Comparison of selected analytical techniques for protein sizing, quantitation and molecular weight determination

Goetz, H.; Kuschel, Meike; Wulff, Tanja; Sauber, Christian; Miller, C G; Fisher, Seth; Woodward, Clifford B.

doi:10.1016/j.jbbm.2004.01.007

Cited by 103 publications

(82 citation statements)

References 16 publications

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“…Protein standards, obtained from Sigma, were cytochrome c (12 kDa), carbonic anhydrase (29 kDa), ovalbumin (44 kDa), bovine serum albumin (66 kDa), gamma globulin (157 kDa), and thyroglobulin (670 kDa). Blue dextran and DTT were used to determine the void and total column volumes, respectively, to enable calculation of the distribution coefficient K av according to the equation (19,45) …”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Identification of an l -Rhamnose Synthetic Pathway in Two Nucleocytoplasmic Large DNA Viruses

Chothi

Duncan

Armirotti

et al. 2010

J Virol

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Section: Methodsmentioning

confidence: 99%

“…However, retention times in size exclusion chromatography can be influenced by the shape of the protein (19). We cannot presently draw any definitive conclusion about the 17-kDa species.…”

mentioning

confidence: 95%

Identification of an l -Rhamnose Synthetic Pathway in Two Nucleocytoplasmic Large DNA Viruses

Chothi

Duncan

Armirotti

et al. 2010

J Virol

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“…Proteolytic digestions of mAbs were performed at physiological pH and ionic strength as previously described (1). IgG cleavage was assessed by Agilent Biosizing microcapillary electrophoresis in SDS denaturing solution without reduction of disulfide bonds (31). Purification of mAb fragments used protein A adsorption steps and yielded Fab and F(abЈ) 2 without detectable levels of Fc-containing materials.…”

Section: Proteases and Igg Digestionsmentioning

confidence: 99%

Human Anti-IgG1 Hinge Autoantibodies Reconstitute the Effector Functions of Proteolytically Inactivated IgGs

Brezski¹,

Luongo²,

Petrone³

et al. 2008

The Journal of Immunology

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A number of proteases of potential importance to human physiology possess the ability to selectively degrade and inactivate Igs. Proteolytic cleavage within and near the hinge domain of human IgG1 yielded products including Fab and F(ab′)2 possessing full Ag binding capability but absent several functions needed for immune destruction of cellular pathogens. In parallel experiments, we showed that the same proteolytically generated Fabs and F(ab′)2s become self-Ags that were widely recognized by autoantibodies in the human population. Binding analyses using various Fab and F(ab′)2, as well as single-chain peptide analogues, indicated that the autoantibodies targeted the newly exposed sequences where proteases cleave the hinge. The point of cleavage may be less of a determinant for autoantibody binding than the exposure of an otherwise cryptic stretch of hinge sequence. It was noted that the autoantibodies possessed an unusually high proportion of the IgG3 isotype in contrast to Abs induced against foreign immunogens in the same human subjects. In light of the recognized potency of IgG3 effector mechanisms, we adopted a functional approach to determine whether human anti-hinge (HAH) autoantibodies could reconstitute the (missing) Fc region effector functions to Fab and F(ab′)2. Indeed, in in vitro cellular assays, purified HAH autoantibodies restored effector functions to F(ab′)2 in both Ab-dependent cellular cytotoxicity and complement-dependent cytotoxicity assays. The results indicate that HAH autoantibodies selectively bind to proteolytically cleaved IgGs and can thereby provide a surrogate Fc domain to reconstitute cell lytic functions.

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“…In comparison to other analytical techniques such as SDS-PAGE, capillary gel electrophoresis, or size exclusion chromatography, MALDI MS provides superior mass resolution and accuracy independent of the three-dimensional structure of the analyte. [14][15][16] High accuracy mass information is key, for example, for determining the stoi-chiometry of protein-protein, 3,4,17,18 protein-DNA, 19 or protein-RNA complexes. 20 Exact mass information is also used for the determination of the degree and the kind of post-translational modifications (PTMs), 7 PEGylations, 21 as well as for determining truncation of the primary sequence.…”

Section: Introductionmentioning

confidence: 99%

A New, Modular Mass Calibrant for High-Mass MALDI-MS

et al. 2013

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The application of matrix-assisted laser desorption/ionization mass spectrometry (MALDIMS) for the analysis of high-mass proteins requires suitable calibration standards at high m/z ratios. Several possible candidates were investigated, and concatenated polyproteins based on recombinantly expressed maltodextrin-binding protein (MBP) are shown here to be well suited for this purpose. Introduction of two specific recognition sites into the primary sequence of the polyprotein allows for the selective cleavage of MBP3 into MBP and MBP2. Moreover, these MBP2 and MBP3 oligomers can be dimerized specifically, such that generation of MPB4 and MBP6 is possible as well. With the set of calibrants presented here, the m/z range of 40-400 kDa is covered. Since all calibrants consist of the same species and differ only in mass, the ionization efficiency is expected to be similar. However, equimolar mixtures of these proteins did not yield equal signal intensities on a detector specifically designed for detecting high-mass molecules. and MBP 6 is possible as well. With the set of calibrants presented here, the m/z range of 40-400 kDa is covered. Since all calibrants consist of the same species and differ only in mass, the ionization efficiency is expected to be similar. However, equimolar mixtures of these proteins did not yield equal signal intensities on a detector specifically designed for detecting high-mass molecules.

show abstract

Comparison of selected analytical techniques for protein sizing, quantitation and molecular weight determination

Cited by 103 publications

References 16 publications

Identification of an l -Rhamnose Synthetic Pathway in Two Nucleocytoplasmic Large DNA Viruses

Identification of an l -Rhamnose Synthetic Pathway in Two Nucleocytoplasmic Large DNA Viruses

Human Anti-IgG1 Hinge Autoantibodies Reconstitute the Effector Functions of Proteolytically Inactivated IgGs

A New, Modular Mass Calibrant for High-Mass MALDI-MS

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