Practical Protein Chemistry - A. Darbre 1989

Latest methods for solid-phase and liquid-phase amino acid sequence determination
Automated liquid-phase analysis. Advanced procedures
Amino acid sequence analysis of small and hydrophobic peptides

It is well known that automated analysis of such objects often fails due to peptide washout during the extraction of the reaction mixture with Solvents and residual Reagents. Since the greatest losses occur during the washing of the Reactor with ethyl acetate—which removes Quadrol—it was proposed to replace Quadrol with a volatile buffer removed by evaporation in vacuo, thereby eliminating the ethyl acetate wash step from the protocol [77]. Analyzing the complete Amino Acid Sequence of nonpolar Peptides requires additional modifications to both the Procedure and the degradation program.

Peptides containing C-terminal Lys or S-aminoethylcysteine residues were modified with 4-sulfophenyl isothiocyanate to increase peptide hydrophilicity [16, 52].

This reagent attaches to both the N-terminal α-amino group and the side-chain amino groups of the aforementioned Amino Acids. Upon acid Treatment, the first residue is cleaved, while the second is ready to participate in the conventional Edman reaction; the sulfophenylthiocarbamoyl group attached to the side chain of the C-terminal amino acid remains uncleaved. The sole drawback of this technique lies in the incomplete Cleavage of the first residue, which severely reduces the initial yield and introduces a high Background of residual amino acids at the very beginning of the analysis. Furthermore, identifying the first amino acid presents a certain difficulty. It has been reported that the isomer (3-sulfophenyl isothiocyanate) offers certain advantages for this modification [30], yet little is known about its application to date. To increase peptide hydrophilicity, side-chain carboxyl groups can be amidated with 2-amino-1,5-disulfonate [36]. However, the carboxyl activation required for this can lead to blocking of the N-terminal amino group and cause internal cyclization of Asp residues.

Another approach has been proposed to reduce peptide losses during extractions. Following the carbamoylation step, Quadrol is not extracted with solvents, but is left in the reactor to form a hydrophilic film that retains the peptide [25]. However, retaining Quadrol in the reactor is disadvantageous because its addition in each cycle leads to the accumulation of a large amount of salts in the reactor after several cycles.

It was hypothesized that a protein with a blocked N-terminal amino group that does not undergo the Edman reaction could serve as an "anchor" to retain the peptide in the reactor. Parvalbumin was used for this purpose [89].

However, under acid exposure, this protein undergoes random cleavage; new sequences appear, and the background level of PTH-amino acids increases. The retention of small or hydrophobic peptides in the reactor increases significantly in the presence of Polybrene—a synthetic polymer containing positively charged quaternary nitrogen atoms that is inert under sequencing conditions [59, 100]. We utilized it when working with very large peptides containing numerous nonpolar amino acids. Good results were obtained for some peptides; at the same time, we observed no significant effect from Polybrene addition when working with other large fragments or intact Proteins. Moreover, when using Polybrene, we noted a decrease in initial yields (see also Chapter 15).