title: Human Basal Tear Peptidome Characterization by CID, HCD, and ETD Followed by in Silico and in Vitro Analyses for Antimicrobial Peptide Identification.
abstract: Endogenous peptides are valuable targets in the analysis of biological processes. The tear film contains proteins and peptides released by the tear duct mucosal cells, including antimicrobial peptides involved in the protection against exogenous pathogens; however, the peptide content of the tear liquid remains poorly characterized. We analyzed naturally occurring peptides isolated from human basal tears. Mass spectrometry analysis of endogenous peptides presents a number of drawbacks, including size heterogeneity and nonpredictable fragmentation patterns, among others. Therefore, CID, ETD, and HCD methods were used for the characterization of the tear peptide content. The contribution of DMSO as an additive of the chromatographic solvents was also evaluated. We identified 157, 131, and 122 peptides using CID-, ETD-, and HCD-based methods, respectively. Altogether, 234 different peptides were identified, leading to the generation of the biggest data set of endogenous tear peptides to date. The antimicrobial activity prediction analysis performed in silico revealed different putative antimicrobial peptides. Two of the extracellular glycoprotein lacritin peptides were de novo synthesized, and their antimicrobial activity was confirmed in vitro. Our findings demonstrate the benefits of using different fragmentation methods for the analysis of endogenous peptides and provide a useful approach for the discovery of peptides with antimicrobial activity.
authors.affiliation: †Proteomics Platform, CIC bioGUNE, CIBERehd, ProteoRed-ISCIII, Bizkaia Science and Technology Park, 48160 Derio, Spain.
authors.forename: Mikel
authors.initials: M
authors.lastname: Azkargorta
authors.affiliation: ‡Bioftalmik Applied Research, Bizkaia Science and Technology Park, 48160 Derio, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Soria
authors.affiliation: ∥Instituto de Biología, Pontificia Universidad Católica de Valparaíso, Avenida Universidad 330, 2373223 Valparaíso, Chile.
authors.forename: Claudia
authors.initials: C
authors.lastname: Ojeda
authors.affiliation: §Núcleo Biotecnológico de Curauma (NBC), Pontificia Universidad Católica de Valparaíso, Avenida Universidad 330, 2373223 Valparaíso, Chile.
authors.forename: Fanny
authors.initials: F
authors.lastname: Guzmán
authors.affiliation: ‡Bioftalmik Applied Research, Bizkaia Science and Technology Park, 48160 Derio, Spain.
authors.forename: Arantxa
authors.initials: A
authors.lastname: Acera
authors.affiliation: †Proteomics Platform, CIC bioGUNE, CIBERehd, ProteoRed-ISCIII, Bizkaia Science and Technology Park, 48160 Derio, Spain.
authors.forename: Ibon
authors.initials: I
authors.lastname: Iloro
authors.affiliation: ‡Bioftalmik Applied Research, Bizkaia Science and Technology Park, 48160 Derio, Spain.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Suárez
authors.affiliation: †Proteomics Platform, CIC bioGUNE, CIBERehd, ProteoRed-ISCIII, Bizkaia Science and Technology Park, 48160 Derio, Spain.
authors.forename: Felix
authors.initials: F
authors.lastname: Elortza
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000494:pharmacology; D006801:Humans; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000494:pharmacology; D020543:Proteome; D013666:Tears / Q000737:chemistry*
chemical_list: D000900:Anti-Bacterial Agents; D010455:Peptides; D020543:Proteome
keywords: CID; ETD; HCD; antimicrobial peptides (AMP); degradome; natural peptides; peptidome; tear; top-down proteomics