Fractionation methods peptides

The development of modem methods, suitable for the analysis of ampholytes in biological fluids, provided means for isolating from urine some chemically better defined fractions containing peptide compounds. The methods used did not, however, exclude the existence of some other forms of combined amino acids in the fractions studied. 

Because of the favorable sorptive properties of the reversed-phase supports, batch adsorption and desorption can be a very effective way to desalt a chromatographed sample or to partially fractionate a peptide mixture during a purification procedure. For example, 1-2 gm of an oc-tadecyl silica packed into a silanized glass or plastic pipette can be used for the batch fractionation of small amounts of a crude peptide extract from tissues, such as the pancreas or pituitary, or from a synthetic experiment. A number of commercial products, such as the Waters Sep-Pak, have found use in this manner 10) as a purification or sample preparation aid. Protocols for batch extraction procedures on alkyl silicas have been discussed 17a,b) and applied to neuropeptides 10, 158, 166) and other hormonal peptides 88, 162, 167, 168). With these methods recoveries of peptides present in a tissue extract are generally higher than those found with classical fractionation techniques due in part to the fact that proteolytic degradation is minimized. 

This fractionation method was subsequently used successfully by other investigators (Bll, D2a, Gl, G64, H39, H40, II, 12, N6, N7, P14, S6-S9, W7). Schrager (SIO) 12 years later severely criticized it and considered that measurement of mucosubstances by determination of their peptide moiety was an improper technique for measuring the gastric juice mucosubstances. He simultaneously estimated the tyrosine and hexosamine contents of the mucoprotein and mucoproteose fractions and found no quantitative correlation between the two. He therefore concluded that tyrosine measurement was not adequate for quantitation of these fractions (see Section 2.2). These results are contradicted by recent data of DeGraef (D2a) obtained on Heidenhain pouches which show very satisfactory correlation between mucoprotein and mucoproteose assay by means of tyrosine and hexosamine quantitation (see Fig. I8a). 

Obviously, incomplete reaction and losses during manipulation prevent the yield of 3-phenyl-2-thiohydantoin from reaching 100%. With each cycle of the Edman method, the yield of product derived from the newly exposed TV-terminus will decrease. In addition, small amounts of the 3-phenyl-2-thiohydantoins corresponding to earlier positions in the sequence will be formed as a consequence of incomplete reaction at each cycle. If the fraction of peptide that reacts with phenyl isothiocyanate and gives the relevant 3-phenyl-2-thiohydantoin is x, then the yield at any stage is... 

It is likely that adsorption methods may be useful for fractionating larger peptides, which cannot readily be separated by other methods... 

Many of the methods considered above have not been extensively used for the fractionation of peptides, so that it is impossible to know how far they may be applied and which are the most effective methods. Also it is to be expected that considerable improvements in these techniques will take place and that other new methods will be devised in the near future. 

Some of the substances that have been separated by this method are given in papers referred to by Morris and Morris (1964) amino acids, peptides (particularly those having molecular weights ranging from 500 to 5000), polypeptide antibiotics, proteins (including enzymes), carbohydrates (although for most compounds in this chemical class other fractionation methods are much more frequently applied), purines, pyrimidines, nucleic acid derivatives, tRNA s that are specific for various amino acids, organic acids, steroids, lipids, antibiotics that are not peptides, porphyrins, pterins, vitamin B,2 and other vitamins, lipoic acid, and alkaloids. The countercurrent-distribution procedure of Holley et al. (1965) is widely used, sometimes with modifications. Korte et al. (1965) have separated three isomers of tetrahydrocannabinol. 

In proteome studies, the process usually begins with a highly complex mixture of proteins, typically hundreds of unknown species. This requires one or several protein fractionation steps such as 1-D or 2-D gel electrophoresis or LC separation of the proteins and peptides prior to MALDI-MS analysis. The different types of fractionation methods can be combined in several ways for example, SDS-PAGE followed by LC separation of in-gel-digested peptides. In contrast to ESI, LC-separation techniques cannot be directly coupled to the MALDI instruments. Instead, the LC-separated peptide or protein fractions are spotted onto the MALDI-target, and then analyzed in an offline approach. 

The first edition of this book was written as a practical guide for scientists who are required to fractionate proteins, peptides, and polynucleotides. This edition includes many of the same topics, updated for discoveries of the past ten years. It also includes sections on new areas that have emerged from the field, such as the use of specialized detection systems. Many of the authors of these chapters are the pioneers who first attempted to use HPLC in the investigation of biomolecules in their areas of interest. If they as a group had not dared to try this technique in the 1970s, it would not be the fundamental method of protein and peptide characterization it is today. 

The second method also relies on site-specific chemical modification ofphosphoproteins (Oda et al., 2001). It involves the chemical replacement of phosphates on serine and threonine residues with a biotin affinity tag (Fig. 2.7B). The replacement reaction takes advantage of the fact that the phosphate moiety on phosphoserine and phosphothreonine undergoes -elimination under alkaline conditions to form a group that reacts with nucleophiles such as ethanedithiol. The resulting free sulfydryls can then be coupled to biotin to create the affinity tag (Oda et al., 2001). The biotin tag is used to purify the proteins subsequent to proteolytic digestion. The biotinylated peptides are isolated by an additional affinity purification step and are then analyzed by mass spectrometry (Oda et al., 2001). This method was also tested with phosphorylated (Teasein and shown to efficiently enrich phosphopeptides. In addition, the method was used on a crude protein lysate from yeast and phosphorylated ovalbumin was detected. Thus, as with the method of Zhou et al. (2001), additional fractionation steps will be required to detect low abundance phosphoproteins. 

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