Amino Acid Analysis
Amino acid analysis has been available
at this facility since 1996. After several chemistries were explored, a pre-column system
has been adopted which appears to provide a stable environment for amino acid
analysis on the Beckman
166 HPLC System. The AQC (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate) chemistry produced
by Waters Corporation provides an acceptable balance of column lifetime and sensitivity,
thus permitting routine amino acid analysis on low pmol amounts of material. Non-standard amino
acids may be analyzed in the Purdue Proteomics Facility (See below for more information).
Amino Acid services (hydrolysis and analysis) usually require about 2 business days. Send an e-mail to the Purdue Proteomics Facility for information about special requirements.
The estimated amount of protein required for an accurate analysis is 0.1 to 5.0 µg
(4 to 20 pmols), based on an average molecular weight of 25,000. For peptides, the requirement
is much smaller: 0.02 to 1.0 µg (200 to 1,000 pmols), based on an average molecular
weight of 1,000.
When analyzing proteins blotted to PVDF, mixed results are obtained. Of several factors that influence the quality of the compositions, protein "concentration" is perhaps the most important. For PVDF-bound proteins, if possible, around 3 times the amounts suggested above (0.3 to 5.0 µg) are requested. The more protein per unit area of membrane, the better the results will be, since the PVDF contributes to higher background levels.
Generally, pure samples are required. The presence of salts, buffers, or detergents is deleterious. Amines (primary or secondary) will react with the carbamate, adversely affecting results. Salts can alter the pH of the sample, causing the derivitization to either be incomplete or simply to fail. Additionally, significant levels of glycerol or carbohydrates are problematic. The glycerol is nonvolatile and attracts moisture (acid); the carbohydrates char, decomposing to ash and taking the sample with them. If it is impossible to clean up your sample, an attempt can be made other desalting methods, such as precipitation or reverse phase HPLC cleanup.
To enable efficiency in the facility, samples for amino acid analysis will be processed ONLY on the first and third Tuesday of every month. A minimum of 5 samples will be required to trigger each biweekly run. If the run will not occur because of insufficient numbers of sample, the requesting researcher will be notified of the delay. Rates are based on the operator and equipment use required for these analyses.
Submitting Your Samples
Submission forms can be found on the wall outside the lab OR you can download
the Amino Acid Analysis Request Form by clicking here.
The form requests a valid account number (or a P.O. number for non-campus submissions), your name,
your P.I., your phone number, and information about the sample.
Bring or send in campus mail your sample to the PPF (Room B054 in the Hansen Building).
Leave the form and your sample inside the PPF with a laboratory technician. Turnaround times vary due to demand, but it is generally one week.
There is some interest in identifying post-translational modifications using amino acid analysis (AAA). HydroxyPro and two Methyl Lysines in a modified system have been characterized. The modified system is usable for HyPro. If you have a special residue to identify and think the problem is amenable to AAA, by all means, bring it to the PPF. Please note that a reactive amine is required for derivatization using the carbamate.
Samples to be hydrolyzed are placed into a 6 x 50 mm corning tube (which has been fired at 525 degrees C for a minimum of 6 hours) and dried in a rotary evaporator. For hydrolysis, the tubes are placed into a Waters hydrolysis vessel, and 250 µL 6 N HCl with 0.1% phenol is added. The vessel is flushed 3 times with Nitrogen and evacuated for 2 minutes (at a pressure <25 µm). The vessel is heated for 22-24 hours at 110 ± 2 degrees C in a convection (mechanical) oven. The tubes are removed, wiped to remove excess acid, and dried in a speed-vac for 30 minutes. Following removal of the acid, the samples are derivatized according to the vendor's recommendations (Waters, Inc.) using 20 mM HCl, Borate buffer, and AQC reagent in acetonitrile (1:3:1, v/v/v). For analysis, the samples are transferred to autosampler vials equipped with micro inserts (National Scientific, Inc.) and heated gently for several minutes to complete conversion of di-Tyr products to mono-Tyr AQC derivatives. Analysis of samples is performed using a Beckman HPLC (126 pump, 166 Detector, 507 autosampler, and "System Gold" data system) equipped with a Waters AccQTag amino acid analysis column and buffers. The gradient program is essentially the one recommended by Waters (with only small adaptions), and the column heater temperature is 37 degrees C. Calibration is performed using an amino acid mixture obtained from Pierce (Pierce Std H), which is processed in parallel with the unknown samples.
With well-prepared samples, the average internal error is generally between 5 and 10%. This
figure is for a constellation of amino acids, excluding Cysteine (Cys) and Tryptophan (Trp). Tryptophan
is totally destroyed in hydrolysis, and Cys is normally unreliable, unless special hydrolysis
conditions are used. However, the PPF has recently employed a method to convert Cysteine
to Cysteic Acid by hydrolyzing the samples in the presence of DMSO. Under these conditions,
several other amino acids are affected, primarily Histidine (His) and Tyrosine (Tyr). Therefore,
the sample must also be hydrolyzed by conventional methods to obtain accurate determination for
the other 18 amino acids.
Once the results of the analysis have been obtained, a spreadsheet summarizing raw data, mole percent, grams recovered, and error (if possible) will be composed. Information for control samples is included, if pertinent.