Top-down sequencing

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. 

Top-down sequence analysis of whole glycoprotein ions using CID and ion/ion proton transfer in a quadrupole ion trap MS [66], This approach eliminated gas-phase deglycosylation of N-linked oligosaccharide in ribonuclease B, and the glycosylation site was identified to be Asn-Leu-Thr at residues 34-36 [66],... 

More recently, it has been demonstrated that the reagent anions used for either ETD or proton transfer can be derived from the same neutral compound. The radical anions used for ETD, [M]- , are converted into even-electron proton transfer reagent anions. [M + H]-, by changing the potential on the methane Cl source.1 9 This voltage switch can be acheived in milliseconds allowing for rapid sequential ion—ion reactions and opens up the possibility of top-down sequencing of intact proteins in RF ion traps. 

Top-down sequence analysis of the intact protein via ion-ion reactions 484... 

A top-down sequence analysis approach has been developed in which sequence information on intact glycoproteins is obtained.121 First, the charge state of the ES-ionized protein is optimized through ion-ion reactions with [M - F and [M - CF3] anions. The CID spectrum of that precursor ion is well endowed with abundant sequence-specific ions, which are used to identify glycosylation sites. The top-down approach has the advantage that additional time- and sample-consuming purification and proteolytic cleavage experiments can be avoided. 

In cases where the predicted mass calculated from the amino acid sequence does not match the observed mass, top-down sequencing is used to determine sites of posttranslational modifications as seen in Fig. 5. 

Top-down sequencing of the denatured and reduced protein was analyzed by direct infusion using a Exactive Hybrid Quadrupole-Orbitrap mass spectrometer with a resolving power of 70,000. 

Top-down sequencing used ProSightLite to determine the pyroglutamate on the N-terminus. 

Top-Down Sequence Analysis of the Whole Protein via Ion-Ion Reactions In favorable cases, multiply charged ESI-produced ions of an intact glycoprotein can be subjected to CID in a quadrupole ion trap to identify the glycosylation sites [86]. The charge state of the protein ions can be manipulated by an ion-ion reaction with [M - F ] and [M - CFs ] ions derived from perfluoro-l,3-dimethylcyclohexane. The charge reduction of the CID product ions by ion-ion reactions also simplifies the MS/MS spectrnm and facilitates... 

All the synthesis steps mentioned above can be executed either automatically or interactively. The programmed ASIC emulator can be inserted directly into the overall mechatronic system to facilitate real-time test runs. In the following, all main synthesis steps will be described in the same top-down sequence as they are implemented through the synthesis script. Throughout the rest of the chapter, the terms ASIC emulator (board), rapid prototyping board, and target architecture will be used synonymously. 

---