Mass spectrometry and structural characterization of 2S albumin isoforms from Brazil nuts (Bertholletia excelsa) F. Javier Moreno * , John A. Jenkins, Fred A. Mellon, Neil M. Rigby, James A. Robertson, Nikolaus Wellner, E.N. Clare Mills Institute of Food Research, Norwich Research Park, Colney Lane, Norwich NR4 7UA, UK Received 15 April 2003; received in revised form 3 September 2003; accepted 13 November 2003 Available online 1 December 2003 Abstract Proteomic approaches have been used to characterise the main 2S albumin isoforms from Brazil nuts (Bertholletia excelsa). Whilst most isoforms ( f 10 discrete protein species) exhibited molecular masses of around 12 kDa with a high amino acid sequence homology, important charge heterogeneity was found, with pIs varying between 4.6 and 6.6, with one z 7.0. Proteomic analysis showed that these corresponded to a total of six National Center for Biotechnology Information (NCBI) accessions and that three isoforms had been purified to homogeneity corresponding to gi/384327, 112754 and 99609. The latter sequence corresponds to an isoform, previously only identified at the nucleotide sequence level, had a slightly higher molecular weight (13.4 kDa), and with noticeable differences in the primary structure. Proteins corresponding to six different NCBI accessions were identified, the heterogeneity of which had been increased by posttranslational processing. Evidence was found of cyclization of the N-terminal glutamine residue in two isoforms, together with ragged C-termini, indicative of carboxypeptidase activity within the vacuole following posttranslational processing. No evidence of glycosylation was found. Circular dichroism (CD) and Fourier transform-infrared (FT-IR) spectroscopy indicated all the studied isoforms were predominantly a-helical in nature, but that the Mr 13 400 species was structurally distinct, with a higher proportion of a-helical structure. D 2003 Elsevier B.V. All rights reserved. Keywords: 2S albumin; Brazil nut; Allergen; Proteomic 1. Introduction 2S albumins are water-soluble seed storage proteins that are widely distributed in dicotyledonous plants. Those of Brazil nut (Bertholletia excelsa) are exceptionally rich in the sulfur amino acids, containing about 18% methionine and 8% cysteine [1]. Encoded by a multigene family, the 2S albumins of Brazil nut are subjected to a posttranslational proteolytic processing. Thus, they are synthesized as a single larger precursor polypeptide of Mr 18 000, which is cleaved to generate a polypeptide of Mr 15 000. This is subsequently processed to a polypeptide of Mr 12 000 and eventually to the 9000 and 3000 subunits linked by disulfide bridges found in the mature form [2]. The amino acid composition [3,4], protein and nucleotide sequences coding for some of Brazil nut 2S albumin isoforms have been reported [2,5–8]. Despite differences in their subunit structure and synthe- sis, 2S albumins are considered to be structurally homolo- gous, compact globular proteins with a conserved skeleton of cysteine residues [9]. The global fold of a 2S albumin from rapeseed (napin) has been determined by nuclear magnetic resonance (NMR) [10], together with that of the recombinant 2S albumin from castor bean, and sunflower seed SFA-8 [11]. They all adopt a structure comprising a bundle of five a-helices held together by four disulfide 1570-9639/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2003.11.007 Abbreviations: NCBI, National Center for Biotechnology Information; NMR, nuclear magnetic resonance; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; IEF, isoelectric focusing gel electro- phoresis; MES, 2-(N-morpholino) ethane sulfonic acid; DTT, dithiothreitol; IFR, Institute of Food Research; JIC, John Innes Centre; MALDI, matrix- assisted laser desorption ionization; TOF, time-of-flight; ESI, electrospray- ionization; MS, mass spectrometry; RP-HPLC, reversed-phase HPLC; CD, circular dichroism; FT-IR, Fourier transform-infrared; FSD, Fourier self- deconvolution; BSA, bovine serum albumin * Corresponding author. Tel.: +44-1603-255-200; fax: +44-1603-507- 723. E-mail address: javier.moreno@bbsrc.ac.uk (F.J. Moreno). www.bba-direct.com Biochimica et Biophysica Acta 1698 (2004) 175 – 186