Mass spectrometry protected peptides

P. Roepstorff, K. Norris, S, Severinsen, K. Brunfeldt, Mass spectrometry of peptide derivatives. Temporary protection of methionine as sulfoxide during permethylation FEBS Lett. 9 235-238(1970)... 

However, interpretation of, or even obtaining, the mass spectrum of a peptide can be difficult, and many techniques have been introduced to overcome such difficulties. These techniques include modifying the side chains in the peptide and protecting the N- and C-terminals by special groups. Despite many advances made by these approaches, it is not always easy to read the sequence from the mass spectrum because some amide bond cleavages are less easy than others and give little information. To overcome this problem, tandem mass spectrometry has been applied to this dry approach to peptide sequencing with considerable success. Further, electrospray ionization has been used to determine the molecular masses of proteins and peptides with unprecedented accuracy. 

The TCR 110-76 peptide on the other hand could neither be dissolved with full side chain protection nor could its purity be defined by mass spectrometry after deprotection of the side chain due to its insolubility. This peptide was therefore used without purification. 

The purity of protected amino acids is especially important for the synthesis of longer peptides. Standard techniques such as melting point determination, nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and optical rotation are effective means of characterization. The optical purity can also be evaluated by high-performance liquid chromatography (HPLC) after derivatization with Marfey s reagent [216,217]. The advanced Marfey method refers to analysis by mass spectrometry after derivatization with Marfey s reagent [218-221]. Purification of side-chain protected amino acids by recrystallization is usually sufficient. 

The characterization of a peptide after purification is necessary to establish whether the desired peptide and not some structural modification has been isolated. Characterization of peptide molecules is not always straightforward owing to the particular type of structural complexity that these molecules present. Characterizations are best realized by mass spectrometry, especially in the fast atom bombardment (FAB) mode [132-136], although electrospray and matrix-assisted laser desorption-time of flight (MALDI-TOF) modes can also be useful, as well as by nuclear magnetic resonance (NMR) spectroscopy. In the latter technique, the interpretation of the NMR spectra of large protected peptides can be complicated. One-dimensional spectra are not normally sufficient and more sophisticated two- and even three-dinlen-... 

The dried, partially protected crude peptide is dissolved in DMF-pyridine (4 1 v/v) and treated with DMF-SO3 (125-fold molar excess) in the presence of 1,2-ethanedithiol (100-fold molar excess). After about 15 h at room temperature, the solution is applied to a size exclusion chromatography column and eluted with DMF. The target compound is collected and the solvent evaporated to dryness. After lyophilization, the crude compound is treated with a 90% TFA-based reagent at 4°C. The reaction time of this step is optimized by monitoring the acidolytic treatment of a small aliquot of the O-sulfated peptide and analysis of the synthetic products by analytical HPLC and mass spectrometry. 

The techniques in solid-phase organic chemistry complement those from solid-phase peptide assembly. Synthesis is performed on resins using anchoring linkages and orthogonal protection schemes. Molecules are cleaved from the solid support, purified (see Chapter 18), and then analyzed by high-performance liquid chromatography or mass spectrometry (see Chapters 19 and 20). Reactions can be performed in a simple apparatus that... 

Truncated sequences, core sequences, incompletely synthesized peptides. Imperfect conversion during acylation or deprotection of the temporary protecting group in solid-phase synthesis may lead to mismatch sequences that lack some amino acids and truncated sequences (core sequences). They occur, when the accessibility or reactivity of the peptide bound to the solid phase is insufficient difficult sequences). Truncated sequences may be classically identified and quantified by a modified Edman degradation, also called preview analysis. Alternatively, mass spectrometry provides efficient tools for the identification of truncated sequences. 

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