Steven A. Cohen

Amino acid analysis utilizing phenylisothiocyanate derivatives

Advances in liquid chromatography have brought about the development of new techniques in amino acid analysis which take full advantage of precolumn derivatization procedures. Using phenylisothiocyanate as the reagent, detection limits under 1 pmol can be routinely achieved, allowing the analysis of submicrogram protein samples. Analysis times as short as 10 min for samples after hydrolysis and 1 h for physiologic samples are possible. Accurate, reproducible quantitation of amino acids can be obtained from complex matrices such as plasma, urine, feed, and food samples. This level of performance and flexibility gives the analyst the first realistic alternative to ion-exchange analysis without compromising desirable features of the traditional methodology.

Applications of amino acid derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Analysis of feed grains, intravenous solutions and glycoproteins.

Primary and secondary amines are rapidly labelled by 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate to form highly fluorescent asymmetric urea derivatives which are readily amenable to analysis by liquid chromatography. Derivatization consists of a simple, one-step procedure, and the resulting labelled amines can be analyzed without further cleanup. The adducts are extremely stable with no discernible loss in response after storage for one week at room temperature, making the reagent an ideal candidate for pre-column amino acid analysis. Chromatographic methods for protein hydrolysates have been developed for the analysis of samples containing many unusual amino acids including a number of cysteine derivatives, collagen hydrolysates containing hydroxyproline and hydroxylysine, performic acid oxidized samples and glycoprotein hydrolysates containing glucosamine and galactosamine. Samples with potentially interfering matrix components such as hydrolyzed feed grains and intravenous solutions are readily analyzed and are quantified with average per cent relative standard deviations in the 1-2% range. Comparative data on these samples are in good agreement with either ion-exchange amino acid analysis or label information.

Compositional Protein Analysis Using 6-Aminoquinolyl-N-Hydroxysuccinimidyl Carbamate, a Novel Derivatization Reagent

Publisher Summary This chapter discusses compositional protein analysis using 6-aminoquinolyl- N -hydroxysuccinimidyl carbamate (AQC)—a novel derivatization reagent. Today many standard protein chemistry protocols—such as Edman degradation and peptide mapping—can be accomplished with low or even subpicomole sample amounts through the use of modem liquid chromatography. Amino acid analysis has been assisted in this drive for higher sensitivity by the use of an array of precolumn derivatization reagents that yield easily detected labels. The synthesis of a novel activated carbamate, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC), forms the basis for a precolumn derivatization method and provides very accurate amino acid analysis of hydrolyzed peptides and proteins. A rapid reaction with amino acids forms highly stable asymmetric urea compounds with good fluorescence characteristics. A dramatic shift in fluorescence emission maximum for the amino acid adducts compared to the major reagent peak allows the direct injection of the sample reaction mixture with no interference from excess reagent. The chapter describes the application of AQC to the analysis of protein and peptide hydrolyzate samples, with an emphasis on the accuracy and reproducibility of the compositional data.

Analysis of derivatized peptides by capillary electrophoresis

Thirteen synthetic prothrombin leader peptides differing only in C terminus were analyzed by capillary electrophoresis. These peptides were derivatized using the novel, fluorescent derivatizing agent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Derivatized peptides were detected using fluorescence and ultraviolet absorption signals, and underivatized peptides were detected using ultraviolet absorbance at 185 nm. Data are presented which compare the analysis of the derivatized and underivatized peptides.

Amino Acid Analysis of Submicrogram Hydrolyzate Samples

Understanding the mechanisms by which diseases occur is a complex process, and often involves the isolation and characterization of minute quantities of rare proteins from living tissue. Amino acid analysis is a vital tool for the elucidation of the structure of such molecules, but today’s researchers must operate at or beyond the utmost limits of conventional methods. In February, 1984 (1) we reported on a new approach to precolumn derivatization and analysis of amino acids which appeared to satisfy all the needs of protein chemists. The approach was based upon the formation of a phenylthiocarbamyl (PTC) derivative of the amino acids (2). The method was shown to be rapid, efficient, sensitive, and specific for the analysis of primary and secondary amino acids in protein hydrolyzates. The method allows for the rapid, bonded phase separation with ultraviolet (UV) detection at 254 nm of the common amino acids with 12 minute analysis time and a one picomole sensitivity for a standard amino acid sample.

Post-run Analysis of Proteins Purified by Capillary Electrophoresis with Membrane Fraction Collection

Publisher Summary This chapter explores the post-run analysis of proteins purified by capillary electrophoresis with membrane fraction collection. Twenty-second injections of the five protein mixture were loaded onto the capillary, separated by CZE, and collected onto the rotating Immobilon-P membrane supports. Protein concentrations tested ranged from 2.5 ng to 2 μg/ml. Following electrophoresis, the dried membranes were wet with methanol and rinsed with Milli-Q ® water. Proteins collected with the membrane fraction collector following CZE can be visualized at the low picogram level using antigen-specific immunodetection procedures. This level of sensitivity is comparable to that obtained using nonselective, on-capillary detection at 185nm. Membrane-bound samples are readily amenable to reversed phase high-performance liquid chromatography amino-acid analysis following vapor-phase hydrolysis and derivatization with AQC. As little as 100 ng of bound sample can provide useful information with average errors less than 20%. Higher sample amounts can provide routine analysis with average errors ca. 10%.

Amino Acid Analysis Using Pre-Column Derivatization with Phenylisothiocyanate: Matrix Effects and Tryptophan Analysis

We have recently reported1–3 on a new methodology for amino acid analysis (AAA) that relies on the reaction of free amino acids with Edman’s reagent, phenylisothiocyanate (PITC), to form the phenylthiocarbamyl (PTC) derivatives, which are subsequently separated and quantitated using a reverse phase gradient liquid chromatography (LC) system for analysis. This work extended the methodology for AAA using PITC introduced several years ago by Tarr and co-workers4–5, and appeared concurrently with a report by Heinrikson and Meredith6 describing similar methodology for AAA. The method was shown to be reliable, accurate, and extremely rapid (12 minute) with a maximum one picomole detection limit for each amino acid. This new method provides improved capabilities in comparison with conventional ion-exchange, post-column ninhydrin reaction systems. In addition, linear response in the 1020131000 pmol range was demonstrated as well as a high degree of reproducibility1. This system has been recently commercialized under the name PICO·TAG™ Amino Acid Analysis System3.