Novo Sequencing of Peptides

MATTHEW T. OLSON, JONATHAN A. EPSTEIN, and ALFRED L. YERGEY  

National Institute of Child Health and Human Development, NIH, Bethesda, MD, USA 

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. 

The local approaches tend to be somewhat less computationally intensive in that they filter the spectral data in some fashion prior to any evaluation of candidate sequences. The various local approaches [15,16,5,10,17] then employ 

Perkins, D.N., Pappin, D.J., Creasy, D.M. and Cottrell, J.S., Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis, 20, 3551-3567 (1999). 

---

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

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. 

On the basis of the complementarity of CID/IRMPD and ECD/ETD, Zubarev et alul have concluded that de novo sequencing of peptides using these two fragmentation techniques in conjunction with high mass accuracy113,114 can be achieved with >95% reliability. They have furthermore stated that it is... 

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. 

Because of this sequestration of the proton to the basic sites in the peptides, MALDI often gives rise to poor MS/MS fragmentation efficiency of peptide ions. Therefore, the applicability of MALDl-MS/MS for the de-novo sequencing of peptides was initially limited. This kindled interest in the development of alternative... 

Yergey AL, Coorssen JR, Backlund PS, Blank PS, Humphrey GA, Zimmerberg J, Campbell JM, Vestal ML. De novo sequencing of peptides using MALDI/TOF-TOF. J Am Soc Mass Spec-trom. 2002 13(7) 784-91. 

---