Partial characterization of a novel amphibian hemoglobin as a model for graduate student investigation on peptide chemistry, mass spectrometry, and atomic force microscopy

Graduate students in chemistry, and in biological and biomedical fields must learn the fundamentals and practices of peptide and protein chemistry as early as possible. A project-oriented approach was conducted by first-year M.Sc and Ph.D students in biological sciences. A blind glass slide containing a cellular smear and an aqueous cellular extract were offered to the students. Qualitative and quantitative cell morphological parameters were analyzed by atomic force microscopy. The fractionation of the aqueous extract was conducted by reversed-phase chromatography followed by analysis of the isolated and partially purified proteins and peptides by mass spectrometry (MS). The proteins were treated by peptidases and the obtained peptide fragments were sequenced by de novo MS/MS, together with peptides already present in the extract. The most abundant protein fractions were identified as the alpha and beta chains of hemoglobin from an amphibian of the Leptodactylus genera. Two of the peptides sequenced by the students were synthesized by the solid-phase methodology, one of those being obtained by the split-and-pool library synthesis method. Thus, the students were able to learn some advanced principles and practices of protein chemistry and bionanotechnology in a 6-weeks project-oriented approach.