Amino acid analysis is the golden standard for accurate quantification of proteins. The amino acid analysis service is quite straightforward: First a hydrolysis of an accurate amount of sample using strong acid, and subsequently detection and quantification of individual amino acids.
The analysis provides quan...
Do you need accurate protein quantification?
Amino acid analysis is the golden standard for accurate quantification of proteins. The amino acid analysis service is quite straightforward: First a hydrolysis of an accurate amount of sample using strong acid, and subsequently detection and quantification of individual amino acids.
The analysis provides quantitative data for all amino acids except tryptophan and cysteine that are degraded during the hydrolysis. Also, we determine asparagine as aspartic acid, and glutamine as glutamic acid.
Our amino acid analysis service consists of:
Your choice between a single or triplicate amino acid assay. We recommend analysis in triplicates, whenever you need a very accurate protein concentration determination.
Comparison of results against your protein sequence.
The analysis process includes protein hydrolysis, HPLC analysis and data calculation. We base the Alphalyse procedure on the Ph. Eur and USP. Note that we determine asparagine as aspartic acid, and glutamine as glutamic acid.
Apparatus
We perform the analysis on a BioChrom 30 amino acid analyser. It makes ion-exchange chromatography with post-column derivatization using ninhydrin. We can thus monitor all amino acids except Proline (440 nm) at 570 nm.
Protein hydrolysis – Vapour phase
First, we place the sample in a 0.5 mL hydrolysis tube and dry it down. We then place the tube in a vessel with an acid hydrolysis solution (6M HCl containing, 0.1% phenol, 0.1% thioglycolic acid). The vessel is flushed with nitrogen. After evacuation (<10 mBar) the vessel is placed at 110°C for 24 hours. After hydrolysis the test sample is dried in vacuum. Next, we dissolve the sample in loading buffer (sodium citrate pH 2.20) including a fixed amount of sarcosine (Sar). We use this as an internal standard to correct for changes in reagent stability and flow rates. However, it also makes sure that the variation for each sample does not exceed 5%.
Calibration of Instrument
We analyze a standard mixture of amino acids at 5 different concentrations levels (double determination). This shows the linearity within the measurement range 625 to 10000 pmol). Next, a plot shows the peak area versus the known concentration of each standard solution. It passes when the r-value of the calibration is better than 0.999 (proline is accepted at 0.995 due to lower signal/noise). The instrument is linear to 10000 pmol and usually up to ~50 nmol. Depending on the amino acid residue, the signal goes into saturation around 100 nmol.
Accuracy & Precision
For each batch run we include a duplicate analysis of a NIST BSA quality control standard. We use the results to determine the precision and the accuracy of the test. The precision should be below 5% and the accuracy within 15%.
NIST BSA (double determination) is analyzed for each sequence. Based on that, we then calculate a compensation factor to correct for ninhydrin activity. We correct each amino acid in the sample by this compensation factor.
Data from the amino acid analysis service
Unknown protein samples: If there is no protein sequence, we now calculate the total protein amount based on the amount of each amino acid (in pmol), multiplied by the mass of the given amino acid residue. Please note that since the content of cysteine and tryptophan is not included, the obtained value is underestimated by approximately 2.7% (average Cys and Trp content in proteins in the Swiss-Prot database).
Known protein samples: If we know the protein sequence, we then divide the measured AA amount in pmol by the number of each specific amino acid in the sequence providing a pmol protein. We use the average of these numbers in pmol to calculate the amount of protein in µg with the molecular weight of the protein.
The calculations against known amino acid sequence is then made. We use a best linear fit based on all detected amino acids. It is modified for each experiment not to include amino acids with greater than 5 per cent variation.
Sample preparation
Sample requirements for quantification analysis
You may submit samples in liquid form or as solid material.
Sample Purity
We can analyze most samples that contain proteins and peptides. Avoid very high salt concentrations. Do not use buffer that contain high amounts of primary amines, e.g. TRIS or glycine. Because it may interfere with the ninhydrin reaction and thus provide false quantification data of specific amino acids. Avoid fingerprints and dust in the sample. These contain large amounts of human proteins that will obscure the results.
Sample Amount
Provide >100 microliters of liquid material at minimum concentration of 0.1 microgram per microliter. Solid material should be provided in sufficient amount for accurate weighing of samples at our facilities (mg material).
Related services
You can combine this analysis with other analyses such as:
