Practical Protein Chemistry - A. Darbre 1989

The latest methods for solid-phase and liquid-phase determination of amino acid sequences
Solid-Phase Analysis: The Latest Approaches
Edman Degradation

Preliminary Cleavage of Peptides attached to solid-phase Supports is carried out almost exclusively by the Edman method using Reagents and conditions similar to those for liquid-phase analysis. A Structure/133.html">Discussion of these techniques can be found in the literature cited in Section 16.2.1.

The main advantage of solid-phase analysis chemistry lies in The flexibility of choosing buffers, Solvents, wash regimes, reagents, various additives, and Temperature. Sample losses due to extraction, as well as substance volatility or solubility during analysis, impose very minor constraints on operating parameters, except when peptides are attached via adsorption bonds or when reactions are conducted in a gas-solid phase system.

When working with both liquid-phase and solid-phase Methods, equally high standards of purity must be maintained for the Reagents and solvents used. While the presence of harmful impurities and Side Reactions cannot be completely eliminated, their impact is significantly reduced by employing minimal volumes and concentrations of reagents. Solid-phase reactions can be carried out in a continuous flow of fresh reagents with the removal of by-products; this advantage is easily implemented in miniaturized systems where Heat transfer (via preheating of reagents) presents no problem.

16.2.4.1. Reagent Coupling Reactions. In this reaction forming phenylthiocarbamoyl peptides (PTC-peptides) or their analogues, buffers play no role in film formation or peptide retention; very few hydrogen ions are released or consumed during the reaction, allowing it to be performed using very low buffer concentrations. While it is tempting to enhance the efficiency of the reagent-polypeptide interaction through various additives (Denaturing Agents, reaction catalysts, impurity scavengers), the potential Introduction of side processes must be carefully weighed when adopting these innovations. Scavengers (e.g., primary amines) used at molar concentrations exceeding those of the peptides (but not the isothiocyanate) can be useful in Microscale analysis, as they help remove aldehydes and other highly reactive impurities. The upper concentration limit of amines is further dictated by the requirement that their presence must not trigger side reactions similar to those occurring after the 'capping' of the support surface by PTC groups.

The correct choice of reaction reagent is a fundamental condition for success. The preference for aqueous media over non-aqueous ones is partly driven by the need to solvate attached peptides of varying composition. While Water facilitates solvation, it also induces the Hydrolysis of the isothiocyanate, leading to subsequent side reactions. An open-minded approach to new proposals should be maintained when addressing this issue.

16.2.4.2. Cleavage Reactions. TFA has proven to be the best cleavage agent found to date. It is easier to purify than higher-molecular-weight perfluoroacids, and its volatility is advantageous for the recovery and Subsequent transformation of the cleaved products (anilinothiazolinones, ATZ).

In principle, other strong acids such as BF₃ can also be used. A decisive argument for or against a particular cleavage method is the yield of Ser obtained as PTH or another derivative suitable for identification.

In the solid-phase method, The amino acid thiazolinone (ATZ) is typically washed off the Column with the same acid used to effect the ATZ cleavage. Because TFA is a strong eluting solvent, at this cycle stage it strips many adsorbed substances from the resin, which end up in solution alongside the ATZ and contaminate it.

With small reaction column dimensions, the collected ATZs are easily cooled immediately after formation, thereby protecting them from degradation. Cleavage is usually performed at elevated temperatures for a duration sufficient to release the most refractory residues (e.g., Pro [15, 41]). Alternatively, cyclization and cleavage can be carried out using acid vapors acting on the dry bound peptide [42, 56]. After displacing the acid vapors with an inert gas, the ATZs can be washed out with a solvent of lower elution strength than TFA, resulting in fewer contaminants in the solution.

Acid-mediated cleavage of ATZs should be completed before their elution from the column (or performed in several steps with intermediate column dryings). Under these conditions, highly reactive ATZs become concentrated on the support surface at elevated temperatures. Because they are prone to degradation when maintained under these conditions, the Selection of the ATZ cleavage regime must be guided by rigorous control of experimental parameters.

Water content in the TFA must be strictly controlled. If the acid contains too little water, it remains in equilibrium with a small amount of trifluoroacetic anhydride. The latter can react with the PTC-peptide to yield an insoluble by-product [8]. The anhydride may be retained on the support surface via adsorption forces involving potentially reactive trifluoroacetyl derivatives of various Structural elements of the support (e.g., OH and NH groups [91]). Upon The addition of buffer (during the reagent coupling stage in the next cycle), these derivatives block the N-terminal amino acid of the peptide.

Conversely, an excessively high water content in TFA leads to an unacceptably high rate of internal peptide bond cleavage and reduces The rate of ATZ release. It remains unclear what water content represents a reasonable compromise, highlighting The Need for convenient and reliable methods to determine water content in TFA.

16.2.4.3. Conversion Reactions. Amino Acids cleaved via the solid-phase method as ATZs are typically converted into PTH-Amino Acid Derivatives using the same Procedures as in liquid-phase analysis. Some of the latest developments in the detection and quantification of PTH-amino acid derivatives are outlined in Section 16.3.3. A new conversion method, utilized in both solid-phase and manual sequencing, was recently proposed: rapid aminolysis of thiazolinones (either in the dry state or in solution) with primary alkylamines to yield alkylamides of PTC-amino acids [1, 50, 51, 57]. These reactions require no heating or extraction. Upon completion, excess amine is removed under a stream of nitrogen. In their chromatographic behavior and UV absorbance, alkylamides resemble PTH-amino acids. Methodological details are provided in [51]. Several interesting conversion reactions are briefly described in [81]. 5-Methylthiazolinones (MTAs) can be O-acetylated with acetic anhydride and subsequently identified by GLC. MTAs can also be cleaved with aqueous ammonia to yield the amides of the parent amino acids.

16.2.4.4. Identification and Quantification. The sensitivity of Sequence Determination can be increased by employing modified reagents during the coupling stage. This chapter does not cover methods involving radioactive labels or intensely colored isothiocyanates, as these have evolved primarily within the framework of manual or automated liquid-phase sequencing methodology.

The Use of [³⁵S]PITC in automated solid-phase analysis has been described [17, 18]. Although the best experiments claimed the capability to detect picomole quantities of PTH-amino acids, routine practice generally operated at the ≥100 pmol level (Waxdall, personal communication). The primary challenges are, first, the relatively low specific radioactivity of the available reagent and, second, the dilution of the reagent within the large dead volumes of the Reactor and delivery system.

An improved delivery scheme for [³⁵S]PITC has been proposed [44]. To drastically reduce column volume, a substantial improvement in solid-phase support properties is necessary. These innovations should also lower the radioactive Background, which becomes a severe issue when working with picomole quantities of peptide. We believe that future Prospects for analyzing extremely small amounts of substance using radioactive isothiocyanates will prove far more favorable for the solid-phase method than for liquid-phase determinations. The sensitivity of solid-phase analysis can also be significantly enhanced without resorting to expensive and hazardous radiolabeled reagents. A manual solid-phase method utilizing 1N,N-dimethylaminoazobenzene-4'-isothiocyanate

(DABITC) [21] (Ch. 14) has been described. This reagent yields intensely colored thiohydantoin derivatives (DABTH). Recently, these derivatives were separated and quantified via HPLC at the 5–10 pmol level [24, 107]; DABTH-amino acid derivatives absorb at a wavelength of 436 nm. The application of this method in automated solid-phase analysis has been reported [46]. In each cycle, following the coupling reaction between DABITC and the polypeptide, an additional reaction with more reactive PITC is performed to achieve complete 'capping' of unreacted N-terminal amino groups. The resulting PTH-Amino acids are colorless and do not interfere with the identification of the colored thiohydantoins [23]. In our view, performing the reaction within a column (whether manually or automatically) offers significant advantages over the original test-tube reaction protocol [21]. We have successfully performed manual solid-phase sequencing (using PITC) on small amounts of peptide-support placed atop a tiny piece of Glass wool in the constricted tip of a Pasteur pipette. The pipette was shortened at both ends; the resulting micro-column was heated inside a test tube placed in a propylene glycol bath, and column washing was facilitated by applying a slight positive pressure of nitrogen.