Electrophoresis molecular mass determination

M. A. Cevalloa, C, Navarro-Duque, M. Vaiela-JuKa, and A. C. Alagon. Molecular mass determination and assay of venom byaluronidases by sodium dodecyl sulfate-poly sciylaraide gel electrophoresis. Tcvcwm 50 925 (1992). 

Mass spectrometry is more than 100 times more accurate than gel electrophoresis in molecular mass determination, and obtaining the data for sequence analysis requires only a fraction of the time needed for Edman degradation. Moreover, mass spectrometry is well suited for analysis of posttranslational modifications, which cannot be determined by Edman degradation. 

The membrane-bound H. saccharovorum ATPase can be purified to a specific activity of 3.6(imol Pj/min/mg protein [46]. The native enzyme (Mr 350000) is composed of four subunits. Molecular-mass determinations in the presence of sodium dodecylsulfate (SDS) provide Mr estimates of 87 000, 60 000, 29 000, and 20 000 for the subunits, designated I, II, III, and ly respectively [23], SDS gel electrophoresis overestimates the molecular masses of acidic proteins [70] and more accurate values can be obtained by electrophoresis in the presence of cetyltrimethylammonium bromide [71]. When this is done, the resulting values for Mr agree with the those obtained using SDS [46]. 

Tsuji, K. Sodium dodecyl sulfate polyacrylamide gel- and replaceable polymer-filled capillary electrophoresis for molecular mass determination of proteins of interest. J. Chromatogr. A, 666, 294, 1994a. 

Photo system I (PS I) preparations from barley contain polypeptides with apparent molecular masses of 82 (PSI-A and PSI-B), 18 (PSI-D), 16 (PSI-E), 14, 9.5 (PSI-H), 9 (PSI-C), 4, and 1.5 kDa (PSI-I) as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (1, 2). The nomenclature used for the subunits is described in more detail by Moller et al. (3). In this paper we report the characterization of a cDNA clone for the PsaE gene encoding the 16-kDa polypeptide PSI-E. The molecular mass of the mature polypeptide is 10,821 Da when deduced from the nucleotide sequence. To test whether the discrepancy between the molecular mass determinations could be due to post-translational modification of the polypeptide, the isolated polypeptide was analyzed by plasma desorption mass spectrometry. It has previously been established that the N- and C-terminal amino acid residues of the PSI-E polypeptide are not modified (4). 

Besides affinity chromatography, capillary zone electrophoresis can easily be coupled to an ESI high-performance RTOF mass spectrometer. The high accuracy achievable in molecular mass determination by newly developed reflectron mass spectrometers makes the assignment of isomeric glycoproteins feasible [93] and can furthermore be a powerful tool to discriminate between different serologically relevant molecules. 

Rothe, GM, Determination of Molecular Mass, Stoke radius. Frictional Coefficient and Isomer-Type of Non-denatured Proteins by Time-Dependent Pore Gradient Gel Electrophoresis, Electrophoresis 9, 307, 1988. 

SDS polyacrylamide gel electrophoresis (SDS-PAGE) represents the most commonly used analytical technique in the assessment of final product purity (Figure 7.1). This technique is well established and easy to perform. It provides high-resolution separation of polypeptides on the basis of their molecular mass. Bands containing as little as 100 ng of protein can be visualized by staining the gel with dyes such as Coomassie blue. Subsequent gel analysis by scanning laser densitometry allows quantitative determination of the protein content of each band (thus allowing quantification of protein impurities in the product). 

Figure 11.14 Determination of the relative molecular mass (RMM) of a protein by SDS electrophoresis.

Size-based analysis of SDS-protein complexes in polyacrylamide gels (SDS-PAGE) is the most common type of slab gel electrophoresis for the characterization of polypeptides, and SDS-PAGE is one of the most commonly used methods for the determination of protein molecular masses.117 The uses for size-based techniques include purity determination, molecular size estimation, and identification of posttranslational modifications.118119 Some native protein studies also benefit from size-based separation, e.g., detection of physically interacting oligomers. 

The independent determination of molecular masses by SDS-PAGE is impossible. To estimate the molecular mass of a protein, measure the path of that protein or calculate its Rf value (distance of the protein from origin/distance of electrophoresis front from origin) and compare these values with that of marker proteins, i.e., proteins with independently determined molecular masses. This method is successful only if the protein of interest behaves regularly in SDS-PAGE, i.e., it is totally unfolded by SDS, has a rod-like shape, and the SDS/protein ratio is the same for the unknown and the marker protein. Especially highly hydrophobic proteins and glycoproteins often deviate from these assumptions. 

You have isolated and purified a new enzyme (E) which converts a single substrate (S) into a single product (P). You have determined Mr by gel filtration as 46,400. However, in SDS gel electrophoresis, a molecular mass of 23 kDa was indicated for the single protein band observed. A solution of the enzyme was analyzed in the following way. The absorbance at 280 nm was found to be 0.512. A 1.00 ml portion of the same solution was subjected to amino acid analysis and was found to contain 71.3 nmol of tryptophan. N-terminal analysis on the same volume of enzyme revealed 23.8 nmol of N-terminal alanine. 

Fig. 2. SDS-PAGE. (a) Appearance of proteins after electrophoresis on an SDS polyacrylamide gel. Lane 1, proteins (markers) of known molecular mass lane 2, unpurified mixture of proteins lane 3, partially purified protein lane 4, protein purified to apparent homogeneity (b) determination of the molecular mass of an unknown protein by comparison of its electrophoretic mobility (distance migrated) with those of proteins (markers) of known molecular mass.

Subunit Composition of a Protein A protein has a molecular mass of 400 kDa when measured by gel filtration. When subjected to gel electrophoresis in the presence of sodium dodecyl sulfate (SDS), the protein gives three bands with molecular masses of 180, 160, and 60 kDa. When electrophoresis is carried out in the presence of SDS and dithiothreitol, three bands are again formed, this time with molecular masses of 160, 90, and 60 kDa. Determine the subunit composition of the protein. 

A. R. Ivanov, I. V. Nazimov, and L. Baratova, Determination of biologically-active low-molecular-mass thiols in human blood. II. High-performance capillary electrophoresis with photometric detection, J. Chromatogr. A 895, 167-171 (2000). 

Very precise appraisals of polydispersities due to branch defects can be made by HPLC fractionation, electrophoresis and mass-spectral analyses of the components. The molecular-mass and polydispersity values thus obtained, corroborate branch-defect ranges determined by high-field 13C-NMR spectroscopy. 

To study subunit structure, it is essential to determine the molecular mass of the holoenzyme by gel filtration and/or sucrose density gradient followed by determination of enzymatic activity. The size of the subunits can be determined by sodium dodecyl phosphate-polyacrylamide gel electrophoresis (SDS-PAGE). Put together, this information will show whether the enzyme is a monomer or a polymer and, if the latter, how many subunits make up the holoenzyme and whether there is only one kind of subunit or more than one kind. 

Synthetic polyelectrolytes can be separated by capillary electrophoresis applying the same rules derived for the electrophoresis of biopolymers. In the reptation regime, determination of the molecular mass and polydispersity of the polyelectrolytes is possible. Introduction of chromophores facilitates the detection of non-UV-absorbing polymers. Indirect detection techniques can probably be applied when analytes and chromophores of similar mobilities are available. 

Giavalisco, P., Nordhoff, E., Lehragh, H., Gobom, j., Klose, j., Mueller, M., Egelhoeer, V., Theiss, D., Seitz, H. (2003). Extraction of proteins from plant tissues for two-dimensional electrophoresis analysis A calibration method that simplifies and improves accurate determination of peptide molecular masses by MALDl-TOE MS. Electrophoresis 24, 207-216. 

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