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.
Bibliographical noteFunding Information:
This work was supported by start-up funds to Wendell P. Griffith from the University of Toledo , and by a grant from the deArce Memorial Endowment Fund . U. uncia (Snow leopard) and P. tigris altaica (Amur tiger) packed red blood cells were generously donated by the Toledo Zoo. We acknowledge the NSF MRI Program (Award # 0923184 , PI: WPG) for funding the acquisition of the MALDI-TOF/TOF mass spectrometer. We would also like to thank Dr. Bryant Leif Hanson for assistance with the use of the X-ray crystallography equipment as well as useful advice and discussions. We are grateful to the University of Toledo Arts and Sciences Instrumentation Center, where all X-ray diffraction and MALDI-TOF/TOF-MS data were collected.
Copyright 2012 Elsevier B.V., All rights reserved.
- Amino acid sequence
- Amur tiger hemoglobin
- Mass spectrometry protein sequencing
- Snow leopard hemoglobin
- X-ray crystallography