Peptide ion fragmentation

Until several years ago, gas-phase fragmentation was viewed exclusively as a working tool of the top-down HX-MS measurements, even though sevraal attempts had been made ovct a decade ago to incorporate peptide ion fragmentation in the workflow of the hottom-up HX-MS measurements as well [34, 35]. However, earlier attempts to supplement proteolysis in solution with fragmentation in... 

TOF mode and reflector TOF mode for the observation of labile modifications [45] (differences between linear and reflectron TOFs in the detection of metastable ions are detailed in Chapter 2). It may also be useful to try negative- and positive-ion modes, as certain species ionize more efficiently in the negative-ion mode [46]. An alternative to the MALDl-MS/MS analysis of modified peptides is electron-transfer dissociation (ETD)-based ESl-MS/MS, where the modification is retained during peptide ion fragmentation, making the assignment of the modification site more straightforward. 

Cui, W., Thompson, M. S., Reilly, ]. P. (2005). Pathways of peptide ion fragmentation induced by vacuum ultraviolet light. Journal of The American Society for Mass Spectrometry, 15(8), 1384-1398. 

Figure 2.3. A. Mass spectrometer consisting of an ionization source, a mass analyzer and an ion detector. The mass analyzer shown is a time-of -flight (TOF) mass spectrometer. Mass-to-charge (m/z) ratios are determined hy measuring the amount of time it takes an ion to reach the detector. B. Tandem mass spectrometer consisting of an ion source, a first mass analyzer, a collision cell, a second mass analyzer and a detector. The first mass analyzer is used to choose a particular peptide ion to send to the collision cell where the peptide is fragmented. The mass of the spectrum of fragments is determined in the second mass analyzer and is diagnostic of the amino acid sequence of the peptide. Figure adapted from Yates III (2000).

Figure 2.5. Tandem mass spectrometry. A. A peptide mixture is electrosprayed into the mass spectrometer. Individual peptides from the mixture are isolated (circled peptide) and fragmented. B. The fragments from the peptide are mass analyzed to obtain sequence information. The fragments obtained are derived from the N or C terminus of the peptide and are designated "b" or "y" ions, respectively. The spectrum shown indicates peptides that differ in size by the amino acids shown.

Brown, R. S. Lennon, J. J. Sequence-specific fragmentation of matrix-assisted laser-desorbed protein/peptide ions. Anal. Chem. 1995,67,3990-3999. 

Figure 6.5. Nomenclature of peptide ions resulting from backbone fragmentation.

Fig. 5. Fragmentation nomenclature of peptides. Bond breakages of all bonds of the peptide backbone have a systematic name (I). When fragmenting multiply charged peptide ions the peptide bond breaks preferentially since it is among the most labile bonds and only relatively low collision energies are involved (II).

Continuous ion series are often generated when multiply charged peptide ions are fragmented. The problem in de novo sequencing with electrospray tandem mass spectrometry lies in minimizing the error rate of the interpretation. There are two different approaches to this problem ... 

It would be complicating if not impossible having to obtain sequence information from such a spectrum without rules to follow. [138-141,146,147] The most abundant ions obtained from the fragmenting peptide ion usually belong to six series named a, b, and c if the proton (charge) is kept in the A-terminus or x, y, and z, respectively, where the proton is located in the C-terminal part. Within each se-... 

Example The reduced sample consumption of nanoESI allows for the sequencing of the peptides (Chap. 9.4.7) obtained by tryptic digestion of only 800 fmol of the protein bovine semm albumin (BSA, Fig. 11.6). [66] The experiment depicted below requires each of the BSA-derived peptide ions in the full scan spectrum to be subjected to fragment ion analysis by means of CID-MS/MS on a triple quadrupole instmment (Chaps. 2.12 and 4.4.5). 

Fig. 11.6. Peptide sequencing by nanoESI-CID-MS/MS from a tryptic digest of bovine serum albumin (BSA) 800 fmol of BSA were used, (a) Eull scan spectrum, (b) fragmentation of the selected doubly charged peptide ion at m/z 740.5. Adapted from Ref. [66] by permission. Nature Publishing Group, 1996.

Scheme 1 The Expected Fragmentation Pattern of Protonated Peptide Ions and the Nomenclature of the Amino Acid Sequence Fragment Ions for Determining Amino Acid Sequences...

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