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Determination of peptide lengths and their relative abundances in complex mixtures


A unique, semi-quantitative method has been developed to determine peptide lengths and their relative abundances (in mol% or ųg%) in (very) complex peptide mixtures like protein hydrolysa­tes [1].

In short, the method is based on algorithms applied to the quantitative data obtained from automated Edman degradation of the sample, usually for 16 cycles, and amino acid analysis (both of a hydrolyzed and a non-hydrolyzed sample) of the peptides and free amino acids extracted from the intact dairy products or other foods. The analysis results in a graphical presentation of the relative occur­rence versus length of peptide.

Until now, we have used this method to compare different protein hydrolysa­tes of unknown composition concerning the relative occur­rence of peptides ranging in length from 1 - 15 amino acids (Mw range 0.1 - 1.5 kDa). It is possible to extend this region to peptides of 30-40 amino acid residu­es, although the reliabi­lity of the quantification may be somewhat lower. The presence of longer fragments, however, will be recog­nized/detec­ted, and their molar percentage determined. This procedure has also been applied successfully to mixtures containing only 15% protein material (dry mass basis), and in the presence of sugars, lipids, fats, minerals, salts and vitamins.

Because the method and the algorithms applied contain certain assump­tions (e.g. an equal efficiency of extraction for all fragments; contribu­tions of Cys, Trp are not taken into account and N-terminally blocked fragments are of minor importance) it should be noted that, in our opinion, the method is suitable for com­parison of different samples treated in the same way or to categorize them, more than that it pretends to give an exact reflection of the true situa­tion.
To find out to what extend the results reflects the actual situation and for 'fine-tuning' the method, experiments with samples of known composition have been performed. The results of four such experiments on different mixtures of peptides and free amino acids (mix 1, 2, 3 and 4) are presented in the figure below.

As can be seen, there is a pretty good agreement between the experimental and theoretical values. The lengths of the peptides are given as 1-2, 2-3, 3-4 residues, etc., rather than simply 1,2, 3 etc. The reason is that peptides of length n will give a certain contribution to the determi­na­tion of peptides with length n-1, but no significant con­tribu­tion to the determination of peptides of length n+1 or n+2, etc., or to peptides of length n-2, n-3, etc, as can be seen in the Figure of Mix 4. This contribution, unfortu­nately, is variable (it depends on the specific amino acid sequence of the peptide, among other factors) and cannot be quantified exactly. Thus, a pentapeptide gives a 'false' signal at length '4' (4-5), as can be seen in the figure of Mix 4; addition of the two experimental values for peptides with length '4' (4-5) and '5' (5-6) in Mixture 4 gives a good correlation between the experimental and theoreti­cal values.

Summarizing, the method is suitable for com­parison of different samples treated in the same way or to categorize them. Experiments with samples of known composition indicate a good correlation between theoretical and experimental relative values; however, one should be careful with absolute data deduced from the (semi-quantitative) relative values.
Furthermore, it should be noted that in case of the presence of S-S-linked peptides, the length of each individual polypeptide chain will be determined, as will be the case for most, otherwise linked, fragments.
Apart from giving information about the relative distribu­tion profile; the amino acid analysis data also provides quantitative data about both the free amino acid content and the protein content. There­fore, this method is very suitable for comparative studies of protein hydrolysates, for example present in food or feed applica­tions, especially for mixtures containing short peptides (< 15 amino acids). It does not suffer from the disadvantages of the GPC analysis or the copper-Sephadex method [2] , which yields only a very rough indication of the peptide profile.








Determination of peptide lengths and their rela­tive abundance (in mol%) in four different mixtures of peptides of known
composition. The X-axis (peptide length) is divided into groups (1-2, 2-3, 3-4, etc. instead of 1, 2, 3 etc.) because a
peptide with length n adds a 'false' signal to the experimental determination (shad­ed bars) of peptides of length n-1 as
can be seen for Mix 4. The the­oretical values (hatched bars) are plotted on the same axis, although they refer to peptide
lengths of exactly 1, 2, 3, etc1.

 

©Parts of the Figures and text have been published in Ref. 1.

[1]  Siemensma, A.D., Weijer, W.J. and Bak, H.J. (1993) Trends in Food Sci. Technol. 4, 16-21
[2]  Rothenbühler, E., Waibel, R. and Solms, J. (1979) Anal. Biochem. 97, 367-375.