title: Native top-down electrospray ionization-mass spectrometry of 158 kDa protein complex by high-resolution Fourier transform ion cyclotron resonance mass spectrometry.
abstract: Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) delivers high resolving power, mass measurement accuracy, and the capabilities for unambiguously sequencing by a top-down MS approach. Here, we report isotopic resolution of a 158 kDa protein complex, tetrameric aldolase with an average absolute deviation of 0.36 ppm and an average resolving power of ~520 000 at m/z 6033 for the 26+ charge state in magnitude mode. Phase correction further improves the resolving power and average absolute deviation by 1.3-fold. Furthermore, native top-down electron capture dissociation (ECD) enables the sequencing of 168 C-terminal amino acid (AA) residues out of 463 total AAs. Combining the data from top-down MS of native and denatured aldolase complexes, a total of 56% of the total backbone bonds were cleaved. The observation of complementary product ion pairs confirms the correctness of the sequence and also the accuracy of the mass fitting of the isotopic distribution of the aldolase tetramer. Top-down MS of the native protein provides complementary sequence information to top-down ECD and collisonally activated dissociation (CAD) MS of the denatured protein. Moreover, native top-down ECD of aldolase tetramer reveals that ECD fragmentation is not limited only to the flexible regions of protein complexes and that regions located on the surface topology are prone to ECD cleavage.
authors: Department of Biological Chemistry, David Geffen School of Medicine and ‡Department of Chemistry and Biochemistry, University of California , Los Angeles, California, 90095, United States.
authors: Huilin
authors: H
authors: Li
authors: Jeremy J
authors: JJ
authors: Wolff
authors: Steve L
authors: SL
authors: Van Orden
authors: Joseph A
authors: JA
authors: Loo
mesh_terms: D017357:Cyclotrons; D005583:Fourier Analysis; D013058:Mass Spectrometry / Q000379:methods; D046912:Multiprotein Complexes / Q000032:analysis / Q000737:chemistry*; D017433:Protein Structure, Secondary; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D046912:Multiprotein Complexes