Practical Protein Chemistry - A. Darbre 1989

Methods for Solid-Phase Amino Acid Sequence Analysis
Discussion
Microscale Structure Analysis

With the advent of HPLC and highly sensitive flow-through detectors, micro-scale analysis enabling the identification of picomole quantities of substances has become a reality. The sensitivity of microanalysis is limited by derivative purity, stability during and after Cleavage, and the presence of impurities. High solvent purity is a mandatory requirement; to eliminate any possibility of contamination, the Sequencer must, firstly, feature minimal dead volumes along with non-corrosive Valves and fittings, and secondly, maintain a consistently high level of technical performance. The limiting factor in liquid-phase analysis is the quality of Polybrene, whereas in solid-phase analysis, it is the quality of the Supports. A further complication arises from significant variations in the yields of individual derivatives. Some thiohydantoins are cleaved in high yields and exhibit good stability, while others are present in very small amounts because they are either poorly cleaved or degraded post-cleavage; in either case, they are difficult to detect against a Background of by-products. The technical condition of the instrument must be carefully monitored to ensure reliable results in analyses where There is a risk of signal suppression by background noise, even when minimized. Instrument operability can be verified by running a blank test (omitting the peptide or protein). Fractions collected from each cycle should be checked for contamination by analyzing large aliquots of the fraction. To establish a calibration curve for the instrument, control analyses are performed using standard peptide or protein samples. It is worth noting that Isolation Methods used for peptide and Protein Purification heavily influence sample solubility, meaning they play a critical role when assessing the feasibility of Edman Degradation for structural analysis. Trace amounts of buffer salts (such as ammonium acetate) or amines reduce the cleavage efficiency of ATZ/PTH-Amino Acid Derivatives and cause partial blocking of the N-terminal Amino Acids of Peptides. This factor becomes decisive when analyzing very small quantities of peptides; to prevent irreversible N-terminal blocking, the isolation of Peptides and Proteins should be carried out under reducing conditions.

12.6.4.1. Edman Degradation Using Labeled Reagents. Several Procedures have been described in the literature that can significantly reduce sample consumption. One such approach utilizes radioactively labeled PITC [6, 8, 9, 27]. Work should be performed exclusively with high-purity reagents, as impurities present in the starting PITC, as well as those generated during cleavage, compromise the reliable identification of PTH-amino acid derivatives. The level of radioactive background also depends on the tightness of valve seals and threaded connections. Structural analysis of in vivo labeled peptides and proteins proceeds much more cleanly. The challenges associated with labeling proteins or reagents are discussed in reference [44].

12.6.4.2. Analysis Using DABITC. An efficient approach to manual micro-sequencing has been described in which PITC is replaced by its analogue

4-N,N-dimethylaminoazobenzene-4'-isothiocyanate (DABITC) [12]. The method is highly sensitive—amino acid derivatives can be readily identified at the picomole level by TLC and quantified by HPLC [13, 37]. Detailed procedures are outlined in Chapter 14. For solid-phase microanalysis, a dual-coupling method utilizing both PITC and DABITC has been developed [25]. An improved version of this method has recently been proposed, yielding unambiguous results when working with very small sample amounts [52]. Acknowledgments. I would like to thank A. Lehmann for his assistance in preparing this chapter, as well as K. Ashman and P. Rolling for reading the manuscript prior to publication.