Characterization of cytochrome c variants with high-resolution FTICR mass spectrometry: correlation of fragmentation and structure.
The dissociation of cytochrome c ions (15+ charge state) generated by electrospray ionization has been studied by Fourier transform ion cyclotron resonance mass spectrometry (FTICR) using a sustained off-resonance irradiation/collision-induced dissociation (SORI-CID) technique. Over 95% of the fragment ions can be accurately assigned (to better than 10 ppm), yielding information on the primary sequences of the various proteins. Up to four stages of mass spectrometry (MS4) have been achieved without the need for quadrupole excitation/collisional cooling of the product ions. The subtle structural differences among the cytochrome c variants (from bovine, tuna, rabbit, and horse) are clearly reflected in their fragmentation patterns: replacing 3 out of 104 residues of the cytochrome c is shown to dramatically change the dissociation pattern. Of particular importance are a variety of results indicating that the dissociation of the cytochrome c's is influenced by higher-order structure and charge location, in addition to the primary structure (i.e., sequence). No fragmentation is observed in the region between residues 10-20 and little dissociation between residues 70-90. This is most likely due to the interactions of the heme group with the polypeptide chain, and such a heme "footprinting" pattern is analogous to the protein conformation in solution. These studies demonstrate that electrospray ionization-FTICR using SORI-CID can be a useful tool to probe not only the small differences in the primary sequences of proteins but also suggest the potential for probing their higher-order structures and yielding information not readily available from H/D exchange or circular dichoism studies.
Wu
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Chemical Sciences Department, Pacific Northwest Laboratory, Richland, Washington 99352, USA.
Van Orden
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Cheng
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Bakhtiar
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R
Smith
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RD
D000595:Amino Acid Sequence; D000818:Animals; D017357:Cyclotrons; D003574:Cytochrome c Group / Q000032:analysis*; D005583:Fourier Analysis; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D007527:Isoenzymes / Q000032:analysis*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis
D003574:Cytochrome c Group; D007527:Isoenzymes; D010446:Peptide Fragments