Correlation of electrophoretic mobilities from capillary electrophoresis with physicochemical properties of proteins and peptides

Rickard, Eugene C.; Strohl, M.M.; Nielsen, R.G.

doi:10.1016/0003-2697(91)90379-8

Cited by 236 publications

(136 citation statements)

References 20 publications

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“…Further, they indicated that, after some simplifications and transformations of Equation (3), a relationship similar to Equation (2) is obtainable. On the other hand, if K 2 (K 3 , Equation (4) becomes similar to the correlation observed by Rickard et al [115], where…”

Section: Determination Of Absolute Mobility and Of Its Relation To Chsupporting

confidence: 87%

Capillary electrophoretic analysis of proteins and peptides of biomedical and pharmacological interest

Righetti

2001

Biopharm & Drug Disp

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Capillary electrophoresis (CE) is an automated approach to electrokinetic separations that has had a deep impact in all fields of life sciences, including biomedical and biotechnological research and clinical and forensic practice. The present review highlights aspects of peptides and proteins separations, with particular emphasis on macromolecular analytes of biomedical interest. Among the various CE techniques available, a novel methodology is here illustrated consisting in separations in acidic, isoelectric buffers, which have the advantage of protonating the silica wall, thus minimizing interactions of proteinaceous material with the siliceous surface, while allowing delivery of high voltage gradients, due to their low conductivities. The review ends with applications of CE to the analysis of folding/unfolding/refolding/misfolding of proteins, a field which has deep implications in the biomedical arena, since it is connected to a host of disorders, such as prion protein diseases.

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Section: Determination Of Absolute Mobility and Of Its Relation To Chsupporting

confidence: 87%

Capillary electrophoretic analysis of proteins and peptides of biomedical and pharmacological interest

Righetti

2001

Biopharm & Drug Disp

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“…The terms C p and a in Equation 1 are constants. The theoretical value of a varies from 0.3 to 1.0, and depends on the model used to derive the hydrodynamic drag from the molecular weight; typically for globular proteins a % 2/3 (Rickard et al 1991;Basak and Ladisch 1995). Equation 1 expresses the mobility of rungs of a charge ladder by expressing the net charge, Z, as a function of the net charge of the unmodified protein (Z°B ) and the change in charge (n Á DZ) that results from n acetylations.…”

Section: Chemical Modification Of A-amylasementioning

confidence: 99%

Lysine acetylation can generate highly charged enzymes with increased resistance toward irreversible inactivation

et al. 2008

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This paper reports that the acetylation of lysine e-NH 3 + groups of a-amylase-one of the most important hydrolytic enzymes used in industry-produces highly negatively charged variants that are enzymatically active, thermostable, and more resistant than the wild-type enzyme to irreversible inactivation on exposure to denaturing conditions (e.g., 1 h at 90°C in solutions containing 100-mM sodium dodecyl sulfate). Acetylation also protected the enzyme against irreversible inactivation by the neutral surfactant TRITON X-100 (polyethylene glycol p-(1,1,3,3-tetramethylbutyl)phenyl ether), but not by the cationic surfactant, dodecyltrimethylammonium bromide (DTAB). The increased resistance of acetylated aamylase toward inactivation is attributed to the increased net negative charge of a-amylase that resulted from the acetylation of lysine ammonium groups (lysine e-NH 3 + ! e-NHCOCH 3 ). Increases in the net negative charge of proteins can decrease the rate of unfolding by anionic surfactants, and can also decrease the rate of protein aggregation. The acetylation of lysine represents a simple, inexpensive method for stabilizing bacterial a-amylase against irreversible inactivation in the presence of the anionic and neutral surfactants that are commonly used in industrial applications.

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“…5b). The pI values were calculated by summing the pI values of the single amino acids and dividing them by the number of the amino acids (19). The relative distribution of the normalized mycobacterial proteins showed a low number of proteins in the pI range of 7-8, an observation that has also been described for several other organisms (20 -22).…”

Section: Complementary Analysis Of M Tuberculosis By 2-de and Icatmentioning

confidence: 99%

Complementary Analysis of the Mycobacterium tuberculosis Proteome by Two-dimensional Electrophoresis and Isotope-coded Affinity Tag Technology

Schmidt

Donahoe

Hagens

et al. 2004

Molecular & Cellular Proteomics

169

138

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Classical proteomics combined two-dimensional gel electrophoresis (2-DE) for the separation and quantification of proteins in a complex mixture with mass spectrometric identification of selected proteins. More recently, the combination of liquid chromatography (LC), stable isotope tagging, and tandem mass spectrometry (MS/MS) has emerged as an alternative quantitative proteomics technology. We have analyzed the proteome of Mycobacterium tuberculosis, a major human pathogen comprising about 4,000 genes, by (i) 2-DE and mass spectrometry (MS) and by (ii) the isotope-coded affinity tag (ICAT) reagent method and MS/MS. The data obtained by either technology were compared with respect to their selectivity for certain protein types and classes and with respect to the accuracy of quantification. Initial datasets of 60,000 peptide MS/MS spectra and 1,800 spots for the ICAT-LC/MS and 2-DE/MS methods, respectively, were reduced to 280 and 108 conclusively identified and quantified proteins, respectively. ICAT-LC/MS showed a clear bias for high M r proteins and was complemented by the 2-DE/MS method, which showed a preference for low M r proteins and also identified cysteine-free proteins that were transparent to the ICAT-LC/MS method. Relative quantification between two strains of the M. tuberculosis complex also revealed that the two technologies provide complementary quantitative information; whereas the ICAT-LC/MS method quantifies the sum of the protein species of one gene product, the 2-DE/MS method quantifies at the level of resolved protein species, including post-translationally modified and processed polypeptides. Our data indicate that different proteomic technologies applied to the same sample provide complementary types of information that contribute to a more complete understanding of the biological system studied. with mass spectrometry (MS) and have revealed about 1,800 distinct spots separated by 2-DE (1). About 350 of these were identified, and the comparison of the protein patterns of virulent and attenuated strains identified several proteins that are being studied further as potential vaccine candidates (1, 2). More than two million deaths/year and eight million new infections are caused by M. tuberculosis, the bacterium responsible for tuberculosis (3). Genomics, transcriptomics, and proteomics, which form the rationale basis to develop new therapeutic and preventive strategies, have been applied to combat this disease. The complete genome of M. tuberculosis comprises about 4,000 genes, which were classified in six protein classes and 30 subclasses (4).The smallest unit of the proteome, the protein species, is defined by its chemical structure (5). Therefore, each modification of a protein leads to a new protein species. These are successfully resolved by 2-DE if they differ by at least one charge or by at least several hundred daltons in mass. Quantification by 2-DE requires a high degree of pattern reproducibility, which is difficult to achieve in a multistep and parallel procedure.To alleviate limi...

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Correlation of electrophoretic mobilities from capillary electrophoresis with physicochemical properties of proteins and peptides

Cited by 236 publications

References 20 publications

Capillary electrophoretic analysis of proteins and peptides of biomedical and pharmacological interest

Capillary electrophoretic analysis of proteins and peptides of biomedical and pharmacological interest

Lysine acetylation can generate highly charged enzymes with increased resistance toward irreversible inactivation

Complementary Analysis of the Mycobacterium tuberculosis Proteome by Two-dimensional Electrophoresis and Isotope-coded Affinity Tag Technology

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