Differences in the spatial and quantitative reproducibility between two second-dimensional gel electrophoresis systems

Two-dimensional polyacrylamide gel electrophoresis (PAGE), together with 2-D gel electrophoresis (GE) analysis software, is a common technique to analyze a complex proteome. In order to accurately locate the differentially expressed proteins in human pituitary macroadenoma tissues in our long-term research program to clarify the molecular mechanisms of macroadenoma formation, a reproducible separation system is needed. An immobilized pH-gradient dry gel-strip (IPG strip) has been extensively used for first-dimensional isoelectric focusing (IEF), and has achieved a high degree of reproducibility in the IEF direction. For the second dimension (SDS-PAGE), different types of gel systems are available, including horizontal vs. vertical gel systems, and gradient vs. constant-percentage gels. A typical horizontal system is the Multiphor II system that analyzes one gel at a time, using a precast gradient gel (180 x 245 x 0.5 mm), and a typical vertical system is the Dodeca system, which analyzes up to 12 gels at a time, using usually a single-concentration gel (190 x 205 x 1 mm). The present study evaluated the spatial and quantitative reproducibility of the two systems for the separation of the complex human pituitary proteome. PDQuest software was used to analyze the digitized gel-image data, and SPSS statistical software was used to analyze the data. The results demonstrated a high percentage (>99%) of protein-spot matches within each electrophoretic system. The Dodeca gel system demonstrated better between-gel reproducibility for spot position, higher resolution in the Sodium dodecyl sulfate (SDS)-PAGE direction, lower gel background, better spot quality, and higher reproducibility of the spot volume.     

Quantitative and reproducible two-dimensional gel analysis using Phoretix 2D Full

Quantitative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is used to determine changes in individual protein levels in complex protein mixtures. To provide reliable data, the software used for 2-D gel image analysis must provide a linear response over a wide dynamic range of data output. Here, we show that Phoretix 2D Full analysis of 2-D gels stained with colloidal Coomassie Brilliant Blue G-250 can provide a linear measure of changes in protein quantity. We show using a complex mixture of Arabidopsis thaliana proteins, that this is true for essentially all focused proteins, in a data output range greater than three orders of magnitude. An analysis of the factors that affect errors in the results demonstrated that reproducibility of the data is significantly improved by user seeding, whereas it is reduced by use of the background subtraction algorithms.     

Postelectrophoretic staining of proteins separated by two-dimensional gel electrophoresis using SYPRO dyes

While the classical silver stain has been the method of choice for high sensitivity protein visualization on two-dimensional gel electrophoresis (2-D PAGE), post-electrophoretic fluorescent staining with the SYPRO group of dyes has emerged to challenge silver staining for proteome analysis. The latter offers improved sensitivity, higher dynamic range and easy handling. However, most of the published data were derived from analysis of 1-D gel separations. In this work, we have focused on three commercially available fluorescent dyes, SYPRO Ruby, SYPRO Orange and SYPRO Red (Molecular Probes, Eugene, OR, USA) and studied their sensitivity and dynamic range on 2-D PAGE. The use of a multiwavelength fluorescent scanner to image 2-D protein profiles visualized with fluorescent staining is discussed, and a detailed comparison with analysis by silver staining is also provided. These results demonstrate the advantages of using SYPRO dyes, which are in agreement with the literature based on 1-D gel electrophoresis, and give a more realistic understanding of the performance of these fluorescent dyes with 2-D PAGE.     

A new isoelectric focusing system for fast two-dimensional gel electrophoresis using a low-concentration polyacrylamide gel supported by a loose multifilament string.

A new isoelectric focusing (IEF) system for two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) has been proposed. In this system, a super-soft and tough IEF gel was achieved by casting polyacrylamide gel down to 2.0% T using a loose multifilament string (LMS) of nylon as a gel support. The IEF apparatus for the LMS-gel, fabricated from acrylic boards, had a cooling water chamber, and eliminated the need of electrode solutions by directly connecting the two ends of individual gels to platinum electrodes. The carrier ampholyte-generated pH gradients using the new IEF system was stable over a long duration of time and a wide range of voltages, and the IEF time became shorter using a 2.0% T gel than using a 4.0% T gel. Also, the LMS-gels prepared in different runs exhibited excellent reproducibility. The new IEF system was applied to 2-D PAGE of a chicken skeletal muscle extract, and it was found that the protein loading capacity, protein entry into the LMS-gels, and protein transfer efficiency from the first-dimensional to the second-dimensional gels were significantly improved by using a low-concentration (2.5% T) gel. Also, proteins of high molecular weight of more than 200 kDa were observed in the 2-D maps, and therefore the new IEF system has a very good potential to be applied for fast 2-D PAGE of high molecular-weight proteins.     

Alkaline proteins of Bacillus subtilis: first steps towards a two-dimensional alkaline master gel

The genomic sequence of Bacillus subtilis, which is the best studied Gram-positive bacterium, enabled us to obtain a theoretical two-dimensional (2-D) map, demonstrating that about one-third of this proteome has a theoretical alkaline isoelectric point (pI). This represents an important part of the entire proteome, which is not detectable in conventional 2-D gels (pH range 4-7). Sequence analysis revealed that 91% of the ribosomal proteins and a high amount of theoretical membrane proteins should be localized in the alkaline pH range requiring different protein extraction procedures. In order to find the pH range which gives the best resolution results for the alkaline proteins of B. subtilis, immobilized pH gradients (IPGs) with different pH ranges (pH 6-10, 6-11, 4-12, 9-12, and 3-10) were tested and optimized for IPG 4-12. Here we present a version of a first alkaline master 2-D gel for B. subtilis, which is a further complement of the already existing master gel (pH 4-7) in the Sub2D database. Almost 150 spots could be detected and 41 proteins have already been identified.     

Radiochromic gel dosemeter for three-dimensional dosimetry

A gel dosemeter in which ionising radiation causes a colour change was produced by modifying an existing Fricke gel system. This allows a more convenient preparation procedure and gives a better quality dosimetric system for three-dimensional (3-D) dose measurements. The role of three active components of the Ferrous sulphate Xylenol orange Gelatin (FXG) gel dosemeter is quantified with special consideration of their effect on system sensitivity and stability. The optimal composition was found to be 0.5 mM ferrous sulphate, 0.1 mM xylenol orange and 25 mM sulphuric acid. The dose response is linear in the range 0.1–30 Gy. The FXG sensitivity, derived from the gradient of the dose response curve, was found to be ΔA=0.084 cm⁻¹ Gy⁻¹, where A is the optical absorption coefficient at a wavelength of 585 nm, with reproducibility and 24 h stability of better than 5%.     

Lung alveolar proteomics of bronchoalveolar lavage from a pulmonary alveolar proteinosis patient using high-resolution FTICR mass spectrometry

High-resolution Fourier transform ion cyclotron resonance (FTICR) mass spectrometry was developed and applied to the proteome analysis of bronchoalveolar lavage fluid (BALF) from a patient with pulmonary alveolar proteinosis. With use of 1-D and 2-D gel electrophoresis, surfactant protein A (SP-A) and other surfactant-related lung alveolar proteins were efficiently separated and identified by matrix-assisted laser desorption/ionization FTICR mass spectrometry . Low molecular mass BALF proteins were separated using a gradient 2-D gel. An efficient extraction/precipitation system was developed and used for the enrichment of surfactant proteins. The result of the BALF proteome analysis show the presence of several isoforms of SP-A, in which an N-non-glycosylierte form and several proline hydroxylations were identified. Furthermore, a number of protein spots were found to contain a mixture of proteins unresolved by 2-D gel electrophoresis, illustrating the feasibility of high-resolution mass spectrometry to provide identifications of proteins that remain unseparated in 2-D gels even upon extended pH gradients. Yu Bai and Dmitry Galetskiy both contributed equally to this work.     

凝胶过滤/离子交换全二维液相色谱系统的构建与评价

基于蛋白质的尺寸及带电性质,将凝胶过滤色谱(GFC)与离子交换色谱(IEC)两种分离模式结合,采用双捕集柱接口构建了GFC/2×IEC二维液相色谱(2-D LC)分离系统,同时考虑离子交换色谱分离蛋白质对等电点范围的限制,进一步结合中心切割平行柱的方法实现对蛋白质的全二维分离。为与后续蛋白质在线酶解、多肽分离及质谱鉴定匹配,系统中采用常规柱以保证蛋白质质谱鉴定对样品量的要求,3种常规分离柱分别选用凝胶过滤色谱柱TSK-GEL G3000SW_(XL)(300 mm×7.8 mm,5μm)、强阴离子交换色谱柱Hypersil SAX(100 mm×4.6 mm,10μm)和强阳离子交换色谱柱Hypersil SCX(100 mm×4.6 mm,10μm)。最终以酵母细胞蛋白质提取液为样品,对构建的二维系统加以评价,在总蛋白质浓度13.5 mg/mL、上样体积100μL的条件下,将第一维分离等时间切割17次,并将切割馏分全部导入第二维继续分离,二维系统在148 min内获得的总峰容量达到884。说明所构建的系统可以用于蛋白质的在线全二维分离。     

Impact of deglycosylation methods on two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectrometry for proteomic analysis

Glycosylation is a common post-translational modification that can add complexity to the proteome of many cell types. We used enzymatic and chemical methods of deglycosylation to treat a heavily glycosylated exoproteome sample from the filamentous fungus Trichoderma reesei. Deglycosylated samples were resolved on one-dimensional (1-D) and two-dimensional (2-D) gels in order to determine the effect of deglycosylation on the electrophoresis patterns and on the ability to identify proteins by peptide mass matching using matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis of in-gel tryptic digests. We found that deglycosylation of the protein sample resulted in different protein patterns on 1-D and 2-D gels, reduced the complexity of gel patterns, and enhanced the protein identification of some proteins via MALDI-TOF-MS. Deglycosylation with trifluoromethanesulfonic acid (TFMS) was found to be more effective than enzymatic treatments. These deglycosylation techniques may be employed in whole proteome analysis to locate glycosylated proteins and assist in their identification by MS.     

Optimized conditions for diluting and reusing a fluorescent protein gel stain

This study elucidates the optimum conditions at the minimum cost for using SYPRO Ruby protein gel stain. It deals with the effects of gel fixation and staining times, as well as dilution and reuse of SYPRO Ruby protein gel stain in one-dimensional (1-D) gels. Signal strength and dynamic range were highest in gels that were fixed thoroughly before staining, followed by overnight staining. Using the optimized protocol, dilution or reuse of the stain reduces the dynamic range and signal intensity. Sensitivity remains high if the stain is reused up to two times, but signal intensity is reduced up to 2.5-fold in twice used stain. Sensitivity also remains high if the stain is diluted 1:2 in water, but signal intensity is reduced up to 6-fold. Of the two options, reuse or dilution, reuse better retains signal intensity and dynamic range.     

Effect of staining reagent on peptide mass fingerprinting from in-gel trypsin digestions: a comparison of SyproRuby and DeepPurple

As the new fluorescent stains such as SyproRuby and DeepPurple are getting widespread recognition for proteome analyses by the traditional 2-D gel method, it becomes important to test the feasibility of these stains with respect to staining reproducibility, protein quantitation, and compatibility of the stain with downstream MS. The binding of epicocconone, active ingredient of DeepPurple, to one of the primary cleavage sites of trypsin (lysine residue) raises the possibility of incomplete cleavage and interference with PMF. However, the current study tests and concludes that the DeepPurple stain can result in increased peptide recovery compared to SyproRuby stain and can improve MS-based identification of lower intensity proteins spots.     

Identification by mass spectrometry of two-dimensional gel electrophoresis-separated proteins extracted from lager brewing yeast

As two-dimensional (2-D) electrophoresis allows the separation of several hundred proteins in a single gel, this technique has become an important tool for proteome studies and for investigating the cellular physiology. In order to take advantage of information provided by the comparison of proteome pictures, the mass spectrometry technique is the way chosen for a rapid and an accurate identification of proteins of interest. Unfortunately, in the case of industrial yeasts, due to the high level of complexity of their genome, the whole DNA sequence is not yet available and all encoded protein sequences are still unknown. Nevertheless, this study presents here 30 lager brewing yeast proteins newly identified with matrix assisted laser desorption/ionization-time of flight (MALDI-TOF), tandem mass spectrometry (MS/MS) and database searching against the protein sequences of Saccharomyces cerevisiae. The identified proteins of the industrial strain correspond to proteins which do not comigrate with known proteins of S. cerevisiae separated on 2-D gels. This study presents an application of the MS technique for the identification of industrial yeast proteins which are only homologous to the corresponding S. cerevisiae proteins.     

Identification of new regional marker proteins to map mouse brain by 2-D difference gel electrophoresis screening

To screen for new region-specific protein markers we compared the proteome maps of the primary visual and somatosensory areas V1 and S1 in mouse brain using 2-D difference gel electrophoresis (2-D DIGE). Twenty-three protein spots showed a statistically significant difference in expression level between V1 and S1, with 52% appearing more abundantly in V1. Twenty-six proteins were mass spectrometrically identified in 22 spots. To assess the validity of this list of potential areal markers generated by 2-D DIGE, the effective area-specific distribution profile of creatine kinase brain subtype (CKB), a protein with a clearly higher expression level in S1, was monitored with in situ hybridization. The mRNA expression profile of CKB displayed a clear area-specific distribution, which allowed demarcation of S1 and its topographical borders with neighboring neocortical areas. This proteomic study demonstrates the innovative application of 2-D DIGE and MS to select new regional markers for neuroscience research.     

Automated chip-based nanoelectrospray-mass spectrometry for rapid identification of proteins separated by two-dimensional gel electrophoresis

We report a method using a fully automated chip-based nanoelectrospray system for two-dimensional (2-D) gel sample analyses with mass spectrometric detection. The automated nanoelectrospray system, consisting of the NanoMate and electrospray ionization (ESI) chip, serves as both an autosampler and nanoESI source. This infusion system aspirates samples from a 96-well plate using disposable pipette tips and then delivers these samples sequentially to an ESI chip. This chip is a fully integrated monolithic device consisting of a 10x10 array of nozzles. The automated nanoelectrospray system is easily controlled through software, permitting the user to select the number of samples to be analyzed, the volume of sample to aspirate, the spray voltage, and analysis time. The system offers all the advantages of conventional nanoelectrospray plus automated, high-throughput analyses without analyte carryover. The system was used for a protein identification study of 2-D gel spots of both Escherichia coli and yeast crude cell extracts. The identification of 50 spots from E. coli crude cell extract and 27 spots from yeast extract is presented, demonstrating the powerful combination of the automated nanoESI system, the Thermo Finnigan LCQ Deca ion-trap mass spectrometer, and SEQUEST search software. In addition, the effects of silver staining and colloidal Coomassie blue staining of 2-D gel spots on the detection sensitivity and protein sequence coverage are compared and discussed. Furthermore, the comparison results using the multiwell microscale preparation kit versus manual extraction for in-gel samples are presented.     

Characterization of long-term dose stability of N-isopropylacrylamide polymer gel dosimetry

In this study, the detailed characteristics, including spatial uniformity, dose distributions, inter-batch variability, reproducibility, and long-term temporal stability, of N-isopropylacrylamide (NIPAM) polymer gel dosimeter were investigated. A commercial 10x fast optical computed tomography scanner (OCTOPUS^TM-10×, MGS Research, Inc., Madison, CT, USA) was used to measure NIPAM polymer gel dosimeter. A cylindrical NIPAM gel phantom that measured 10 cm × 10 cm was irradiated via a single-field treatment plan with a field size of 4 cm × 4 cm. The maximum standard deviation of spatial uniformity for NIPAM gel was less than 0.29 %. The average standard deviation among the three batches of gel dosimeters was less than 1 %. The gamma pass rate could reach as high as 96.76 % when a 3 % dose difference and a 3 mm dose-to-agreement criteria were used. The long-term measurement of irradiated NIPAM gel dosimeter indicated that the dose maps attained a gradually stable value 15 h post-irradiation and remained stable until 72 h post-irradiation. The gamma pass rate could achieve a maximum value between 24 and 72 h post-irradiation. The edge enhancement effect that occurred around the irradiated region was observed 72 h post-irradiation. Thus, the results from this study suggest that NIPAM gel dosimeter should be measured approximately 24 h post-irradiation to reduce the occurrence of the edge enhancement effect.     

Two-dimensional difference gel electrophoresis applied for analytical proteomics: fundamentals and applications to the study of plant proteomics

The present review reports the principles, fundamentals and some applications of two-dimensional difference gel electrophoresis for analytical proteomics based on plant proteome analysis, also emphasizing some advantages of 2-D DIGE over 2-D PAGE techniques. Some fluorescent protein labeling reagents, methods of protein labeling, models of 2-D DIGE experiments, and some limitations of this technique are presented and discussed in terms of 2-D DIGE plant proteomes. Finally, some practical applications of this technique are pointed out, emphasizing its potentialities in plant proteomics.     

Recent advances in capillary separations for proteomics

The sequencing of several organisms' genomes, including the human's one, has opened the way for the so-called postgenomic era, which is now routinely coined as "proteomics". The most basic task in proteomics remains the detection and identification of proteins from a biological sample, and the most traditional way to achieve this goal consists of protein separations performed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Still, the 2-D PAGE-mass spectrometry (MS) approach remains lacking in proteome coverage (for proteins having extreme isoelectric points or molecular masses as well as for membrane proteins), dynamic range, sensitivity, and throughput. Consequently, considerable efforts have been devoted to the development of non-gel-based proteome separation technologies in an effort to alleviate the shortcomings in 2-D PAGE while reserving the ability to resolve complex protein and peptide mixtures prior to MS analysis. This review focuses on the most recent advances in capillary-based separation techniques, including capillary liquid chromatography, capillary electrophoresis, and capillary electrokinetic chromatography, and combinations of multiples of these mechanisms, along with the coupling of these techniques to MS. Developments in capillary separations capable of providing extremely high resolving power and selective analyte enrichment are particularly highlighted for their roles within the broader context of a state-of-the-art integrated proteome effort. Miniaturized and integrated multidimensional peptide/protein separations using microfluidics are further summarized for their potential applications in high-throughput protein profiling toward biomarker discovery and clinical diagnosis.