Reliable sequence determination of ribosome- inactivating proteins by combining electrospray mass spectrometry and Edman degradation

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Abstract
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 pI 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 spectrometry hsDNA herring sperm DNA CPase carboxypeptidase HSer homoserine LC liquid chromatography MAP Mirabilis antiviral protein isolated from Mirabilis jalapa roots MAP‐S RIP isolated from Mirabilis jalapa seeds RIP ribosome‐inactivating protein RP‐HPLC reversed‐phase high‐performance liquid chromatography saporin‐S RIP isolated from Saponaria officinalis seeds u mass unit. The standard one‐ or three‐letter code has been used for the amino acids.