Peptide mapping of complex proteins at the low-picomole level with capillary electrophoretic separations

Abstract

A variety of different peptide-mapping schemes are presented, with emphasis on the development of procedures which can be done with limited quantities (i.e. 5 pmol) of protein. Results are obtained from model proteins which contain disulfide bonds, which must be broken prior to fragmentation of the protein. A reaction involving the simultaneous use of tributylphosphine and 2-methylaziridine to reduce and alkylate the disulfide bonds is employed, due to favorable attributes of these reagents for the scaled-down procedure. The traditional performic acid oxidation reaction to cleave cystine groups is also successfully used with low-picomole quantities of protein. Three different protein digestion reagents are used: trypsin, chymotrypsin, and cyanogen bromide. Each reagent produces a unique mixture of peptides. Capillary electrophoresis is used to separate the peptides, offering high separation efficiencies, short analysis times, and compatibility with small sample sizes. In addition to the conventional use of UV detection for underivatized peptides, laser-induced fluorescence detection is employed in conjunction with an arginine-selective derivatization reaction. This latter procedure for derivatization and detection offers an alternative peptide-mapping mode, in which only the arginine-containing peptides are detected, and is useful in simplifying the peptide maps of large proteins.

Screening and characterization of biopharmaceuticals by high‐performance capillary electrophoresis with laserinduced native fluorescence detection

“…The need for low concentration and mass sensitivity in peptide mapping is necessary to minimize disturbance of the culture media or purification process during sampling and allow the expressed protein to be sequenced even if it is present at very low concentrations. The results of Cobb and Nowtny [28,29]show that the sequencing of 2 pmol(4 VM) of Ij-casein using a reactor column filled with a trypsin-immobilized agarose gel followed by analysis with microcolumn HPLC or HPCE is attainable. However, the poor sensitivity of absorbance detectors and the required sample handling prevented a further decrease in sample size.…”

“…However, the poor sensitivity of absorbance detectors and the required sample handling prevented a further decrease in sample size. Although LIF detection of labeled peptides after digestion has been attempted, derivatization necessitates dilution, thereby increasing the initial sample size compared to no derivatization [29]. Recently, Amankwa and Kuhr [30] have succeeded in carrying out digestion reactions in 50 pm ID trypsin-immobilized open tubes and, hence, opened up the feasibility of performing online digestion and separation.…”

On‐line protein digestion and peptide mapping by capillary electrophoresis with post‐column labeling for laser‐induced fluorescence detection

“…The detection of peptide fragments can be enhanced by precolumn derivatization with fluorescent tags [22]. However, labeling peptides with fluorophores will change the peptides' mobility, and therefore, the peptide map is not comparable with that obtained by UV or mass spectrometry detection.…”

Separations of proteins and proteolytic digests of proteins by capillary electrophoresis on superox‐coated open tubular columns

“…Tryptic digests were separated by reversed phase HPLC, isoelectric focusing, and CE in modified capillaries at a pH of 2.7, with favorable results for the peptides, although closely related proteins could not be separated. Cobb and Novotny [28,29] developed procedures for the peptide mapping of complex proteins at the low-picomole level. Immobilized enzymes in an agarose gel minireactor allowed digestion of as little as 50 ng of protein.…”