Amino acid analysis has been offered at our facility since 1996. Finally, 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 Corp. provides an acceptable balance of column lifetime and sensitivity permitting routine amino acid analysis on low pmol amounts of material.
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.
We have had mixed results when analyzing proteins blotted to PVDF. Of several factors which influence the quality of the compositions, protein "concentration" is perhaps the most important. We request that for PVDF-bound proteins, you supply around 3 times the amounts requested above (0.3 to 5.0 µg), if possible. The more protein per unit area of membrane, the better the results will be since the PVDF contributes to higher background levels.
We also require generally pure samples. The presence of salts, buffers, or detergents are deleterious. Amines (primary or secondary) will react with the carbamate, adversely affecting results. While salts can alter the pH of the sample causing the derivitization to be incomplete or simply fail. Additionally, significant levels of glycerol or carbohydrates are problematic - the glycerol is nonvolatile and attracts moisture (acid) and carbohydrates char, decomposing to ash taking the sample with them. If it is impossible to clean up your sample, we can attempt other desalting methods, such as precipitation or reverse phase HPLC cleanup.
Bring or mail your sample to our laboratory (Room 404 in the Hansen Building). 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 noncampus submissions), your name, your P.I., your phone number, and information about the sample. Drop the form and your sample inside the lab with either Mary or Michele. Turnaround times vary due to demand, but it is generally one week.
| Analysis and Hydrolysis | $40.00 per sample |
| Analysis Only | $30.00 per sample |
With well-prepared samples we will generally obtain between 5 and 10% average internal error. 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, we have 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 we have obtained the results of our analysis, we will compose a spreadsheet summarizing raw data, mole percent, grams recovered, and error (if possible). Information for control samples is included, if pertinent.
There is some interest in identifying post-translational modifications using amino acid analysis (AAA). We have characterized HydroxyPro, and two Methyl Lysines in a modified system. 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 over. 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 that 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.