title: Proteomic strategy for identifying mollusc shell proteins using mild chemical degradation and trypsin digestion of insoluble organic shell matrix: a pilot study on Haliotis tuberculata.
abstract: A successful strategy for the identification of shell proteins is based on proteomic analyses where soluble and insoluble fractions isolated from organic shell matrix are digested with trypsin with the aim of generating peptides, which are used to identify novel shell proteins contained in databases. However, using trypsin as a sole degradative agent is limited by the enzyme's cleavage specificity and is dependent upon the occurrence of lysine and arginine in the shell protein sequence. To bypass this limitation, we investigated the ability of trifluoroacetic acid (TFA), a low-specificity chemical degradative agent, to generate clusters of analyzable peptides from organic shell matrix, suitable for database annotation. Acetic acid-insoluble fractions from Haliotis tuberculata shell were processed by trypsin followed by TFA digestion. The hydrolysates were used to annotate an expressed sequence tag library constructed from the mantle tissue of Haliotis asinina, a tropical abalone species. The characterization of sequences with repeat motifs featured in some of the shell matrix proteins benefited from TFA-induced serial cutting, which can result in peptide ladder series. Using the degradative specificities of TFA and trypsin, we were able to identify five novel shell proteins. This pilot study indicates that a mild chemical digestion of organic shell matrix combined with trypsin generates peptides suitable for proteomic analysis for better characterization of mollusc shell matrix proteins.
authors.affiliation: UMR BOREA (Biologie des Organismes et Ecosystèmes Aquatiques), MNHN/CNRS 7208/IRD 207, Muséum National d'Histoire Naturelle CP 26, 43 rue Cuvier, 75231, Paris Cedex 05, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Bédouet
authors.forename: Arul
authors.initials: A
authors.lastname: Marie
authors.forename: Sophie
authors.initials: S
authors.lastname: Berland
authors.forename: Benjamin
authors.initials: B
authors.lastname: Marie
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Auzoux-Bordenave
authors.forename: Frédéric
authors.initials: F
authors.lastname: Marin
authors.forename: Christian
authors.initials: C
authors.lastname: Milet
mesh_terms: D060105:Animal Shells / Q000737:chemistry*; D000818:Animals; D005109:Extracellular Matrix / Q000737:chemistry*; D008974:Mollusca / Q000378:metabolism*; D010865:Pilot Projects; D020543:Proteome / Q000032:analysis* / Q000737:chemistry*; D040901:Proteomics / Q000379:methods; D012995:Solubility; D014357:Trypsin / Q000737:chemistry*
chemical_list: D020543:Proteome; D014357:Trypsin