High-throughput sodium dodecyl sulfate polyacrylamide gel electrophoresis by linking conventional gel plates with automated liquid handler via Gel Adaptor

The automation of SDS-PAGE required substantial investment. To lower this barrier, we devised a cost-effective, simple, and general method where samples are loaded on SDS-PAGE gels held by a newly devised "Gel Adaptor" on a general automated liquid handler. In this method, the liquid handler loads samples on the SDS-PAGE gels held at an angle of 80 degrees on the Gel Adaptor so that the samples can be vertically loaded on the narrow paths of the wells of the slanted gels. This method allows the automated liquid handler to load 144 samples on SDS-PAGE gels in approximately 10 min, greatly reducing the time and cost for high-throughput SDS-PAGE analyses.     

Adaptive contrast enhancement of two-dimensional electrophoretic protein gel images facilitates visualization, orientation and alignment

2-DE is a powerful technique to discriminate post-translationally modified protein isoforms. However, all steps of 2-DE preparation and gel-staining may introduce unwanted artefacts, including inconsistent variation of background intensity over the entire 2-DE gel image. Background intensity variations limit the accuracy of gel orientation, overlay alignment and spot detection methods. We present a compact and efficient denoising algorithm that adaptively enhances the image contrast and then, through thresholding and median filtering, removes the gray-scale range covering the background. Applicability of the algorithm is demonstrated on immunoblots, isotope-labeled gels, and protein-stained gels. Validation is performed in contexts of (i) automatic gel orientation based on Hough transformation, (ii) overlay alignment based on cross correlation and (iii) spot detection. In gel stains with low background variability, e.g. Sypro Ruby, denoising will lower the spot detection sensitivity. In gel regions with high background levels denoising enhances spot detection. We propose that the denoising algorithm prepares images with high background for further automatic analysis, without requiring manual input on a gel-to-gel basis.     

Sequence‐Specific DNA Alkylation Targeting for Kras Codon 13 Mutation by Pyrrole–Imidazole Polyamide seco‐CBI Conjugates

Hairpin N‐methylpyrrole‐N‐methylimidazole polyamide seco‐CBI conjugates 2 – 6 were designed for synthesis by Fmoc solid‐phase synthesis, and their DNA‐alkylating activities against the Kras codon 13 mutation were compared by high‐resolution denaturing gel electrophoresis with 225 base pair (bp) DNA fragments. Conjugate 5 had high reactivity towards the Kras codon 13 mutation site, with alkylation occurring at the A of the sequence 5′‐ACGTCACC A ‐3′ (site 2), including minor 1 bp‐mismatch alkylation against wild type 5′‐ACG C CACC A ‐3′ (site 3). Conjugate 6 , which differs from conjugate 5 by exchanging one Py unit with a β unit, showed high selectivity but only weakly alkylated the A of 5′‐ACGTCACC A ‐3′ (site 2). The hairpin polyamide seco‐CBI conjugate 5 thus alkylates according to Dervan′s pairing rule with the pairing recognition which β/β pair targets T–A and A–T pairs. SPR and a computer‐minimized model suggest that 5 binds to the target sequence with high affinity in a hairpin conformation, allowing for efficient DNA alkylation.     

Myofibroblasts are important contributors to human hepatocellular carcinoma: Evidence for tumor promotion by proteome profiling

Liver cancer typically occurs with a background of chronic fibrosis, characterized by the accumulation of myofibroblast‐like cells. We performed 2D‐PAGE‐based comparative analyses with the aim to identify proteins expressed in human hepatocellular carcinoma (HCC) tissue but not in neighboring healthy liver tissue, and to make out which cell types are responsible for the expression of proteins most characteristic for HCC. LC‐MS/MS analysis of the most striking spots identified proteins that were mainly related to myofibroblast‐like cells. To gain more insights into the role of these cells in their contribution to HCC, we isolated myofibroblast‐like cells as well as hepatocytes, both derived from HCC tissues, and subjected them to proteome profiling based on shotgun experiments. Comparative analysis, also referring to proteome profiles of other cell types previously investigated by us, pointed again to a marked contribution of myofibroblast‐like cells to HCC. Intriguingly, secretome analysis of these cells identified several growth factors that may act as tumor promoters and several proteins that have been described as potential biomarkers for HCC including dickkopf‐1, connective tissue growth factor, and CXCL1. Other biomarker candidates presently identified in the secretome of myofibroblasts, including lipocalin‐1 and pappalysin‐1, may be selected for future clinical validation. The identification of myofibroblast‐like cells as important source of tumor‐promoters may open new avenues to therapeutic intervention by targeting these stroma cells in addition to the cancer cells.     

Peptide fractionation by acid pH SDS-free electrophoresis

SDS-free polyacrylamide gel electrophoresis is an effective alternative approach to peptide fractionation. Here we describe a discontinuous buffer system at acid pH that improves the separation of acidic peptides from tryptic digestion. MOPS and chloride act as trailing and leading ions, respectively, in this system, while histidine operates as counterion and buffers all solutions. In these electrophoretic conditions, peptides with pI below 5.5 migrate with low overall electrophoretic mobilities but high differences from one another, which allows for their efficient resolution. In silico analysis of several proteomes shows that the acid pH system allows a peptide simplification of 2.5-fold with respect to the total peptide mixture, and still a proteome coverage of about 95% is achievable. A straightforward method with a protocol including proteomic studies was achieved for SDS-PAGE of proteins, enzyme treatment and further peptide fractionation by SDS-free acid PAGE.     

Glycoproteomic analysis and molecular modeling of haptoglobin multimers

Extra-thiol groups on the α-subunit allow haptoglobin (Hp) to form a variety of native multimers which influence the biophysical and biological properties of Hp. In this work, we demonstrated how differences of multimeric conformation alter the glycosylation of Hp. The isoform distributions of different multimers were examined by an alternative approach, i.e. 3-D-(Native/IEF/SDS)-PAGE, which revealed differences in N-glycosylation among individual multimers of the same Hp sample. Glycomic mapping of permethylated N-glycan indicated that the assembled monomer and multimeric conformation modulate the degree of glycosylation, especially the reduction in terminal sialic acid residues on the bi-antennary glycan. Loss of the terminal sialic acid in the higher order multimers increases the number of terminal galactose residues, which may contribute to conformation of Hp. A molecular model of the glycosylated Hp multimer was constructed, suggesting that the effect of steric hindrance on multimeric formation is critical for the enlargement of the glycan moieties on either side of the monomer. In addition, N241 of Hp was partially glycosylated, even though this site is unaffected by steric consideration. Thus, the present study provides evidence for the alteration of glycan structures on different multimeric conformations of Hp, improving our knowledge of conformation-dependent function of this glycoprotein.     

Quantitative analysis of dissociation of LDH by high pressure native PAGE

ABSTRACT

High pressure native polyacrylamide gel electrophoresis has been designed to visualize the dissociation/association process of protein complexes. This paper reports this methodology in more quantitative way by inspecting pressure dissociation of pig heart lactate dehydrogenase, a tetrameric protein, which was extensively investigated in spectroscopic methods. We observed the change of electrophoresis pattern with pressure up to 150?MPa. By optimizing the buffer system and careful image analysis of the stained gels, we quantified all the dissociates in the process of pressurization. We discussed the characteristics of our methodology by comparing the result with the previously reported.     

Physical interpretation of the L(r) parameter in the theory for the gel electrophoresis of partially denatured DNA

Partial strand melting of dsDNA during gel electrophoresis typically results in an abrupt reduction of mobility. Several DNA analysis technologies are based on this phenomenon. Inspired by the de Gennes' theory for the reptation of branched polymers in gels, Lerman et al. (Ann. Rev. Biophys. Bioeng. 1984, 13, 399-423) proposed a mathematical expression to predict this reduced mobility. The latter contains only two parameters: the average number of denatured bases p (which can be obtained using a theory for DNA melting) and a constant L(r). However, there is confusion in the literature regarding the physical interpretation of L(r) and little is known about its dependence upon experimental parameters. The purpose of this short communication is to derive an explicit equation for the parameter L(r) from the de Gennes theory of reptation. Our derivation shines light on the meaning of L(r), clarifies the scope of the underlying approximations, and makes predictions about the dependence of L(r) upon the gel pore size and the persistence length of ssDNA.     

Proteomic profiling of human colon cancer cells treated with the histone deacetylase inhibitor belinostat

The anticancer drug belinostat is a hydroxamate histone deacetylase inhibitor that has shown significant antitumour activity in various tumour models and also in clinical trials. In this study, we utilized a proteomic approach in order to evaluate the effect of this drug on protein expression in the human colon cancer cell line HCT116. Protein extracts from untreated HCT116 cells, and cells grown for 24 h in the presence of 1 and 10 μM belinostat were analysed by 2‐D gel electrophoresis. Proteins were visualized by colloidal Coomassie blue staining and quantitative analysis of gel images revealed 45 unique differentially expressed proteins that were identified by LC‐MSMS analysis. Among these proteins, of particular interest are the downregulated proteins nucleophosmin and stratifin, and the upregulated proteins nucleolin, gelsolin, heterogeneous nuclear ribonucleoprotein K, annexin 1, and HSP90B that all were related to the proto‐oncogene proteins p53, Myc, activator protein 1, and c‐fos protein. The modulation of these proteins is consistent with the observations that belinostat is able to inhibit clonogenic cell growth of HCT116 cells and the biological role of these proteins will be discussed.     

Sample pooling in 2-D gel electrophoresis: a new approach to reduce nonspecific expression background

Protein expression alterations unrelated to an investigated phenotype are accumulated in most cell line models during establishment. Performing a whole proteome screening of lymphoma cell lines, we established a method to reduce the influence of protein expression unrelated to the distinct investigated phenotype. In 2-D PAGE, the comprehensive analysis of a large number of protein spots would be simplified by pooling cell line samples of the investigated phenotype. Applying this pooling approach, unrelated alterations of single samples are 'muted' by dilution. Analysing two different lymphoma subtypes (follicular and mantle cell lymphoma) by this method, spots originating in only single cell lines were reduced by 72% (650/900), whereas even modestly altered expression of protein spots detected in all lines were reliably detected in the pooled protein gels. We conclude that our pooling approach is a preferable approach to reliably detect a common protein expression pattern and may even allow proteomic analysis of clinical samples with limited amounts of sample material, even with minimal cell numbers as low as 1 x 10(6).