four amino acid residues are different from the amino acid sequence of MAP, isolated from the roots of the same plant, i.e. Leu34 (Glu), Ile161 (Leu), Asp185 (Glu) and Asp191 (Glu) (in parentheses, the residues present in MAP). The reported approach can provide rapid and reliable sequence screening in the analysis of homologous proteins, including the presence of disulphide bridges. Copyright © 2001 John Wiley & Sons, Ltd. Abbreviations: ESMS electrospray mass spectrometryhsDNA herring sperm DNACPase carboxypeptidaseH Ser homoserineLC liquid chromatographyMAP Mirabilis antiviral protein isolated from Mirabilis jalapa rootsMAP‐S RIP isolated from Mirabilis jalapa seedsRIP ribosome‐inactivating proteinRP‐HPLC reversed‐phase high‐performance liquid chromatographysaporin‐S RIP isolated from Saponaria officinalis seedsu mass unit. The standard one‐ or three‐letter code has been used for the amino acids.