Fragmentation efficiency

It is often neglected that the first step of de novo sequencing is data acquisition. The quality of the spectrum or spectra used for sequencing is the most critical parameter of the entire procedure. First of all, the mass spectrometer should be well calibrated and tuned. If it can operate in different modes, the one with the highest possible mass accuracy and resolution should be applied. If the experimenter has more spectrometers to choose from, the one with the highest mass accuracy and resolution should be used, provided it shows good fragmentation efficiency. 

Such modifications can be very helpful when the fragmentation efficiency of a peptide is poor or the number of sequence-specific peptides is limited. 

Dongre A.R., Jones J.L., Somogyi A., and Wysocki G.H. (1996), Influence of peptide composition, gas-phase basicity, and chemical modification on fragmentation efficiency evidence for the mobile proton model, J. Am. Soc. Mass Spectrom. 118, 8465-9374. 

Abbreviations BMT-Ballistic Mortar Test BPT-Ballistic Pendulum Test CCCT-Copper Cylinder Compression Test FET-Fragmentation Efficiency Test FGT-Fragment Gun Test LBCT-L e ad Block Compression Test PCT-PIate Cutting Test PDT-Plate Denting Test ST-Sand Test and TT-Trauzl Test (Lead Block Expansion Test)... 

ECD may also be used in a complementary manner with the newly developed EDD technique. Although the fragmentation efficiency of EDD (average 3.6%) was low compared with ECD (average 15.7%), Kjeldsen etal416 have recently demonstrated that the combination of the two techniques could increase the overall amino acid sequence coverage of proteins and PTM characterization. 

Venhorst J, Nunez S, Kruse CG (2010) Design of a high fragment efficiency library by molecular graph theory. ACS Med Chem Lett 1 499-503... 

As described above, ECD of large polypeptides and proteins (> 20 kDa) is characterized by low fragmentation efficiency. Abundant peaks corresponding to charge-reduced species but few backbone product ions are observed. Electron capture cleaves... 

Figure 8.8 Enhanced ion charging of peptic peptides during HX-ETD analysis. MS spectra of microglobulin peptide 1-9 (IQRTPKIQV) (left) and EED spectra (right). HX-ETD was performed in the absence (top) and in the presence of 0.05% m-NBA (bottom). A shift from the doubly to the triply charged precursor Is apparent resulting in enhanced fragmentation efficiency of the triply charged ion. Reproduced with permission from [47] 2009 American Chemical Society. (See Insert for color representation of the figure.)...

An alternative strategy to increase the ETD fragmentation efficiency is the use of a supplemental collisional activation of intact electron-transfer product species either during or after ETD [60, 61]. Mild subsequent activation of nondissociated ET product species has been shown to increase the ion signal two- to threefold and importantly without inducing H/D scrambling in the selectively deuterium-labeled model peptide PI [54] and in peptic peptides of Factor Vila (FVIIa) [57]. 

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