Fragmentation peptide derivatization

Figure 3.7 Derivatization of peptides by sulfonic acid reagents such as sulfonophenylisothiocyanate (SPITC) enhances peptide fragmentation efficiency in MS/MS. MALDI-TOF-TOF MS/MS of a singly protonated peptide NYELPDQGVITIGAER revealed only few fragment ions (top panel). In contrast, the same peptide derivatized by SPITC generated a strong y-ion series that allowed near-complete interpretation of the...

The versatility of the reagent system in the assay of small peptides is nicely illustrated in Figures 3 through 5. Neurotensin, a polypeptide composed of 13 amino acids, and three fragments from partial hydrolysis were derivatized with NDA/CN and separated by... 

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. 

Most HPLC instruments monitor sample elution via ultraviolet (UV) light absorption, so the technique is most useful for molecules that absorb UV. Pure amino acids generally do not absorb UV therefore, they normally must be chemically derivatized (structurally altered) before HPLC analysis is possible. The need to derivatize increases the complexity of the methods. Examples of derivatizing agents include o-phthaldehyde, dansyl chloride, and phenylisothiocyanate. Peptides, proteins, amino acids cleaved from polypeptide chains, nucleotides, and nucleic acid fragments all absorb UV, so derivatization is not required for these molecules. 

Amino acid derivatives and symmetric derivatization of (3-cyclodextrin that allows the selective replacement of all the primary hydroxy groups are predominant in literature but compared to a- and y-cyclodextrin, (3-cyclodextrin exhibits a rather low water solubility.174 In view of the methodology of chemoselective ligation, which allows the condensation of completely unprotected peptide fragments in aqueous buffer solution, symmetric derivatization of a- and y-cyclodextrin should be favorable. 28 ... 

The advent of FAB mass spectrometry has allowed the routine molecular weight determination of polar molecules, without derivatization, up to ca 3,000 Daltons, and in exceptional cases, within 1 mass unit to the region of 8,000 Daltons. This advance, coupled with FAB fragmentation, and enzymic digestion techniques, has allowed the rapid solution of a number of problems in protein and peptide chemistry - problems which were hitherto rather difficult to solve. Examples are given. 

Interpretation of the fragmentation spectra can be difficult and time consuming. It can be simplified if the peptide is modified in such a way that one type of fragmentation is favoured. This can be achieved by derivatizing the amino terminal group with N-succinimidyl-2-(3-pyridyl) acetate, which promotes b fragments [59], Modification of the peptide also can make the distinction between C- and N-terminal fragments easier. 

We have implemented scanning methodologies using MALDI-TOF mass spectrometry to partially purified venom from C. striatus and C. ermineus. We have carried out specific derivatizations in order to deduce composition and sequence information. Together with an intact mass these measurements are used to determine whether an ionized species observed in the MALDI mass spectrum corresponds with the intact protonated molecule of a previously characterized conotoxin. The information obtained from derivatizations is also important when the ionized species does not correspond with the intact mass of peptides of known sequence. In that case, post source decay of the native and derivatized species may help assign the fragment ions. 

These derivatizations are highly selective, and may thus allow PSD measurements to be carried out on peptides after modification. Such a protocol would significantly enhance our ability to derive sequence information from PSD spectra, because the mass shifts observed in fragments help locate the particular residue within the peptide, and also confirm assignments of fragments as arising from N- or C-terminal regions. In addition to derivatizations that may modify the C- and N-termini and the derivatization of tyrosine residues, we have carried out oxidation of methionine residues with sufficient specificity to enable measurement of PSD spectra. 

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