Novo Sequencing

Most problems in de novo sequencing are caused by the high number of possible bonds that may be cleaved and because not all recorded fragment ions are 

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 

The other approach to de novo sequencing is based on a systematic treatment of tandem mass spectrometric data and database search. An excellent description of these methods is available in Chapter 9 thus, we refrain from the detailed discussion here. 

As the exploration of the human proteome advances from better known proteins to more and more obscure ones, the significance of de novo sequencing as the primary source of information is hkely to grow. Similarly, the identification of spUce variants, mutations, and modifications calls for increasing number of de novo investigations. 

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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. 

If the instrument allows for multistage fragmentation, the MS3 spectra of the most intense MS/MS fragment ions should also be acquired. There is never too much data in a de novo sequencing experiment. Even if the MS2 spectrum provides the complete sequence, data obtained from additional stages can still be used for sequence validation. In case of three-dimensional ion-traps that suffer from low mass cut-off, MS" spectra are indispensable to cover low mass fragments. For further explanation of this phenomenon, please refer to Chapter 3. 

The main principles of de novo sequencing will be discussed here using a few d spectra that were acquired using an ion trap mass spectrometer equipped with an electrospray ion source, under low energy collision induced dissociation (CID). 

Here we encountered a typical situation in the de novo sequencing—there is a part of the sequence that is not covered by any ion series. Not all bonds between amino acids are of equal strength, and some of them might be particularly resistant to collisions, which in turn results in the missing mass-shifts (and missing residues). 

If we include this peak, the complete sequence of the peptide will be H-FGGFTGAR(K/Q)SA. This identification is still not complete and, unfortunately, not the most reliable. This example shows the common situation that de novo sequencing often cannot provide the entire sequence. There are only indirect clues supporting the... 

Example 3. The last example covers the biggest peptide obtained from a tryptic digestion of an unknown protein. Although there are better ways to identify such peptides than de novo sequencing, this example allows us to present some additional hints that can be used in sequencing strategies. 

Enzymatic Digestion. If the peptide to be sequenced is in a pure form or can be isolated and purified, the more classic methods can be used to support de novo sequencing. However, they are unlikely to provide good results when working with mixtures of peptides. Below, several additional techniques are listed. 

The straightforward approach to de novo sequencing sometimes fails, for example, due to the low quality tandem mass spectra. Often it is not caused by the equipment settings or operator s capabilities, but just by unfavorable fragmentation pattern of a given peptide. Among possible approaches to solve such issues is chemical derivatization of peptides. 

The identification of peptides and proteins using MS information can be done in three different fashions (i) de novo sequencing, (ii) library searching, and (iii) sequence collection searching. 

D. De novo Sequencing of Peptides with Mass Spectrometry. 15... 

De novo Sequencing Using MALDI and Postsource Decay Fragmentation... 

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 ... 

Naven, T., Canas, B., Rahman, D., Bardetjones, M., Jeffrey, W., and Pappin, D. (1999). De novo sequencing of peptides by low-energy CID. 47th ASMS Conference, June 13-17, Dallas. 

Smith, 1992). All of these can identify an optimal alignment between the query and either a set of previously studied sequences or a pattern of sequence elements identified as common to a set of previously studied proteins. De novo sequence analysis methods have proved less useful. Although there has been some slow progress in predicting a protein s structure from its sequence, no direct functional predictions methods have been developed. 

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