Project description:Comparative sequence analyses of genome and transcriptome reveal novel transcripts and variants in the Asian elephant Elephas maximus
Project description:We used morphological, ancient DNA and high throughput ancient proteomic analyses to demonstrate that a widely-discussed syntype specimen of E. maximus, a complete foetus preserved in ethanol, is actually an African elephant, genus Loxodonta. Nano-liquid chromatography (nanoLC) coupled with high resolution tandem mass spectrometry (MS/MS) was used to generate a spectra dataset from tryptic peptides and identify, as genus-diagnostic markers, the non-identical homologous ones present in publicly available protein lists for both the two elephant genera. A small, approximately 5×5×3 mm, oesophagus fragment and the terminal portion of a cartilaginous rib of similar size, of 64 and 39 mg respectively (wet weight), were removed from the well preserved, near complete, body of an elephant foetus in a spirit jar, held today at the Swedish Museum of Natural History (NRM) in Stockholm and referred to by Linnaeus (1764) in his description of the Swedish King Adolf Fredrik’s collection. Tryptic peptides were generated using a filter-aided sample preparation (FASP) protocol (Wisniewski et al., 2009). The LC-MS system consisted of an EASY-nLC™system (Thermo Scientific, Odense, Denmark) connected to the Q Exactive (Thermo Scientific, Bremen, Germany) through a nano electrospray ion source. Five uL of each peptide sample were auto-sampled onto and directly separated in a 15 cm analytical column (75 um inner diameter) in-house packed with 3 μm C18 beads (Reprosil-AQ Pur, Dr. Maisch). The settings used were as described for the ‘sensitive’ acquisition by Kelstrup et al. (JPR 11: 3487-3497). Raw files of the two processed samples, generated during spectra acquisition, were merged and searched on a workstation using the MaxQuant algorithm v. 1.2.2.5 and the Andromeda peptide search engine. Trypsin was selected as the proteolytic enzyme and two missed cleavages were allowed. Oxidation (Met and Pro), deamidation (Asn and Gln), acetylation (K), Gln->pyro-Glu (N-term Q), and Glu->pyro-Glu (N-term E) were selected as variable amino-acid modifications. Carbamidomethylation was selected as fixed modification. Default values were used for precursor and fragment ions mass tolerance. False discovery rate was set at 1%, fragment score cut-off was set at 60 and minimum peptide length was set to 6 amino acids.
