Practical Protein Chemistry - A. Darbre 1989

Latest methods of solid-phase and liquid-phase amino acid sequence determination
Automatic liquid-phase analysis. Advanced techniques
Microscale analysis

In many cases, it is necessary to analyze small amounts of scarce Proteins. Reducing the sample consumption down to 1–10 nmol required significant improvements to existing Methods. For example, the Modification of the liquid-phase Sequencer involved the Structure/175.html">Implementation of a stable high-Vacuum system, The Development of novel extraction techniques, The Use of specially designed hermetic reagent-delivery Valves with zero dead volumes, and the integration of an automated converter into the instrument setup [108–110]. These enhancements were subsequently integrated with a high-efficiency system for purifying Reagents and Solvents, along with the use of Polybrene [47]. In a later study, using only 200 pmol of the sample, The sequence of 47 residues of sperm whale Myoglobin was successfully determined, with stepwise yields of 95.5%. To improve the operational parameters of the liquid-phase sequencer, a novel instrument design was developed [48] (see also Chapter 17). This apparatus enabled the determination of extended sequences using 20 pmol of protein or 200 pmol of Peptides. Attempts to further increase sensitivity using all the aforementioned techniques yielded limited success, as the problems of sample washout during each Cleavage cycle and the contamination of PTH-Amino Acids by process-generated impurities remained unresolved.

The use of highly sensitive radioisotope-based techniques helped overcome some of these challenges. A radioactive label can be introduced either into the amino acids (biosynthetically) or into the reagents used for sequence analysis. Proteins labeled biosynthetically in vivo or in vitro must be purified using micromethods; in many cases, a product suitable for analysis can be obtained via immunochemical purification. Various laboratories have successfully analyzed radiolabeled proteins using as little as 0.1–1.0 pmol of material [79, 103]. The primary limitation of this approach is that not all Proteins can be labeled in vivo or in tissue culture due to their low Abundance and/or slow synthesis rates.

An example of using radiolabeled reagents is described in work [54]. In each cleavage cycle, the protein is treated first with [³⁵S]PITC and then with "cold" PITC. This method enabled the determination of ~20 amino acid residues using 5–15 pmol of protein. Section 16.2.4.4 discusses the application of this approach in solid-phase sequence analysis [17, 18]. The drawback of this method, applicable to both liquid-phase and conventional solid-phase sequencers, is the high reagent consumption required to cover the large surface area of the sequencer Reactor. In turn, this leads to a high radioactive Background level when analyzing very small sample quantities, making the analysis both costly and hazardous.

Recently, the application of HPLC in micro-scale Amino Acid Sequence analysis has been steadily expanding (Chapter 6). This technique allows for the isolation of microgram quantities of peptides in solutions suitable for direct loading into the sequencer reactor [112]. Mass spectrometry serves as another crucial complementary method for analyzing short peptides [12, 60, 73]; its exceptionally high sensitivity is likely to drive its widespread adoption and preference.