A high throughput dimer screening assay for monoclonal antibodies using chemical cross-linking and microchip electrophoresis

SummaryFibrinogen and fibrin related chains in reduced human plasma as well as the bonds interlinking partially cross-linked fibrin from plasma clots have been studied by means of 1D- and 2D electrophoresis and Western blotting. Immunovisualization of reduced plasma or partially cross-linked fibrin with monoclonal antibodies specific for the α-chains or the γ-chains have shown that several bands represent material belonging to both chains. In order to decide whether these bands constitute αγ-chain hybrids or superimposed α- and γ-chain dimers, the cross-linked material was separated according to both isoelectric point (pI) and molecular weight (MW) using Pharmacia’s Multiphor II system. Western blotting of the second dimension gels revealed that partially cross-linked fibrin contains αsγt-chain hybrids and γ- polymers, in addition to the well-known γ-dimers and α-polymers. The main αsγt-chain hybrid has a pI between that of the α- and the γ-chains, a MW of about 200 kDa and contains Aα-chains with intact fibrinopeptide A (FPA). It was also observed that soluble fibrinogen/fibrin complexes as well as partially cross-linked fibrin contain degraded α-dimers with MWs close to the γ-dimers. These findings demonstrate that factor XIII-catalyzed cross-linking of fibrin is a more complex phenomenon than earlier recognized.

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Chemical cross-linking detects association of insulin receptors with four different class I human leukocyte antigen molecules on cell surfaces

Diabetes ◽

10.2337/diabetes.42.4.619 ◽

1993 ◽

Vol 42 (4) ◽

pp. 619-625 ◽

Cited By ~ 4

Author(s):

J. Reiland ◽

M. Edidin

Keyword(s):

Human Leukocyte Antigen ◽

Human Leukocyte ◽

Insulin Receptors ◽

Class I ◽

Cross Linking ◽

Cell Surfaces ◽

Leukocyte Antigen ◽

Chemical Cross Linking

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Statistical Force-Field for Structural Modeling Using Chemical Cross-Linking/mass Spectrometry Distance Constraints

10.26434/chemrxiv.6030563 ◽

2018 ◽

Author(s):

Allan J. R. Ferrari ◽

Fabio C. Gozzo ◽

Leandro Martinez

Keyword(s):

Mass Spectrometry ◽

Amino Acid ◽

Force Field ◽

Protein Structures ◽

Protein Structure Determination ◽

Structural Modeling ◽

Cross Linking ◽

Amino Acid Residues ◽

Distance Constraints ◽

Chemical Cross Linking

<div><p>Chemical cross-linking/Mass Spectrometry (XLMS) is an experimental method to obtain distance constraints between amino acid residues, which can be applied to structural modeling of tertiary and quaternary biomolecular structures. These constraints provide, in principle, only upper limits to the distance between amino acid residues along the surface of the biomolecule. In practice, attempts to use of XLMS constraints for tertiary protein structure determination have not been widely successful. This indicates the need of specifically designed strategies for the representation of these constraints within modeling algorithms. Here, a force-field designed to represent XLMS-derived constraints is proposed. The potential energy functions are obtained by computing, in the database of known protein structures, the probability of satisfaction of a topological cross-linking distance as a function of the Euclidean distance between amino acid residues. The force-field can be easily incorporated into current modeling methods and software. In this work, the force-field was implemented within the Rosetta ab initio relax protocol. We show a significant improvement in the quality of the models obtained relative to current strategies for constraint representation. This force-field contributes to the long-desired goal of obtaining the tertiary structures of proteins using XLMS data. Force-field parameters and usage instructions are freely available at http://m3g.iqm.unicamp.br/topolink/xlff <br></p></div><p></p><p></p>

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