Monitoring protein expression in whole-cell extracts by targeted label- and standard-free LC-MS/MS

Targeted quantification of proteins is a daily task in biological research but often relies on techniques such as western blotting that are only barely quantitative. Here we present a broadly applicable workflow for protein quantification from unpurified whole-cell extracts that can be completed in less than 3 d. Without prefractionation or affinity enrichment, a whole-cell extract is trypsin-digested in an acetonitrile-containing ammonium carbonate buffer and high-molecular-weight compounds are removed by filtration. A normalization strategy, which involves endogenous reference proteins, facilitates the determination of relative changes in protein expression without requiring isotope labeling or standard addition. On a triple-quadrupole mass spectrometer, we demonstrate standard-free quantification of yeast proteins present over five orders of magnitude and present at ≥500 copies per cell. Liquid chromatography/multiple reaction monitoring (LC-MRM)-based proteomics is therefore a next-generation alternative to western blotting, as it allows simultaneous and reliable quantification of multiple endogenous proteins without the need for enrichment, isotope labeling or use of antibodies.     

A Defined Methodology for Reliable Quantification of Western Blot Data

Chemiluminescent western blotting has been in common practice for over three decades, but its use as a quantitative method for measuring the relative expression of the target proteins is still debatable. This is mainly due to the various steps, techniques, reagents, and detection methods that are used to obtain the associated data. In order to have confidence in densitometric data from western blots, researchers should be able to demonstrate statistically significant fold differences in protein expression. This entails a necessary evolution of the procedures, controls, and the analysis methods. We describe a methodology to obtain reliable quantitative data from chemiluminescent western blots using standardization procedures coupled with the updated reagents and detection methods.     

Expression screen by enzyme-linked immunofiltration assay designed for high-throughput purification of affinity-tagged proteins

High-throughput purification of affinity-tagged fusion proteins is currently one of the fastest developing areas of molecular proteomics. A prerequisite for success in protein purification is sufficient soluble protein expression of the target protein in a heterologous host. Hence, a fast and quantitative evaluation of the soluble-protein levels in an expression system is one of the key steps in the entire process. Here we describe a high-throughput expression screen for affinity-tagged fusion proteins based on an enzyme linked immunofiltration assay (ELIFA). An aliquot of a crude Escherichia coli extract containing the analyte, an affinity-tagged protein, is adsorbed onto the membrane. Subsequent binding of specific antibodies followed by binding of a secondary antibody horseradish peroxidase (HRP) complex then allows quantitative evaluation of the analyte using tetramethylbenzidine as the substrate for HRP. The method is accurate and quantitative, as shown by comparison with results from western blotting and an enzymatic glutathione S-transferase (GST) assay. Furthermore, it is a far more rapid assay and less cumbersome than western blotting, lending itself more readily to high-throughput analysis. It can be used at the expression level (cell lysates) or during the subsequent purification steps to monitor yield of specific protein.     

Cy5 total protein normalization in Western blot analysis

Western blotting is a widely used method for analyzing specific target proteins in complex protein samples. Housekeeping proteins are often used for normalization to correct for uneven sample loads, but these require careful validation since expression levels may vary with cell type and treatment. We present a new, more reliable method for normalization using Cy5-prelabeled total protein as a loading control. We used a prelabeling protocol based on Cy5 N-hydroxysuccinimide ester labeling that produces a linear signal response. We obtained a low coefficient of variation (CV) of 7% between the ratio of extracellular signal-regulated kinase (ERK1/2) target to Cy5 total protein control signals over the whole loading range from 2.5 to 20.0 μg of Chinese hamster ovary cell lysate protein. Corresponding experiments using actin or tubulin as controls for normalization resulted in CVs of 13 and 18%, respectively. Glyceraldehyde-3-phosphate dehydrogenase did not produce a proportional signal and was not suitable for normalization in these cells. A comparison of ERK1/2 signals from labeled and unlabeled samples showed that Cy5 prelabeling did not affect antibody binding. By using total protein normalization we analyzed PP2A and Smad2/3 levels with high confidence.     

蛋白免疫印迹法检测小分子蛋白的实验条件优化研究

目的:蛋白免疫印迹法自发明以来被广泛应用于现代生物学研究中的蛋白质定性和半定量分析。为了提高蛋白免疫印迹法的检测效率,需针对不同蛋白的特性调节相关的实验条件参数。本文旨在探讨免疫印迹法不同参数对小分子蛋白检测效果的影响,从而优化并获得最佳实验条件。方法:比较不同转膜电压和时间、转移缓冲液甲醇含量、不同化学发光剂对小分子蛋白的检测效果。结果:选择20 V、10 min转膜电压和时间所获得的信号显著高于10 V、25 min转膜条件,选择含20%甲醇转移缓冲液所获得的信号显著高于无甲醇转移缓冲液,选择飞克级化学发光剂所获得的信号显著高于纳克级化学发光剂。结论:选用高电压、短时间组合,选择含20%甲醇转移缓冲液和飞克级化学发光剂信号均有助于小分子蛋白免疫印迹检测。     

Proteome analysis of human pancreatic cancer cell lines with highly liver metastatic potential by antibody microarray

Antibody microarrays have been successfully used to determine relative abundance of key proteins in various cancers and other diseases. We have previously showed liver metastatic-related genes between the metastatic pancreatic cancer line (SW1990HM) and its parental line (SW1990). In this study, we searched for potential markers for metastatic progression using antibody microarrays. The SpringBio Antibody Microarrays were used to analysis the different proteomes between SW1990HM and SW1990 cells. A standard ≥2.0-fold cutoff value was used to determine differentially expressed proteins and Western blotting analysis further confirmed the results. Antibody microarrays revealed that 40 proteins were reproducibly altered more than 2-fold between the selected variant and its parental counterpart; 14 of the proteins were up-regulated, and 26 were down-regulated. Most of the up-regulated proteins (7/14) play a role in tumor signal transduction, while a number of down-regulated proteins (10/26) function in cell differentiation; this might be crucial for pancreatic cancer metastasis. Four dysregulated proteins were validated by western blotting in the cell lines. Interestingly, the up-regulation of Glucagon and down-regulation of Prolactin were further confirmed in the culture supernatants by western blotting. These proteomic data are valuable for understanding pancreatic cancer metastasis and searching for potential markers of metastatic progression.     

Novel biotin linker with alkyne and amino groups for chemical labelling of a target protein of a bioactive small molecule

We synthesized a novel linker (1) with biotin, alkyne and amino groups for the identification of target proteins using a small molecule that contains an azide group (azide probe). The alkyne in the linker bound the azide probe via an azide-alkyne Huisgen cycloaddition. A protein cross-linker effectively bound the conjugate of the linker and an azide probe with a target protein. The covalently bound complex was detected by western blotting. Linker 1 was applied to a model system using an abscisic acid receptor, RCAR/PYR/PYL (PYL). Cross-linked complexes of linker 1, the azide probes and the target proteins were successfully visualized by western blotting. This method of target protein identification was more effective than a previously developed method that uses a second linker with biotin, alkyne, and benzophenone (linker 2) that acts to photo-crosslink target proteins. The system developed in this study is a method for identifying the target proteins of small bioactive molecules and is different from photo-affinity labelling.     

Development of a heat-mediated protein blotting method

Western blotting is a significant tool employed for the detection of cell proteins. High-molecular-weight proteins have proven a challenge to detect by western blotting, but proteins even of 100 KDa can still present difficulties in detection. This work reports the development of a heat transfer method that is suitable for both low- and high-molecular-weight proteins. The procedure involves the use of a constant temperature at 78 °C in a dedicated heat transfer module. Through the use of this protocol the neuronal adaptor protein X11α (120 KDa), which prior to this methodology was undetectable endogenously in the neuroblastoma cell line (N2a), was successfully detected in the N2a cell line. The procedure provides a reproducible protocol that can be adapted for other high-molecular-weight proteins, and it provides the advantage that low-molecular-weight proteins are not sacrificed by the methodology.     

Protein purification and analysis: next generation Western blotting techniques

Introduction: Western blotting is one of the most commonly used techniques in molecular biology and proteomics. Since western blotting is a multistep protocol, variations and errors can occur at any step reducing the reliability and reproducibility of this technique. Recent reports suggest that a few key steps, such as the sample preparation method, the amount and source of primary antibody used, as well as the normalization method utilized, are critical for reproducible western blot results.

Areas covered: In this review, improvements in different areas of western blotting, including protein transfer and antibody validation, are summarized. The review discusses the most advanced western blotting techniques available and highlights the relationship between next generation western blotting techniques and its clinical relevance.

Expert commentary: Over the last decade significant improvements have been made in creating more sensitive, automated, and advanced techniques by optimizing various aspects of the western blot protocol. New methods such as single cell-resolution western blot, capillary electrophoresis, DigiWest, automated microfluid western blotting and microchip electrophoresis have all been developed to reduce potential problems associated with the western blotting technique. Innovative developments in instrumentation and increased sensitivity for western blots offer novel possibilities for increasing the clinical implications of western blot.     

New prospects for the epidemiologic and immunologic study of malaria by the Western blotting technic

A new modification in western blotting technique now permits the analysis of micro samples of blood collected from inhabitants of malaria endemic areas using several different antigens of Plasmodium falciparum. Compared to Immuno Fluorescent Assay and to Immuno Enzymology, ELISA (using somatic antigens and exoantigens of P. falciparum) the western blotting method gives a more detailed analysis. It seems to open new prospects for seroepidemiological studies of human malaria and for the selection of antigenic fractions that may allow the preparation of a vaccine.     

A method for the long-term preservation of polyacrylamide gel electrophoresis

A new, reversible method for drying polyacrylamide gel electrophoresis is reported. It was studied using proteins from the B17, B20, B21 and ATCC 8014 strains of Lactobacillus plantarum isolated from the brine of table olives. After electrophoretic analysis, the gels were dehydrated in a 95% ethyl alcohol solution and stored either long-term or for a few days, renatured and then subjected to analyses that included combination staining with Coomassie brilliant blue and silver, and western blotting. The immunological tests and electrophoresis performed with the enzymes β-glucosidase, alkaline phosphatase and peroxidase demonstrated that repeated dehydration and renaturation of the polyacrylamide gels does not denature the proteins. The method is simple to perform, inexpensive and does not require special equipment.     

Colony filtration blotting for screening soluble expression in Escherichia coli

We have developed a screen for detecting E. coli colonies that produce soluble recombinant target proteins at the colony level: the colony filtration (CoFi) blot. Colonies are transferred, induced and lysed on a filter membrane that can separate soluble proteins from inclusion bodies. Upon lysis, the soluble proteins diffuse through the filter membrane and are captured on a nitrocellulose membrane. The nitrocellulose membrane is incubated with antibodies or probes specific for the target protein and are then developed. In the resulting image, colonies expressing soluble protein can easily be identified. This protocol can be used to screen thousands of constructs in a matter of days, making it very suitable for expression libraries. The protocol is robust and flexible with regard to lysis conditions, induction temperatures and strains. The method requires only standard laboratory equipment and is based on immunochemicals used for western blotting. The following protocol describes the screening of a DNA library with detection done using chemiluminescence. Depending on induction temperature, the whole procedure can be performed in <2 d.     

Using gene expression to predict differences in the secretome of human omental vs. subcutaneous adipose tissue

The objective of this study was to characterize differences in the secretome of human omental compared with subcutaneous adipose tissue using global gene expression profiling. Gene expression was measured using Affymetrix microarrays (Affymetrix, Santa Clara, CA) in subcutaneous and omental adipose tissue in two independent experiments (n = 5 and n = 3 independent subjects; n = 16 arrays in total, 2 for each subject). Predictive bioinformatic algorithms were employed to identify secreted proteins. Microarray analysis identified 22 gene probe sets whose expression was significantly different with a fold change (FC) greater than 5 in expression in both experiments between omental and subcutaneous adipose tissue. Using bioinformatic predictive programs 11 of these 22 probe sets potentially coded for secreted proteins. Pathway network analysis of the secreted proteins showed that three of the proteins are part of a common pathway network. These proteins gremlin 1 (GREM1), pleiotrophin (PTN), and secretory leukocyte peptidase inhibitor (SLPI) are expressed respectively 43×, 23×, and 5× in omental adipose tissue relative to subcutaneous adipose tissue as determined by real-time PCR. The presence of GREM1, PTN, and SLPI protein in human adipose tissue was confirmed by western blotting. All three proteins are expressed in the human Simpson-Golabi-Behmel syndrome (SGBS) preadipocyte cell line. The expression of GREM1, PTN, and SLPI changed with the differentiation of the preadipocytes into mature adipocytes. Gene expression coupled with predictive bioinformatic algorithms have identified several genes coding for secreted proteins which are expressed differently in omental adipose tissue compared to subcutaneous adipose tissue proving a valid alternative approach to help further define the adipocyte secretome.     

Design,generation, and testing of mammalian expression modules that tag membrane proteins

The expression of mammalian membrane proteins in laboratory cell lines allows their biological functions to be characterized and carefully dissected. However, it is often difficult to design and generate effective antibodies for membrane proteins in the desired studies. As a result, expressed membrane proteins cannot be detected or characterized via common biochemical approaches such as western blotting, immunoprecipitation, or immunohistochemical analysis, and their cellular behaviors cannot be sufficiently investigated. To circumvent such roadblocks, we designed and generated two sets of expression modules that consist of sequences encoding for three essential components: (1) a signal peptide from human receptor for advanced glycation end products that targets the intended protein to the endoplasmic reticulum for cell surface expression; (2) an antigenic epitope tag that elicits specific antibody recognition; and (3) a series of restriction sites that facilitate subcloning of the target membrane protein. The modules were designed with the flexibility to change the epitope tag to suit the specific tagging needs. The modules were subcloned into expression vectors, and were successfully tested with both Type I and Type III human membrane proteins: the receptor for advanced glycation end products, the Toll‐like receptor 4, and the angiotensin II receptor 1. These expressed membrane proteins are readily detected by western blotting, and are immunoprecipitated by antibodies to their relative epitope tags. Immunohistochemical and biochemical analyses also show that the expressed proteins are located at cell surface, and maintain their modifications and biological functions. Thus, the designed modules serve as an effective tool that facilitates biochemical studies of membrane proteins.     

Biochemical and molecular studies using human autopsy brain tissue

The use of human brain tissue obtained at autopsy for neurochemical, pharmacological and physiological analyses is reviewed. RNA and protein samples have been found suitable for expression profiling by techniques that include RT-PCR, cDNA microarrays, western blotting, immunohistochemistry and proteomics. The rapid development of molecular biological techniques has increased the impetus for this work to be applied to studies of brain disease. It has been shown that most nucleic acids and proteins are reasonably stable post-mortem. However, their abundance and integrity can exhibit marked intra- and intercase variability, making comparisons between case-groups difficult. Variability can reveal important functional and biochemical information. The correct interpretation of neurochemical data must take into account such factors as age, gender, ethnicity, medicative history, immediate ante-mortem status, agonal state and post-mortem and post-autopsy intervals. Here we consider issues associated with the sampling of DNA, RNA and proteins using human autopsy brain tissue in relation to various ante- and post-mortem factors. We conclude that valid and practical measures of a variety of parameters may be made in human brain tissue, provided that specific factors are controlled.     

Direct immunodetection of antigens within the precast polyacrylamide gel

Detection of specific proteins separated by SDS-PAGE is the basis for studying specific antigens. Immunodetection of antigens is commonly performed using Western blotting technique. In this paper we have shown that it is possible to eliminate Western blotting and to detect the antigens directly within the precast polyacrylamide gels by pretreating the gels with 50% isopropanol followed by distilled water treatment. This method would be valuable for large or difficult to transfer proteins.     

Application of SYPRO Ruby- and Flamingo-stained polyacrylamide gels to Western blot analysis

Western blot analysis has been a useful method for analysis of expression levels of specific proteins and is conducted after sodium dodecyl sulfate (SDS) or native polyacrylamide gel electrophoresis without staining the gel. However, when it is necessary to analyze the gel, duplicate polyacrylamide gels usually must be prepared, one of which is stained, leading to the consumption of precious sample. Thus, we developed a convenient and efficient Western blotting method using a stained gel. This simple modification should be beneficial for analyzing samples that are limited in quantity and/or samples for which the stained gel serves as the loading control.