Platform Methods to Characterize the Charge Heterogeneity of Three Common Protein Therapeutics by Imaged Capillary Isoelectric Focusing.

Imaged capillary isoelectric focusing (icIEF) is a gold standard method for characterizing the charge heterogeneity of protein therapeutics. A broad range of protein therapeutics such as monoclonal antibodies, antibody-drug conjugates (ADCs), and fusion proteins are routinely analyzed by icIEF due to its high resolution and high reproducibility. Platform methods, which can be applied without modification to the analysis of different protein therapeutics, save valuable time and resources in method development and quality control. Here, we provide platform methods for icIEF analysis of three classes of protein therapeutics, a biosimilar to the monoclonal antibody trastuzumab, recombinant human erythropoietin (rhEPO), and a fusion protein. The details of sample preparation and separation conditions for each molecule are described in this chapter.

Simple Western: Bringing the Western Blot into the Twenty-First Century.

The Western blot is widely used in the study of protein biochemistry, but it is notoriously labor-intensive, and it is limited in its reproducibility and quantification, among many other challenges. By contrast, capillary-based protein separation and immunodetection, known as Simple Western™, overcomes many of the challenges associated with the traditional Western blot, and it is quickly gaining traction as a replacement for traditional Western blot analysis. The advantages that capillary-based immunoassay offers include ease of use, automation, reproducibility, quantification, and even built-in total protein normalization. In this chapter, we describe protocols for the two basic types of capillary-based immunodetection assays, one by molecular weight separation and the other by charge separation. In both methods, protein samples are separated in the capillary followed seamlessly by immunodetection with chemiluminescent or fluorescent antibodies for highly sensitive and specific detection of target proteins.

2-DE with IPGs.

In order to overcome the limitations of carrier ampholyte generated pH gradients, IPGs were developed in the late 1970s. However, the 2-DE pattern we included in the first publication on IEF with IPGs [Bjellqvist et al., J. Biochem. Biophys. Methods 1982, 6, 317-339] was far from being competitive to O'Farrell's high-resolution 2-DE with carrier ampholytes. Our 2-DE pattern in this article was, more or less, only a proof of principle. It was, however, the beginning of a long journey of stepwise improved 2-DE protocols we developed in our laboratory and summarized in the reviews published in Electrophoresis 1988, 9, 531-546 and in Electrophoresis 2000, 21, 1037-1053. Milestones were the design of the IPG strip, and the "reduction-alkylation equilibration protocol" of IPG strips after IEF for the efficient transfer of proteins from first to second dimension. The protocol of 2-DE with IPGs has been constantly refined, e.g. by the generation of tailor-made IPGs with different pH intervals from the acidic to the basic extremes (pH 2.5-12), and extended separation distances for improved resolution. In the present review, a historical outline from the technical difficulties encountered during the development of 2-DE with IPGs, to the establishment of the actual "standard protocol" will be given, as well as the modified procedures for the separation of very acidic, very alkaline, low-abundance and hydrophobic proteins, followed by a brief discussion of the advantages and technical challenges of gel-based proteomic technologies.

Identification of potential substrate proteins for the periplasmic Escherichia coli chaperone Skp.

The "seventeen kilodalton protein" (Skp) is a predominant periplasmic chaperone of Escherichia coli, which is involved in the biogenesis of abundant outer membrane proteins (OMPs) such as OmpA, PhoE, and LamB. In this study the substrate profile of Skp was investigated in a proteomics approach. Skp was overexpressed in a deficient E. coli strain as a fusion protein with the Strep-tag and captured, together with any host proteins associated with it, from the periplasmic cell extract under mild conditions via one-step Strep-Tactin affinity chromatography. Copurified substrate proteins were then identified by high resolution 2-DE with immobilized pH-gradients, followed by MALDI-TOF MS. Apart from the known Skp substrates, including OmpA and LamB, more than 30 other interacting proteins were detected, especially from the outer membrane, among these FadL and BtuB, and from the periplasm such as MalE and OppA. Thus, Skp does not only serve as a specialized chaperone for a small set of OMPs, but it seems to exhibit a broader substrate spectrum, including soluble periplasmic proteins. These findings should prompt further investigation into the physiological role of Skp and may promote its use for the bacterial production of biochemically active heterologous proteins whose folding requires secretion into the oxidizing milieu of the periplasm.

Sample prefractionation in granulated sephadex IEF gels.

Prefractionation procedures not only aid in reducing sample complexity, but also permit loading of higher protein amounts within the separation range applied to two-dimensional electrophoresis (2-DE) gels and thus facilitate the detection of less abundant protein species. Hence we developed a simple, cheap, and fast prefractionation procedure based on flat-bed isoelectric focusing (IEF) in granulated Sephadex gels, containing chaotropes, zwitterionic detergents and carrier ampholytes. After IEF, up to ten Sephadex gel fractions alongside the pH gradient are obtained, and then applied directly onto the surface of the corresponding narrow pH range immobilized pH gradient (IPG) strips as first dimension of 2-DE. The major advantages of this technology are the highly efficient electrophoretic transfer of the prefractionated proteins from the Sephadex IEF fraction into the IPG strip without any sample dilution, and full compatibility with subsequent 2-DE, because the prefractionated samples have not to be eluted, concentrated or desalted, nor does the amount of the carrier ampholytes in the Sephadex fraction interfere with IEF in IPG strips. This sample prefractionation method has been successfully applied for the separation, detection and identification of low abundance proteins from pro- and eukaryotic samples.

Dual specificities of the glyoxysomal/peroxisomal processing protease Deg15 in higher plants.

Glyoxysomes are a subclass of peroxisomes involved in lipid mobilization. Two distinct peroxisomal targeting signals (PTSs), the C-terminal PTS1 and the N-terminal PTS2, are defined. Processing of the PTS2 on protein import is conserved in higher eukaryotes. The cleavage site typically contains a Cys at P1 or P2. We purified the glyoxysomal processing protease (GPP) from the fat-storing cotyledons of watermelon (Citrullus vulgaris) by column chromatography, preparative native isoelectric focusing, and 2D PAGE. The GPP appears in two forms, a 72-kDa monomer and a 144-kDa dimer, which are in equilibrium with one another. The equilibrium is shifted on Ca(2+) removal toward the monomer and on Ca(2+) addition toward the dimer. The monomer is a general degrading protease and is activated by denatured proteins. The dimer constitutes the processing protease because the substrate specificity proven for the monomer (Phi-Arg/Lys downward arrow) is different from the processing substrate specificity (Cys-Xxx downward arrow/Xxx-Cys downward arrow) found with the mixture of monomer and dimer. The Arabidopsis genome analysis disclosed three proteases predicted to be in peroxisomes, a Deg-protease, a pitrilysin-like metallopeptidase, and a Lon-protease. Specific antibodies against the peroxisomal Deg-protease from Arabidopsis (Deg15) identify the watermelon GPP as a Deg15. A knockout mutation in the DEG15 gene of Arabidopsis (At1g28320) prevents processing of the glyoxysomal malate dehydrogenase precursor to the mature form. Thus, the GPP/Deg15 belongs to a group of trypsin-like serine proteases with Escherichia coli DegP as a prototype. Nevertheless, the GPP/Deg15 possesses specific characteristics and is therefore a new subgroup within the Deg proteases.

A proteome analysis of Corynebacterium glutamicum after exposure to the herbicide 2,4-dichlorophenoxy acetic acid (2,4-D).

The herbicide 2,4-dichlorophenoxy acetic acid (2,4-D) induces a wide spectrum of toxic responses in living organisms. In this study, we analyzed the stress-induced responses of Corynebacterium glutamicum cells on protein level upon treatment with 2,4-D. For this, growing C. glutamicum cells were exposed to sublethal concentrations of 2,4-D, and changes of the gene expression profiles in comparison to non-exposed organisms were analyzed by two-dimensional gel electrophoresis and mass spectrometry. 2,4-D induced the over-expression of at least six C. glutamicum proteins, four of which could be identified by MALDI-TOF-MS. One protein (Cg2521; long-chain acyl-CoA synthetase) was related to the energy metabolism, and two proteins were involved in cell envelope synthesis (Cg2410; glutamine-dependent amidotransferase, and Cg1672; glycosyltransferase). The last induced protein was the ABC type transport system (Cg2695, ATPase component). The newly observed proteins, except for the ABC transport system, were not in general stress-related proteins, but were specifically expressed upon 2,4-D exposure and, therefore, can be used as respective biomarkers. Moreover, since these proteins seem to play a pivotal role in the adaptation of the cell to 2,4-D, they may help to gain deeper insight into the damage mechanisms of 2,4-D induced in the living cell.