Peptides fragmentation rules

In the final section of the chapter we discuss procedures to obtain the amino acid sequence of peptides, which is a vital component of protein identification. The fragmentation rules of peptides and guidelines for interpretation of peptide-mass spectrum are also discussed. By following these systematic steps, de novo sequencing of peptides and hence of a protein can be achieved. 

There are two major approaches to de novo sequencing by mass spectrometry. The first one is based on a number of empirical rules obtained by observing typical peptide fragmentation schemes [20]. Current versions of this approach rely on computerized expert systems that are built on the dozens of empirical rules and factors. These include general observations on the prevalence of certain fragments in spectra produced by the used fragmentation methods and in typical instruments. For example, CAD is known to produce predominantly y- and fc-type ions. There... 

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-... 

Figure 9 Illustration of the 1/3rd rule. The top panel shows multiple charge states of the phosphopantetheinylated active site containing peptide from PKS LovB. The 16+ ion at 854.636m/z and the 15+ ion at 911.636mlz of the peptide were subjected to CID. The middle panel shows that PPant ejection (261 m/z) is clearly detected in the MS/MS or MS2 spectrum resulting from CID of the 16+ ion. The bottom panel shows that PPant ejection resulting from fragmentation of the 15+ ion cannot be detected due to limitations defined by the 1/3rd rule.

M + H]+ ions in which protonation occurs at each of the sites required to trigger cleavage of each peptide bond. It is unlikely that the ionization process produces the required population of isomeric [M + H]+ ions, but that such a population arises via CA-induced intramolecular proton transfer prior to fragmentation. This concept has been called the mobile proton model of fragmentation [29] (note that this is essentially a restatement of Bowie rule no. 3). 

QITs are remarkable instruments because spectra can be obtained, ions stored, and sophisticated MS/MS experiments conducted, all in a cost-effective way (with a small footprint). One limitation, applicable to all ion traps, is the so-called one-third rule, which states that in CID MS/MS it is not possible to detect product ions that are less than about one-third of the value of the precursor ion e.g., for a precursor ion of mJz 900 the detection limit for product ions is miz -300. This limit can be frustrating e.g., it is not possible to detect the immonium ions formed from amino acids during CID of a peptide, nor can QIT be used with CID for certain techniques, such as iTRAQ (Section 3.5.19.3). Similarly to quadmpoles, from which they are derived, QITs have limited resolution. Alternative fragmentation induced by rf pulsing provides a type of scan in which the one-third rule does not apply and low mass ions can be observed. 

There are a number of substantial technical issues involved in de novo sequencing that arise from the fact that peptides do not fragment in an ideal manner. One result of this is that skilled spectral interpreters have devised sets of rules that can be used to convert mass differences between ions of a spectrum into amino acid sequences unfortunately, these rules are complicated and are not always followed. In addition, the fragmentations do not occur in a manner that gives rise to uniform ion intensities. This phenomenon results in spectra that have a substantial range of intensities which, depending on the ionization and mass analyzer used. 

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