Specific ion sequences

There is strong evidence in favour of the point of view 1. in the variants colloid anion + micro cation or colloid cation -f micro anion in the specific ion sequences for reversal of charge or for coacervation or flocculation. We remind the reader for example of the sequences Cs < Rb < K < Na < Li which occur with sulphate colloids and carboxyl colloids (p. 289). Here polarisation phenomena are still in the background and here the largest ion, that is to say, the least hydrated ion, is most suitable for reversal of charge or coacervation. This points strongly therefore to a direct contact between cation and ionised group of the colloid. 

In the suppression of the tricomplex flocculation, gelatin + carrageen + Ca, the same specific ion sequence of the monovalent cations and amons was also found. For the interpretation we must go through the same arguments again bearing in mind that the two complex relations are now ... 

The molecular ions of alkenes have a prevalence for rapid randomisation of their constituent hydrogen and carbon atoms. Specific reaction sequences by which the atoms are randomised can be identified through careful consideration of the kinetics of decompositions at the shortest times (< lOOps) and many FIK studies of alkenes have been made with this objective in mind [223]. 

The DLVO theoiy thus enabled theoretical significance to be given to the valence sequence in coagulation experiments that bad been observed many years earher by Schulze (1882) and Hardy (1900). Although expre ions of this type are useful in a qualitative predictive sense, the implication that there is a simple rule applicable to alt systems must be treated with considerable caution. It must be borne in mind that coagulation is a complicated phenomenon involving quite a range of kinetic and specific ion effects. 

Excellent serrsitivity, as irrdicated by the detection of ca. 7 amol of carbotric airhydrase in a erode extract from htrman blood [79]. Dissociation of 9 amol introduced via CE provided sequence specific ions, enabling identification from a protein database. The 42-Da mass difference observed was attributed to acetylation of the carbotric anhydrase. 

One of the early interesting findings came from the work of Schulten on two isomeric dinucleotides CpA and ApC, using FD-MS. He observed completely different spectra for these two isomeric compounds in terms of the presence of specific ions and the relative intensities of common ions. Generally, unambiguous identification of the sequence of any nucleotide is not possible, exception made of GpU (weak G-origin peaks) and the above mentioned pair of nucleotides, unless additional information is available. 

A general outline of the bottom-up mass spectrometry approach for proteome analysis is presented in Figure 3. In general, the mass spectrometry is performed at the peptide level after digesting the protein to obtain the molecular mass and amino acid sequence-specific ions, which are correlated with similar information in the protein or nucleotide database.7 16 Based upon these measurements, the following approaches have evolved. 

Electron-transfer dissociation (ETD) is a variation of ECD, in which an ion-ion reaction, for example, between an anthracene anion and a multiprotonated peptide cation, is used to transfer an electron to the peptide ion.64,78 Similar to ECD, subsequent fragmentation of the odd-electron ions produces c- and z -type sequence-specific ions. ETD is also better suited for investigating phosphorylation and glycosylation. 

One proposal to increase the visibility of the b and y sequence-specific ions and to provide complete sequence information is to derivatize phosphopeptides.106 Sequence analysis can also be improved by using the /3-elimination chemistry.102,107 As described above in this section, phosphoserine and phosphothreonine can be converted to S-ethylcysteine and 5-ethyl-/3-methylcysteine, respectively, upon the base-catalyzed /3-elimination of the phosphate group, followed by the reaction with ethanethiol. CID of the modified peptides results in more evenly distributed sequence-specific fragment ions. 

As mentioned earlier in the chapter, ECD and ETD both are much more useful for identifying the location of a phosphate group in peptides because the unwelcome loss of the phosphate group from the precursor ion is suppressed, and the abundance of sequence-specific ions is commensurately enhanced.108-111 Also, the sequence of the peptide has little influence on fragmentation of the peptide ion, which is solely driven by free-radical chemistry to produce c- and z -type sequence-specific ions. 

As mentioned earlier, ECD is ideally suited for the analysis of the glycosylated sites in peptides this mild activation process does not require the removal of the carbohydrate chains. In the mass spectrum of intact glycopeptides, the carbohydrate chains remain attached to the sequence-specific ions, thereby providing direct evidence of the glycosylation sites.76... 

A top-down sequence analysis approach has been developed in which sequence information on intact glycoproteins is obtained.121 First, the charge state of the ES-ionized protein is optimized through ion-ion reactions with [M - F and [M - CF3] anions. The CID spectrum of that precursor ion is well endowed with abundant sequence-specific ions, which are used to identify glycosylation sites. The top-down approach has the advantage that additional time- and sample-consuming purification and proteolytic cleavage experiments can be avoided. 

Tandem mass spectrometry for the residue-level exchange information-. Details of individual amino acid residue-level structures can be explored by acquiring the CID-MS/MS spectrum of the peptide segments formed by pepsin digestion of the exchanged protein. As mentioned earlier in this chapter, the b- and y-ion series are the primary sequence-specific ions under CID conditions. The amide hydrogen of a particular residue that is involved in HX can be identified by an increase in the mass of the sequence ion containing that residue. 

---