Characterization of Cytochrome c Variants with High-Resolution FTICR Mass Spectrometry: Correlation of Fragmentation and Structure

Abstract

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.

Activation of large lons in FT‐ICR mass spectrometry

“…Wu et al (1995) studied dissociation of the þ15 charge state of cytochrome c ions from different sources. SORI-CID results in numerous backbone cleavages of the proteins.…”

“…In this review, we will discuss only selected studies that demonstrate the utility of SORI-CID for examining structures and relative stabilities of proteins and protein-ligand complexes in the gas phase Wu et al, 1995;Liu et al, 1996;Wu et al, 1997;Gao et al, 1999). Fragmentation patterns obtained using SORI-CID are very sensitive to small changes in the primary structure of peptides and proteins.…”

“…Fragmentation patterns obtained using SORI-CID are very sensitive to small changes in the primary structure of peptides and proteins. For example, different fragmentation spectra were reported for the þ15 charge state of cytochrome c variants as shown in Figure 14 (Wu et al, 1995). Replacing three out of 104 residues of the cytochrome c dramatically changes the dissociation pattern.…”

Dissociation of different conformations of ubiquitin ions

“…In addition to chemical probes of gas phase ion structure [1][2][3][4][5][6][7][8][9][10][11][12][13][14], a number physical methods are being developed to examine gas-phase peptide and protein ion structure [15][16][17][18][19][20][21][22][23][24][25][26][27], including collisional activation to examine fragmentation pathways [18 -23, 25-27]. A few studies have suggested that ion conformation may influence fragmentation pathways [19,23,27,28]. In this paper, we examine this issue for the fragmentation of a series of compact and elongated conformations of ubiquitin ions.…”

The pH Dependence of Product Ion Spectra Obtained from Precursor Ions with the Same Charge Number in ESI of Carbonic Anhydrase 2

“…To date, tandem mass spectrometry (MS/MS) of proteins, has been reported using several dissociation modes. [12][13][14][15][16][17][18] In the case of CA2, MS/MS analyses have been performed by Fourier transform ion cyclotron resonance mass spectrometry. 13,14) The product ions obtained in this study are consistent with these reports, and they were assigned accordingly.…”