Fractionation of Peptides

It has already been emphasized that a partial hydrolyzate of a protein is a complex mixture of closely related compounds, so that very sensitive 

Since amino acids and peptides contain several differently charged groups, they can be fractionated by methods which make use of differences in isoelectric point or electrophoretic mobility. The various techniques available for such separations have been comprehensively reviewed by Svensson (1948). 

A simple compartment type of apparatus may be used to separate a peptide mixture into basic, neutral and acidic fractions (Gordon et al., 1941, 1943 Sanger and Tuppy, 1951a). Since the simplification of the mixture is usually more important than the yield of peptides, it is often advisable to repeat the ionophoresis on each fraction, and in this way clear cut fractionations may be obtained with very little overlapping. This method is especially useful for separating the basic peptides. By 

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In this approach, the SCX column acts as a large peptide reservoir. With at least four times the loading capacity of RP column, S CX- LC greatly increases the number of digested proteins that can be analyzed and therefore allows detection of low-abundance proteins in the mixture. Distinct fractions of peptides are released from the SCX column onto the RP column with increasing salt concentrations. 

If the identity of the OP-labeled protein is unknown, a tagged OP, for example a biotinylated OP, can be used to identify the protein (Schopfer et ah, 2005). After the identity of the OP-labeled protein is known, identification of the OP-labeled peptide depends on separating it from contaminating peptides. We have found that the OP-labeled peptide is frequently not found by mass spectrometry unless it has been extensively purified. In some cases it is possible to identify the labeled peptide simply by LC-MS-MS, where an enzymatic digest of the isolated protein is subjected to liquid chromatography on a Cl8 nanocolumn and the effluent from the column is electrosprayed directly into the mass spectrometer. For other cases, extensive HPLC puri-flcation of the enzymatic digest is necessary to obtain a purifled fraction of peptides that can be introduced into the mass spectrometer. 

Analysis of Purified Peptide by MALDI-TOF Mass Spectroscopy The purified lyophilized fractions of peptides (and significant side products)... 

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... 

Note CL. renal clearance of agent GFR glomerular filtration rate F. free fraction of peptide in perfusate. 

Zip-Tips (Billerica, Massachusetts) are automation-compatible pipette tips that contain a chromatography sorbent held in the tip with a membrane. These tips allow for microscale solid-phase extraction to purify and preconcentrate samples. Some examples of this approach are sample cleanup step with isoelectric point (pl)-based fractionation of peptides prior to matrix-assisted laser-desorption/short ionization (MALDl) spotting [29] or chip-based ESI analysis of colon adenocarcinoma (Caco-2) screening samples [30]. The preliminary results of induction-based fluidics in which nanoliter volumes... 

The MALDI-TOF MS data indicated that the optimum pH was 7.5. The amount of proteins, such as peak a of Fig. 7, can be estimated fairly accurately by taking the MALDI-TOF MS several times (136). Based on 10 different MALDI-TOF MS measurements, it appeared that about one-half of peptide deformylase was cleaved on incubation with 0.19 equiv of the catalyst for 72 h (Fig. 7), which corresponds to ko of 0.010 h Here, the initial fraction of peptide deformylase complexed with the catalyst cannot exceed 20%. The ko observed when peptide deformylase is fully bound to the catalyst is cat- The lower limit of at is, therefore, estimated as 0.050 h For the myoglobincleaving catalyst Co BU, fecat was 0.022 under the same conditions (126, 127). 

It has already been emphasized (p. 14) that the best stage of hydrolysis at which to attempt the fractionation of peptides is at the point where enzyme action will proceed no farther or when there is a very sharp break in the hydrolysis curve. The disadvantage of a long incubation period is of course that the danger of rearrangements increases (see Linder-str0m-Lang and Ottesen, 1949). 

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