| Popis: |
A novel approach for the chemical modification of proteins and peptides was developed, namely the chemical modification under vacuum (in vacuo) of lyophilized proteins and peptides. This method was used with iodomethane to produce peptides with a permanent positive charge by converting their amino groups to trimethylammonium derivatives for analysis by matrix assisted laser desorption ionization mass spectrometry (MALDI MS). Peptides with such a permanent charge have a higher ionization yield under MALDI MS resulting in a dramatic increase in detection. The signal intensity of the trimethylammonium derivative in MALDI MS is much greater compared to the unmodified peptide. The trimethylammonium derivative of alpha-amino groups of peptides derived from test lyophilized peptides and a lyophilized protein, and an epsilon-amino group of a peptide derived from the solitary lysine residue of the oxidized B-chain of insulin were shown to have a large increase in signal intensity compared to the unmodified peptides. The in vacuo methylation procedure is readily amenable to the use of a combination of isotopes, e.g. 12CH3I and 13CH3I or CH3I and CD3I, yielding a doublet signal to discriminate between peptide and non-peptide signals in the mass spectrum providing an even greater increase in sensitivity. This strategy was employed to isolate an N-terminal peptide derived from an enzymatic digest of human hemoglobin and to facilitate the analysis of spectra. Proteins and peptides lyophilized from slightly alkaline solutions yielded the greatest amount of derivatization. However, a peptide lyophilized under acidic conditions could also be methylated to give an increased MALDI signal. In light of these results a mechanism is proposed where the vacuum facilitates the removal of gaseous counter-ions to drive the reaction to the trimethylammonium derivative. In order to selectively isolate the C-terminal peptides from enzymatic digests of proteins, lyophilized proteins were reacted with iodomethane under conditions where reaction occurred mainly with carboxylate groups. A proof of concept has been demonstrated for a method of general applicability for the determination of the C-terminal sequences of proteins. Separation and isolation of the C-terminal peptides generated from enzymatic digests for further sequencing either by chemical means (N-terminal Edman degradation chemistry) or by tandem mass spectrometry (MS/MS) was accomplished by the use of two dimensional (2D) high voltage paper electrophoresis (HVPE). (Abstract shortened by UMI.) |
