Phenylthiocarbamyl amino acid analysis (PTC-AAA) is an established method for picomole-level quantitative analysis based on the reaction of phenylisothiocyanate (PITC) with amino groups to form phenylthiocarbamyl amino acid derivatives, which are then separated by reversed-phase HPLC and identified and quantified by ultraviolet absor-bance. Since its introduction in the 1980s (Koop et al., 1982; Heinrikson and Meredith, 1984; Tarr, 1986), PTC-AAA has become the most popular precolumn-derivatization approach to AAA and may be used today with greater frequency than the classic ninhydrin postcolumn-derivatization method developed in the 1950s (Moore and Stein, 1949; Moore et al., 1958). During the 1990s, the majority of PTC-AAA has been performed with instrumentation from two vendors. Waters Associates first commercialized the PTC method under the trade name PicoTAG amino acid analysis (Bidlingmeyer et al., 1984, 1986), and PE Applied Biosystems Division introduced instrumentation in 1986 that performs automatic PTC derivatization with on-line HPLC analysis. The PE Applied Biosystems instrument was subsequently modified to also perform automatic on-line HCl hydrolysis. Both companies have provided reliable instrumentation and valuable technical and service support for PTC-AAA. However, most other companies marketing HPLC systems also provide instructions for how to separate PTC amino acids using their respective instrumentation. Protocols in this unit can be used with essentially any HPLC system.
Volatile solvents, e.g., HPLC-grade water (Burdick and Jackson); aqueous 0.1% trifluoracetic acid/acetonitrite; 30% to 50% methanol, ethanol, or acetonitrite; 0.1% to 1% N-ethylmorpholine acetone, pH 8.5; or 0.1% HCl.
Internal standard, e.g., a-aminobutyric acid (optional)
2:1 (v/v) methanol (Burdick and Jackson)/diisopropylethylamine (DIEA; PE Applied Biosystems)
7:2:2 (v/v/v) methanol/DIEA/5% (w/v) phenylisothiocyanate (PITC; Pierce or PE
Applied Biosystems) in heptane (Burdick and Jackson) EDTA-containing transfer buffer compatible with HPLC system, e.g., 29 mM sodium acetate buffer pH 5.2 (Mallinkrodt), containing 0.25 mg/ml K3EDTA (Sigma)
Amino acid Standard H (Pierce)
Peptide or protein standard, e.g., insulin subunits, myoglobin, and lysozyme (Sigma) or 7% (w/v) bovine serum albumin (National Institute for Standards and Technology, formerly National Bureau of Standards)
Vacuum system consisting of: Vacuum pump (e.g., Leybold-Heraeus Trivac D2A) Vacuum gauge (e.g., Fredericks Televac) Vacuum hose and connectors Cold trap and Dewar flask Glass three-way stopcock
Polytetrafluoroethylene (PTFE) 6-mm bore Rotofol valve (Corning) or two-way stopcock Argon (or nitrogen) gas supply consisting of: Tank of prepurified gas Regulator for gas tank Silicone tubing Gradient HPLC system consisting of: Two HPLC pumps Mixing chamber Controller UV detector Manual injection valve
Automatic sample injector (recommended but optional) Recording integrator (e.g., Shimadzu C-R6A Chromatopac) Mininert slide valves (Pierce)
Additional reagents and equipment for preparing samples (see Support Protocols for Sample Preparation), hydrolyzing samples (see Support Protocols for Hydrolysis), performing quantitative RP-HPLC (units 8.2 & 11.6), plotting results to determine linear response ranges, and calculating standard deviations to determine reproducibility
1. Assemble key equipment and accessories close at hand in a dedicated PTC-AAA work area near a fume hood. A PTC-AAA workstation consists of a vacuum system, a fume hood, an argon (or nitrogen) flush system, an oven, freezer, HPLC system, automatic pipettor, 6 x 50-mm glass hydrolysis/derivatization tubes, 40-ml screw-cap vials, and Mininert slide valves.
Waters markets a PicoTAG Work Station (Bidlingmeyer et al., 1986) designed after the manual sequencing and PTC-AAA station developed by Tarr (1986). Original workstation descriptions with schematic diagrams appear in the above articles, and photographs of workstations are also available (Kuhn and Crabb, 1986). The work area can be arranged according to individual preference. The vacuum/argon flush system, 6 x 50-mm tubes, and 40-ml reaction vials with Mininert slide valves particularly facilitate manual acid hydrolysis and PTC derivatization protocols.
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