Ion sequences

Besides sensitive methods for the analysis of proteins, bioinformatics is one of the key components of proteome research. This includes software to monitor and quantify the separation of complex samples, e.g., to analyze 2DE images. Web-based database search engines are available to compare experimentally measured peptide masses or sequence ions of protein digests with theoretical values of peptides derived from protein sequences. Websites for database searching with mass spectrometric data may be found at http //www.expasy.ch/tools, http //prospector.ucsf. edu/ and http //www.matrixscience.com. 

It should be pointed out that FAB, MALDI, and ESI can be used to provide ions for peptide mass maps or for microsequencing and that any kind of ion analyzer can support searches based only on molecular masses. Fragment or sequence ions are provided by instruments that can both select precursor ions and record their fragmentation. Such mass spectrometers include ion traps, Fourier transform ion cyclotron resonance, tandem quadrupole, tandem magnetic sector, several configurations of time-of-flight (TOF) analyzers, and hybrid systems such as quadrupole-TOF and ion trap-TOF analyzers. 

Both collisional activation (in ion traps) and post source decay (in curved field reflectron TOF analyzers) have been used successfully to obtain sequence ions from peptides prepared in situ on the sample holder. Single Dalton mass windows are advantageous for precursor selection, as are realized in ion-trap and trap-TOF configurations. Publicly available search algorithms can be used... 

P. Roepstorff and J. Fohlman, Proposal for a Common Nomenclature for Sequence Ions in Mass Spectra of Peptides. Biomed. Mass Spectrom., 11(1984) 601. 

Roepstorff P. and Fohlman J. (1984), Proposal for a common nomenclature of sequence ions in mass spectra of peptides, Biomed. Mass Spectrom. 11, 601. 

A sample size of approximately 0.1 nanomoles is generally sufficient for molecular weight determination in either the positive or negative ion mode, but does not normally allow the sequence of amino acids to be determined. Larger sample sizes, typically between 1 and 5 nanomoles, afford some sequence information. Sequence ions are observed in the positive and negative ion modes from both N- and C-termini of the peptide and this may enable the complete sequence of the peptide to be determined. 

The abundance of sequence ions is in part dependent on sample size but this is clearly not the only factor since large sample sizes ( 50 nanomoles) often fail to provide extensive sequence information for some peptides. Sequence information is frequently not complete and the absence of either C or N terminal sequence ions does not allow the complete sequence determination. 

The assignments of sequence ions observed in the FAB mass spectrum of CB-l and its methyl ester are given in Table I, and suggested that the sequence of CB-l was ... 

Assignment of the sequence ions in the FAB mass sectra of decapeptide... 

Most of the sequence ions observed in the positive ion (+) mode are cationised by NA+. The base peaks in the positive ion FAB mass spectra of peptides that lack basic functional groups as in CB-1 often correspond to the M of peptides cationised by the formation of adducts with Na+ and/or K+, traces of which are usually present in the matrix. 

Sequence ions observed in the negative ion FAB mass spectra of the above peptides are as follows ... 

B. Ernst, D. R. Muller, and W. J. Richter, False sugar sequence ions in electrospray tandem mass spectrometry of underivatized sialyl-Lewis-type oligosaccharides, Int. J. Mass Spectrom. Ion Process., 160 (1997) 283-290. 

In the early LC-MS-MS studies, CID is performed with low-energy collisions by means of triple-quadrapole instruments. Under these conditions, series of N-terminal b-ions and C-terminal y-ions result from cleavages at the peptide bond and charge retention on either side, although double-charge tryptic peptide ions tend to favour fragmentation towards more abimdant y-ions. From these ladders of sequence ions in the MS-MS spectrum, the amino-acid sequence of the peptide may be derived. This is the bottom-up protein identification approach (Ch. 18.3.1). 

De novo sequencing seldom leads to a complete sequence becanse in most cases the y- and b-ion series are not completely present, some sequence ions might be lost in the noise, and/or ambiguities exist due to additional peaks (neutral losses of CO, HjO, NH3, etc.), and isobaric and isomeric amino acids (Table 17.2). 

A complementary approach to protein identification is based on peptide seqnencing analysis (PSA) by collision-induced dissociation (CID) in MS-MS. Por proteomics studies, the de novo sequencing of MS-MS spectra is too time-consuming. Therefore, algorithms have been developed to either provide antomated MS-MS spectmm interpretation, or perform protein identification by means of a SEQUEST database search [6-7]. In the latter case, the precursor m/z values are matched against a virtual digestion of all the proteins in the database. Sequence ions are predicted for the peptides that match the precnrsor m/z values, and compared with the measured MS-MS data. A correlation score is calculated for each match. 

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