Native electrospray and electron-capture dissociation in FTICR mass spectrometry provide top-down sequencing of a protein component in an intact protein assembly.
The intact yeast alcohol dehydrogenase (ADH) tetramer of 147 kDa was introduced into a FTICR mass spectrometer by native electrospray. Electron capture dissociation of the entire 23+ to 27+ charge state distribution produced the expected charge-reduced ions and, more unexpectedly, 39 c-type peptide fragments that identified N-terminus acetylation and the first 55 amino acids. The results are in accord with the crystal structure of yeast ADH, which shows that the C-terminus is buried at the assembly interface, whereas the N-terminus is exposed, allowing ECD to occur. This remarkable observation shows promise that a top-down approach for intact protein assemblies will be effective for characterizing their components, inferring their interfaces, and obtaining both proteomics and structural biology information in one experiment.
Zhang
Hao
H
Department of Chemistry, Washington University, St. Louis, Missouri 63130, USA.
Cui
Weidong
W
Wen
Jianzhong
J
Blankenship
Robert E
RE
Gross
Michael L
ML
D000426:Alcohol Dehydrogenase / Q000737:chemistry; D000595:Amino Acid Sequence; D005583:Fourier Analysis; D005656:Fungal Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D020539:Sequence Analysis, Protein / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D005656:Fungal Proteins; D021122:Protein Subunits; D000426:Alcohol Dehydrogenase