Low‐SDS Blue native PAGE

Hepatitis B virus (HBV) infection is a major health concern with more than two billion individuals currently infected worldwide. Despite the prevalence of infection, gaining a complete understanding of the molecular mechanisms of HBV infection has been difficult because HBV cannot infect common immortalized cell lines. HepG2.2.15, however, is a well established version of the HepG2 cell line that constitutively expresses HBV. Therefore, comparative proteomics analysis of HepG2.2.15 and HepG2 may provide valuable clues for understanding the HBV virus life cycle. In this study, two-dimensional blue native/SDS-PAGE was utilized to characterize different multiprotein complexes from whole cell lysates between HepG2.2.15 and HepG2. These results demonstrate that two unique protein complexes existed in HepG2.2.15 cells. When these complexes were excised from the gel and subjected to the second dimension separation and the proteins were sequenced by mass spectrometry, 20 non-redundant proteins were identified. Of these proteins, almost 20% corresponded to heat shock proteins, including HSP60, HSP70, and HSP90. Antibody-based supershift assays were used to verify the validity of the distinct protein complexes. Co-immunoprecipitation assays confirmed that HSP60, HSP70, and HSP90 proteins physically interacted in HepG2.2.15 but not HepG2 cells. We further demonstrated that down-regulation of HSP70 or HSP90 by small interfering RNA significantly inhibited HBV viral production but did not influence cellular proliferation or apoptosis. Consistent with these results, a significant reduction in HepG2.2.15 HBV secretion was observed when the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin was used to treat HepG2.2.15 cells. Collectively these results suggest that the interaction of HSP90 with HSP70/HSP60 contributes to the HBV life cycle by forming a multichaperone machine that may constitute therapeutic targets for HBV-associated diseases.

Globulins are an important group of seed storage proteins in dicotyledonous plants. They are synthesized during seed development, assembled into very compact protein complexes, and finally stored in protein storage vacuoles (PSVs). Here, we report a proteomic investigation on the native composition and structure of cruciferin, the 12 S globulin of Brassica napus. PSVs were directly purified from mature seeds by differential centrifugations. Upon analyses by blue native (BN) PAGE, two major types of cruciferin complexes of ∼ 300-390 kDa and of ∼470 kDa are resolved. Analyses by two-dimensional BN/SDS-PAGE revealed that both types of complexes are composed of several copies of the cruciferin α and β polypeptide chains, which are present in various isoforms. Protein analyses by two-dimensional isoelectric focusing (IEF)/SDS-PAGE not only revealed different α and β isoforms but also several further versions of the two polypeptide chains that most likely differ with respect to posttranslational modifications. Overall, more than 30 distinct forms of cruciferin were identified by mass spectrometry. To obtain insights into the structure of the cruciferin holocomplex, a native PSV fraction was analyzed by single particle electron microscopy. More than 20,000 images were collected, classified, and used for the calculation of detailed projection maps of the complex. In contrast to previous reports on globulin structure in other plant species, the cruciferin complex of Brassica napus has an octameric barrel-like structure, which represents a very compact building block optimized for maximal storage of amino acids within minimal space.

The nucleus is a highly structured organelle with distinct compartmentalization of specific functions. To understand the functions of these nuclear compartments, detailed description of protein complexes which form these structures is of crucial importance. We explored therefore the potential of blue native PAGE (BN-PAGE) method for the separation of nuclear protein complexes. We focused on (i) solubility and stability of nuclear complexes under conditions prerequisite for the separation by BN-PAGE, (ii) improved separation of native nuclear protein complexes using 2-D colorless native/blue native PAGE (CN-/BN-PAGE), and (iii) mass spectrometric analysis of protein complexes which were isolated directly from native 1-D or from 2-D CN/BN-PAGE gels. The suitability of BN-PAGE for nuclear proteomic research is demonstrated by the successful separation of polymerase I and polymerase II complexes, and by mass spectrometric determination of U1 small nuclear ribonucleoprotein particle composition. Moreover, practical advice for sample preparation is provided.

Identification and characterization of multi-protein complexes is an important step toward an integrative view of protein-protein interaction networks that determine protein function and cell behavior. The limiting factor for identifying protein complexes is the method for their separation. Blue native PAGE (BN-PAGE) permits a high-resolution separation of multi-protein complexes under native conditions. To date, BN-PAGE has only been applicable to purified material. Here, we show that dialysis permits the analysis of multi-protein complexes of whole cellular lysates by BN-PAGE. We visualized different multi-protein complexes by immunoblotting including forms of the eukaryotic proteasome. Complex dynamics after gamma interferon stimulation of cells was studied, and an antibody shift assay was used to detect protein-protein interactions in BN-PAGE. Furthermore, we identified defined protein complexes of various proteins including the tumor suppressor p53 and c-Myc. Finally, we identified multi-protein complexes via mass spectrometry, showing that the method has a wide potential for functional proteomics.

Analysis of protein complexes is of increasing interest in the field of proteomics. A challenge is to develop methods for monitoring changes in the quantity and subunit composition of protein complexes on a proteome-wide scale. Here, we describe the combination of 1-D blue native polyacrylamide gel electrophoresis (BN-PAGE) with stable isotope labelling of amino acids in cell culture (SILAC) and tandem mass spectrometry (MS/MS). Cleared lysates from normal and perturbed samples, one incorporating heavy stable isotopes and the other light isotopes, are co-separated by blue native PAGE and then analysed and quantitated with MS/MS and appropriate software. This permits the analysis of cytoplasmic complexes. To demonstrate this technique, we explored how the 20S proteasome changes when the Pre9/α3 subunit, the only non-essential subunit of this complex, was deleted. Our results showed that ΔPre9/α3 cells can form the 20S proteasome complex, although with reduced efficiency. This involves an increase in expression of the α4 subunit. Our findings suggest this technique as an approach for the study of quantitative and qualitative differences in protein complexes, from cleared cell lysates.

Proteomic approach to characterize the supramolecular organization of photosystems in higher plants

Cystic fibrosis (CF) is a frequent autosomal recessive disorder caused by mutation of a gene encoding a multifunctional transmembrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), located in the apical membrane of epithelial cells lining exocrine glands. In an attempt to get a more complete picture of the pleiotropic effects of the CFTR defect on epithelial cells and particularly on the membrane compartment, a bidimensional blue native (BN)/SDS-PAGE-based proteomic approach was used on colonic crypt samples from control and CFTR knock-out mice (cftr-/-). This approach overcomes the difficulties of membrane protein analysis by conventional two-dimensional PAGE and is able to resolve multiprotein complexes. Used here for the first time on crude membrane proteins that were extracted from murine colonic crypts, BN/SDS-PAGE allows effective separation of protein species and complexes of various origins, including mitochondria, plasma membrane, and intracellular compartments. The major statistically significant difference in protein maps obtained with samples from control and cftr-/- mice was unambiguously identified as mClCA3, a member of a family of calcium-activated chloride channels considered to be key molecules in mucus secretion by goblet cells. On the basis of this finding, we evaluated the overall expression and localization of mClCA3 in the colonic epithelium and in the lung of mice by immunoblot analysis and immunohistochemistry. We found that mClCA3 expression was significantly decreased in the colon and lung of the cftr-/- mice. In an ex vivo assay, we found that the Ca2+-dependent (carbachol-stimulated) glycoprotein secretion strongly inhibited by the calcium-activated chloride channel blocker niflumic acid (100 microm) was impaired in the distal colon of cftr-/- mice. These results support the conclusion that a ClCA-related function in the CF colon depends on CFTR expression and may be correlated with the impaired expression of mClCA3.

Human embryonic stem cells (hESCs) have great potential for use in developmental biology studies, functional genomics applications, drug screening, and regenerative medicine. A detailed understanding of the molecular mechanisms that are responsible for maintaining the undifferentiated and pluripotent nature of hESCs is essential for their effective therapeutic application. It has become evident that many complex cellular processes are carried out by assemblies of protein molecules (protein complexes). Blue native polyacrylamide gel electrophoresis (BN-PAGE) has been used to separate protein complexes from whole cell lysates. Using BN-PAGE, we resolved cytoplasmic and membrane-associated complexes from hESCs and characterised their composition, stoichiometry, and dynamics by denaturing SDS-PAGE. The reliability of the fractionation was examined by western blot analysis of membrane and cytosolic markers. MALDI TOF/TOF mass spectrometry identified 119 cytosolic and 69 membrane proteins from the BN-PAGE proteome maps. Potential protein complexes were validated by computational prediction of possible protein-protein interactions using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database. Based on BN-PAGE gels and validation by databases, 82 heteromultimeric and 47 homomultimeric protein complexes have been found in hESCs. Resolving some of the protein complexes provided insight into the function of previously uncharacterised complexes in hESCs.

The field of protein residue network (PRN) research has brought several useful methods and techniques for structural analysis of proteins and protein complexes. Many of these are ripe and ready to be used by the proteomics community outside of the PRN specialists. In this paper we present software which collects an ensemble of (network) methods tailored towards the analysis of protein-protein interactions (PPI) and/or interactions of proteins with ligands of other type, e.g. nucleic acids, oligosaccharides etc. In parallel, we propose the use of the network differential analysis as a method to identify residues mediating key interactions between proteins. We use a model system, to show that in combination with other, already published methods, also included in pyProGA, it can be used to make such predictions. Such extended repertoire of methods allows to cross-check predictions with other methods as well, as we show here. In addition, the possibility to construct PRN models from various kinds of input is so far a unique asset of our code. One can use structural data as defined in PDB files and/or from data on residue pair interaction energies, either from force-field parameters or fragment molecular orbital (FMO) calculations. pyProGA is a free open-source software available from https://gitlab.com/Vlado_S/pyproga.

The presence of many salts, such as sodium chloride, can adversely affect the performance of native electrospray ionization mass spectrometry for the analysis of proteins and protein complexes by reducing the overall molecular ion abundances and distributing signal for any given charge state into many cationized forms with various numbers of adducts attached. Several solution additives, such as ammonium bromide, ammonium iodide, and NaSbF(6), can significantly lower the extent of sodium ion adduction to the molecular ions of proteins and protein complexes. For ubiquitin, addition of 25 mM ammonium bromide or ammonium iodide into aqueous solutions also containing 1.0 mM NaCl results in a factor of 72 and 56 increase, respectively, in the relative abundances of the fully protonated molecular ions compared to when these additives are not present. The effectiveness of this method for reducing sodium ion adduction is related to the low proton affinity (PA) values of the anions. Anions with very low PA also have a propensity to adduct as an acid molecule, but these adducts can be readily dissociated from the molecular ions either by activation in the source or subsequently by collisional activation in the mass spectrometer. This method of reducing sodium ion adduction to proteins is simple and requires no experimental modifications, making it an attractive alternative to other methods for desalting proteins prior to mass spectrometry analysis.

SAXS Ensemble Refinement of ESCRT-III CHMP3 Conformational Transitions

Characterization of protein higher-order structures and dynamics is essential for understanding the biological functions of proteins and revealing the underlying mechanisms. Top-down mass spectrometry (MS) accesses structural information at both the intact protein level and the peptide fragment level. Native top-down MS allows analysis of a protein complex's architecture and subunits' identity and modifications. Top-down hydrogen/deuterium exchange (HDX) MS offers high spatial resolution for conformational or binding interface analysis and enables conformer-specific characterization. A microfluidic chip can provide superior performance for front-end reactions useful for these MS workflows, such as flexibility in manipulating multiple reactant flows, integrating various functional modules, and automation. However, most microchip-MS devices are designed for bottom-up approaches or top-down proteomics. Here, we demonstrate a strategy for designing a microchip for top-down MS analysis of protein higher-order structures and dynamics. It is suitable for time-resolved native MS and HDX MS, with designs aiming for efficient ionization of intact protein complexes, flexible manipulation of multiple reactant flows, and precise control of reaction times over a broad range of flow rates on the submicroliter per minute scale. The performance of the prototype device is demonstrated by measurements of systems including monoclonal antibodies, antibody-antigen complexes, and coexisting protein conformers. This strategy may benefit elaborate structural analysis of biomacromolecules and inspire method development using the microchip-MS approach.

Efficient and non-denaturing membrane solubilization combined with enrichment of membrane protein complexes by detergent/polymer aqueous two-phase partitioning for proteome analysis

Chloroplast Research Methods: Probing The Targeting, Localization And Interactions Of Chloroplast Proteins.

Alteration of mitochondrial protein complexes in relation to metabolic regulation under short-term oxidative stress in Arabidopsis seedlings