Complete sequence determination of hemoglobin from endangered feline species using a combined ESI-MS and X-ray crystallography approach

We present the first complete amino acid sequences of Snow leopard (Uncia uncia) and Amur tiger (Panthera tigris altaica) hemoglobin (Hb) determined using a combined ESI MS and X-ray crystallography approach. Using mass spectrometry alone, we were able to achieve 81%, 90%, 87% and 72% sequence coverage for the α-, and β-globins of Snow leopard Hb and the α-, and β-chain of Amur tiger Hb, respectively. The incomplete mass spectrometry-determined sequences were used to process X-ray diffraction data of Snow leopard and Amur tiger Hb crystals solved to 2.1 Å and 1.8 Å, respectively. Using tandem mass spectrometry in concert with the electron density maps from X-ray structures, we were able to not only achieve 100% coverage of all amino acid sequences, but to also distinguish between isomeric (leucine and isoleucine) and isobaric (glutamine and lysine) residues. These results demonstrate the synergy between mass spectrometry and X-ray crystallography for the determination of full amino acid sequence coverage of abundant proteins.

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► Previously unknown sequences for Snow leopard and Amur tiger hemoglobin are presented.
► 100% sequence coverage was obtained through a combination of ESI MS and X-ray crystallography.
► The manuscript illustrates the synergy between these 2 complementary techniques.
► The incomplete sequence obtained from MS was used to aid in the solution of the crystal structures.
► Electron density maps allowed the differentiation of isomeric residues and completion of sequence.