Trifluoroacetyl permethyl derivatives

A novel mass spectrometric method has been described that permits the identification of the C-terminal peptide of a protein (68). The technique involves the incorporation of into all a-C02H groups released during enzyme-catalysed partial hydrolysis e. g. with trypsin) of the protein in 0-enriched water ( 0 0 50 50). Individual peptides were analyzed by GC/EIMS as their trifluoroacetyl permethyl derivatives and the C-terminal peptide was characterized as the one that did not incorporate (The non-... 

Several factors affect the volatility and stability of a peptide derivative, not least of these being the number and nature of the constituent amino acids. Heterocyclic and aromatic amino acids reduce volatility while those containing sulphur tend to decrease the thermal stability. Small naturally occurring peptides which are not derived from proteins often contain only aliphatic amino acids which lack functional groups in the side chains. Peptides of this type of up to about ten amino acids, after conversion to suitable derivatives, are amenable to analysis by mass spectrometry, e.g. [164]. A variety of derivatives has been reported and include N-trifluoroacetyl peptide esters [136,165], N-acetyl peptide esters [166-168], aromatic N-acyl peptide esters [169-172], and per-methylated N-acyl peptides [173]. The principal modes of the electron impact induced fragmentation of these peptide derivatives are well established and have been summarised in recent reviews [174,175]. Although the spectra of the permethylated derivatives [176] are perhaps the simplest and easiest to interpret and are now frequently used, the N-acyl peptide esters have been widely and successfully employed. 

The approach has been employed to determine the amino acid sequence in E, coli alanine t-RNA synthetase. After partial hydrolysis, the peptide mixtures were converted to their methyl esters, N-trifluoroacetylated, reduced with B2D6 and converted to their O-TMS derivatives (9). These compounds possess good GC properties and yield simple, yet informative fragmentations. A variety of derivatives have been explored for sensitive sequence analysis of small peptides and a new permethylation procedure has been described for trifluoroacetylated peptides at the 2 —10 nmol level. Mixtures of these derivatives, ideally of di- to hexa-peptides are readily analyzed by GC/MS 66). 

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