Protein ion fragmentation

In recent years, a novel approach to protein identification emerged, called top-down sequencing. Here the entire nondigested protein is analyzed. Apart from accurate MW measurement, the protein ion is fragmented by the electron capture dissociation (ECD) method (see Chapter 3). This provides in-depth information on the sequence of protein. Such analysis can be performed only with FTICR instruments (see Section 2.2.6) that ensure high resolution and accuracy but, at the same time, they are exceptionally expensive. However, as very large ions are analyzed, even the high accuracy of FTICR is sometimes not sufficient, and it is recommended that such analyses are accompanied by more traditional bottom-up approaches. 

Kaltashov I.A., Eyles S.J. Crossing the phase boundary to study protein dynamics and function combination of amide hydrogen exchange in solution and ion fragmentation in the gas phase. J. Mass Spectrom. 2002, 37, 557-565. 

The protein identification or sequence determination of a protein can be achieved using two different approaches top-down [22, 23] and bottom-up [24], A top-down experiment involves high-resolution measurement of an intact molecular weight and direct fragmentation of protein ions by tandem mass spectrometry (MS/MS) [25], This approach surveys an entire protein sequence with 100% coverage. Post-translational modifications such as glyco-... 

Smith et al. [26-28] demonstrated that by a further increase of the nozzle-skimmer potential difference the internal energy of the ions can be increased and fragmentation of the multiple-charge protein ions can be induced as a result of colhsion-induced dissociation (CID). This is called in-source CID in this text. 

Initial results with the fragmentation of multiple-eharge protein ions in FT-ICR-MS were achieved, either by in-source CID [159], sustained off-resonance irradiation (SORI) [160], or infrared multiphoton dissoeiation (IRMPD) [161], and later on with electron-capture dissociation [109-110]. 

FT-ICR-MS enables the identification of unknown proteins with hmited enzymatic cleavage and eventually in-cell fragmentation via a variety of ion-fragmentation techniques and high-resolution m/z determination of the fragment ions. This is the top-down protein identification approach [163] (Ch. 18.3.5). The introduction of ECD was an important development in this respecf as it allows incell fragmentation of the complete protein [110]. 

To this end, different ion fragmentation tools have been characterized with respect to phosphopeptide fragmentation, e.g., electron-capture dissociation (ECD) [31] and infrared multiphoton dissociation (IRMPD) [32]. An application of ECD in PTM analysis is the top-down protein characterization (Ch. 18.3.5) of carbonic anhydrase [33]. IRMPD is applied in the study on protein kinase C phosphorylation [30]. Both ECD and IRMPD were applied in a subsequent nano-ESI-FT-ICR-MS study on protein kinase A phosphorylation [34]. Combined ECD and IRMPD for multistage MS-MS in FT-ICR-MS was applied for phosphopeptide characterization [35]. ECD provides backbone cleavages (c- and z -ions) without H3PO4 loss, whereas in IRMPD the loss of H3PO4 is prominent and only a few backbone cleavages (b- andy-ions) are observed cf. Ch. 17.6.1). 

The results for amino acids show that the main pyrolysis products are similar to the ion fragmentation that takes place upon electron impact. For the case of peptides and proteins, most of the effort has been done in obtaining ions of large fragments in order to account as much as possible for the protein structure [24]. Several mass spectra of substituted diketopiperazines obtained from peptide pyrolysis are shown in Figures 3.6.6 a to 3.6.6 f. The fragments generated in their mass spectra are similar to some small molecules obtained in amino acid pyrolysis. 

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