The human heart proteome: Two-dimensional maps using narrow-range immobilised pH gradients

The analysis of complex proteomes is undertaken using a variety of techniques and technologies such as 2-DE, surface-enhanced laser desorption ionisation, and various types of MS. In order to overcome the complexities of protein expression in discrete proteomes, sample fractionation has become an important aspect of proteomic experiments. The use of narrow-range IPGs (nrIPGs) is of special importance using the 2-DE proteomics workflow, since an enhanced visualisation of a given proteome is achieved through an improved physical separation and resolution of proteins. The work described in this paper presents a series of protein maps of the human heart left ventricle proteome that have been generated using nrIPGs for the first, IEF, dimension of 2-DE. A total of 374 gel spots were excised from seven different pH gradients, covering the range pH 3-10, giving rise to a total of 388 identifications from 110 unique proteins. Using Gene Ontologies (GOs), the identified proteins were found to be associated with 97 types of GO Process, 144 types of GO Function, and 54 types of GO Component. It is hoped that the maps presented in this paper will be of use to other researchers for reference purposes.     

Proteome database of unsensitized CD4 positive T lymphocytes in T cell receptor transgenic mice

We established a two-dimensional electrophoresis (2-DE) mapping database of splenic CD4 T cells prepared from I-A(d)-restricted ovalbumin (OVA)(323-339) specific T cell receptor (TCR) transgenic mice (OVA23-3). First we examined the purification of CD4 T cells and found that the high purity of cells produced more accurate protein maps. The first dimension utilized narrow-range immobilized pH gradients (IPGs), pH 4.0-5.0, pH 4.5-5.5, pH 5.0-6.0, and pH 5.5-6.7. Approximately 1300 spots were detected by silver staining. Detection was performed by in-gel tryptic digestion of the spots, matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) technology and database searches via the world wide web (WWW). We have so far identified 255 proteins on 2-DE gels of whole cell lysates. This is the first construction of a proteome database for murine unsensitized CD4 T lymphocytes. To examine this further, 2-DE mapping was utilized for splenic CD4 T cells from another TCR transgenic mouse strain (DO11.10 TCR transgenic mice). Mapping patterns were found to be almost identical to those from CD4 T cells from OVA23-3 mice. These results indicated that the 2-DE maps in this study could be used for mouse CD4 T cells to examine protein changes in cells given certain stimuli.     

Sample complexity reduction for two-dimensional electrophoresis using solution isoelectric focusing prefractionation

Despite its excellent resolving power, 2-DE is of limited use when analyzing cellular proteomes, especially in differential expression studies. Frequently, fewer than 2000 protein spots are detected on a single 2-D gel (a fraction of the total proteome) regardless of the gel platform, sample, or detection method used. This is due to the vast number of proteins expressed and their equally vast dynamic range. To exploit 2-DE unique ability as both an analytical and a preparative tool, the significant sample prefractionation is necessary. We have used solution isoelectric focusing (sIEF) via the ZOOM IEF Fractionator (Invitrogen) to generate sample fractions from complex bacterial lysates, followed by parallel 2-DE, using narrow-range IPG strips that bracket the sIEF fractions. The net result of this process is a significant enrichment of the bacterial proteome resolved on multiple 2-D gels. After prefractionation, we detected 5525 spots, an approximate 3.5-fold increase over the 1577 spots detected in an unfractionated gel. We concluded that sIEF is an effective means of prefractionation to increase depth of field and improve the analysis of low-abundance proteins.     

Proteome analysis using isoelectric focusing in immobilized pH gradient gels followed by mass spectrometry

Over the past several years, a large effort has been focused on improvements of two-dimensional (2-D) gel electrophoresis-based proteomics technology, and on development of novel approaches for proteome analysis. Here, we describe the application of an alternative strategy for the analysis of complex proteomes. The strategy combines isoelectric focusing in immobilized pH gradient strips (in-gel IEF), mass spectrometry (MS), and bioinformatics. A protein mixture is separated by in-gel IEF, and the entire strip is cut into a set of gel sections. Proteins in each gel section are digested with trypsin, and the tryptic peptides are subjected to liquid chromatography-nanoelectrospray-quadrupole ion-trap tandem mass spectrometry (LC-ESI-MS/MS). The LC-ESI-MS/MS data are used to identify the proteins through searches of a protein sequence database. Using this in-gel IEF-LC-MS/MS strategy, we have identified 127 proteins from a human pituitary. This study demonstrates the potential of the in-gel IEF-LC-MS/MS approach for analyses of complex mammalian proteomes.     

液相等电聚焦结合双向凝胶电泳分离碱性蛋白

在蛋白组学研究中, 经典的双向凝胶电泳法(2-DE)对碱性蛋白及低丰度蛋白的分离存在技术障碍, 但预分离技术的应用可弥补其缺陷. 液相等电聚焦可有效地分离富集复杂蛋白样品. 碱性胶条用于2-DE可极大地提高蛋白上样量和凝胶分辨率. 将上述两种技术相结合用于碱性蛋白质和低丰度蛋白质的分离鉴定, 可使碱端区域双向凝胶图谱质量显著提高, 蛋白点更清晰且点数增多, 质谱鉴定确信度提高, 碱性蛋白和低丰度蛋白质谱鉴定成功率提高, 对于蛋白组学研究具有一定的意义.     

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.     

Evaluation of several two-dimensional gel electrophoresis techniques in cardiac proteomics

Two-dimensional gel electrophoresis (2-DE) is currently the best method for separating complex mixtures of proteins, and its use is gradually becoming more common in cardiac proteome analysis. A number of variations in basic 2-DE have emerged, but their usefulness in analyzing cardiac tissue has not been evaluated. The purpose of the present study was to systematically evaluate the capabilities and limitations of several 2-DE techniques for separating proteins from rat heart tissue. Immobilized pH gradient strips of various pH ranges, parameters of protein loading and staining, subcellular fractionation, and detection of phosphorylated proteins were studied. The results provide guidance for proteome analysis of cardiac and other tissues in terms of selection of the isoelectric point separating window for cardiac proteins, accurate quantitation of cardiac protein abundance, stabilization of technical variation, reduction of sample complexity, enrichment of low-abundant proteins, and detection of phosphorylated proteins.     

Comprehensive proteome analysis of mouse liver by ampholyte-free liquid-phase isoelectric focusing

In this study, ampholyte-free liquid-phase IEF (LIEF) was combined with narrow pH range 2-DE and SDS-PAGE RP-HPLC for comprehensive analysis of mouse liver proteome. Because LIEF prefractionation was able to reduce the complexity of the sample and enhance the loading capacity of IEF strips, the number of visible protein spots on subsequent 2-DE gels was significantly increased. A total of 6271 protein spots were detected after integrating five narrow pH range 2-DE gels following LIEF prefractionation into a single virtual 2-DE gel. Furthermore, the pH 3-5 LIEF fraction and the unfractionated sample were separated by pH 3-6 2-DE and identified by MALDI-TOF/TOF MS, respectively. In parallel, the pH 3-5 LIEF fraction was also analyzed by SDS-PAGE RP-HPLC MS/MS. LIEF-2-DE and LIEF-HPLC could obviously improve the separation efficiency and the confidence of protein identification, which identified a higher number of low-abundance proteins and proteins with extreme physicochemical characteristics or post-translational modifications compared to conventional 2-DE method. Furthermore, there were 207 proteins newly identified in mouse liver in comparison with previously reported large-scale datasets. It was observed that the combination of LIEF-2-DE and LIEF-HPLC was effective in promoting MS-based liver proteome profiling and could be applied on similar complex tissue samples.     

Mass spectrometric imaging of immobilized pH gradient gels and creation of "virtual" two-dimensional gels

We have developed a matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) based technique for the detection of intact proteins directly from immobilized pH gradient gels (IPGs). The use of this technique to visualize proteins from IPGs was explored in this study. Whole cell Escherichia coli extracts of various loadings were separated on IPGs. These IPGs were processed to remove contaminants and to achieve matrix/analyte cocrystallization on the surface of the gel. Mass spectra were acquired by scanning the surface of the gel and were assimilated into a "virtual" two dimensional (2-D) gel. This virtual 2-D gel is analogous to a "classical" 2-D gel, except that the molecular weight information is acquired by mass spectrometry rather than by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This mass spectrometry (MS) based technology exemplifies a number of desirable characteristics, some of which are not attainable with classical two-dimensional electrophoresis (2-DE). These include high sensitivity, high reproducibility, and an inherently higher resolution and mass accuracy than 2-D gels. Furthermore, there is a difference in selectivity exhibited between virtual 2-D gels and classical 2-D gels, as a number of proteins are visible in the virtual gel image that are not present in the stained gels and vice versa. In this report, virtual 2-D gels will be compared to classical 2-D gels to illustrate these features.     

Analysis of plasma protein adsorption onto polystyrene particles by two-dimensional electrophoresis: comparison of sample application and isoelectric focusing techniques

Two-dimensional electrophoresis (2-DE) was previously established for analysis of plasma protein adsorption patterns on particulate carriers for intravenous drug targeting. This study addresses a possible effect of polymeric particles on protein separation in the first dimension, e.g., hindrance of protein entry into the gel or interaction of particles with the gel matrix. Polystyrene beads of mean diameter 100, 200 and 1000 nm were used as model carriers. Two different separation techniques were performed in the first dimension of 2-DE to study possible interactions of the beads with the different gel matrices, i.e., carrier ampholytes (CA) and immobilized pH gradients (IPGs). Comparison of gels obtained from samples including the particles from samples separated from the polystyrene beads showed no noteworthy differences. Therefore, a negative effect of the particles can be excluded, and particle separation from the sample is not necessary. Another goal of this study was the transfer of analytical protocols for isoelectric focusing from CA to IPGs with regard to enhanced reproducibility, faster sample processing, and easier handling. Transfer from CA to IPGs was carried out successfully and showed improved resolution of basic proteins. In contrast to that, lower amounts of a few high molecular mass proteins were detected, especially when sample application cups were employed. A qualitative change in the obtained protein pattern was not observed. Increased entry of high molecular weight proteins was achieved by in-sample rehydration instead of using sample cups.     

Combined cup loading, bis(2-hydroxyethyl) disulfide, and protein precipitation protocols to improve the alkaline proteome of Lactobacillus hilgardii

Despite the large number of papers dealing with bacterial proteomes, very few include information about proteins with alkaline pI's, because of the limits inherent in 2-DE technology. Nonetheless, analyses of in silico proteomes of many prokaryotes show a bimodal distribution of their proteins based on their pI's; the most crowded areas lying between pI 4-7 and 9-11. The aim of the present research was to set up a general, simple, and standardizable 2-DE protocol suitable for studying the alkaline proteome of Lactobacillus hilgardii, a Gram-positive bacillus isolated from wine. The method has also been tested on a Gram-negative bacterium able to degrade aromatic pollutants, Acinetobacter radioresistens S13. Optimization of the method was mainly focused on improving protein extraction and IEF (pI 6-11) separation protocols. Concerning IEF, different methods for sample loading (in-gel rehydration and cup loading), and different reducing agents (DTT and bis(2-hydroxyethyl) disulfide (HED)) were tested and compared. The proposed protocol was found to resolve efficiently alkaline proteins from both of our Lactobacillus and Acinetobacter strains, in spite of their different external layers, thus, enabling a more comprehensive study of their proteomes.     

Acid-labile surfactant improves in-sodium dodecyl sulfate polyacrylamide gel protein digestion for matrix-assisted laser desorption/ionization mass spectrometric peptide mapping

Mass spectrometry (MS) together with genome database searches serves as a powerful tool for the identification of proteins. In proteome analysis, mixtures of cellular proteins are usually separated by sodium dodecyl sulfate (SDS) polyacrylamide gel-based two-dimensional gel electrophoresis (2-DE) or one-dimensional gel electrophoresis (1-DE), and in-gel digested by a specific protease. In-gel protein digestion is one of the critical steps for sensitive protein identification by these procedures. Efficient protein digestion is required for obtaining peptide peaks necessary for protein identification by MS. This paper reports a remarkable improvement of protein digestion in SDS polyacrylamide gels using an acid-labile surfactant, sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate (ALS). Pretreatment of gel pieces containing protein spots separated by 2-DE with a small amount of ALS prior to trypsin digestion led to increases in the digested peptides eluted from the gels. Consistently, treatment of gel pieces containing silver-stained standard proteins and those separated from tissue extracts resulted in the detection of increased numbers of peptide peaks in spectra obtained by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOFMS). Hence the present protocol with ALS provides a useful strategy for sensitive protein identification by MS.     

Prefractionation techniques in proteome analysis: the mining tools of the third millennium

The present review deals with prefractionation protocols used in proteomic investigation in preparation for mass spectrometry (MS) or two-dimensional electrophoresis (2-DE) map analysis. Briefly, reported methods focus on cell organelle differential centrifugation and on chromatographic approaches, to continue in extenso with a panoply of electrophoretic methods. In the case of chromatography, procedures useful as a prefractionation step, including affinity, ion-exchange, and reversed-phase resins, revealed several hundreds of new species, previously undetected in unfractionated samples. Novel chromatographic prefractionation methods are also discussed such as a multistaged fractionation column, consisting in a set of immobilized chemistries, serially connected in a stack format (an assembly of seven blocks), each capable of harvesting a given protein population. Such a method significantly simplifies the complexity of treated samples while concentrating species, all resulting in a larger number of visible proteins by MS or 2-DE. Electrophoretic prefractionation protocols include all those electrokinetic methodologies which are performed in free solution, essentially all relying on isoelectric focusing steps (although some approaches based on gels and granulated media are also discussed). Devices associated with electrophoretic separation are multichamber apparatus, such as the multicompartment electrolyzers equipped with either isoelectric membranes or with isoelectric beads. Multicup device electrophoresis and several others, exploiting the conventional technique of carrier ampholyte focusing, are reviewed. This review also reports approaches for sample treatments in order to detect low-abundance species. Among others, a special emphasis is made on the reduction of concentration difference between proteins constituting a sample. This latter consists in a library of combinatorial ligands coupled to small beads. Such a library comprises hexameric ligands composed of 20 amino acids, resulting in millions of different structures. When these beads are impregnated with complex proteomes (e.g., human sera) of widely differing protein compositions, they are able to significantly reduce the concentration differences, thus greatly enhancing the possibility to evidence low-abundance species. It is felt that this panoply of methods could offer a strong step forward in "mining below the tip of the iceberg" for detecting the "unseen proteome".     

How many proteins can be identified in a 2DE gel spot within an analysis of a complex human cancer tissue proteome?

Two‐dimensional gel electrophoresis (2DE) in proteomics is traditionally assumed to contain only one or two proteins in each 2DE spot. However, 2DE resolution is being complemented by the rapid development of high sensitivity mass spectrometers. Here we compared MALDI‐MS, LC‐Q‐TOF MS and LC‐Orbitrap Velos MS for the identification of proteins within one spot. With LC‐Orbitrap Velos MS each Coomassie Blue‐stained 2DE spot contained an average of at least 42 and 63 proteins/spot in an analysis of a human glioblastoma proteome and a human pituitary adenoma proteome, respectively, if a single gel spot was analyzed. If a pool of three matched gel spots was analyzed this number further increased up to an average of 230 and 118 proteins/spot for glioblastoma and pituitary adenoma proteome, respectively. Multiple proteins per spot confirm the necessity of isotopic labeling in large‐scale quantification of different protein species in a proteome. Furthermore, a protein abundance analysis revealed that most of the identified proteins in each analyzed 2DE spot were low‐abundance proteins. Many proteins were present in several of the analyzed spots showing the ability of 2DE‐MS to separate at the protein species level. Therefore, 2DE coupled with high‐sensitivity LC‐MS has a clearly higher sensitivity as expected until now to detect, identify and quantify low abundance proteins in a complex human proteome with an estimated resolution of about 500 000 protein species. This clearly exceeds the resolution power of bottom‐up LC‐MS investigations.     

Microscale solution IEF combined with 2-D DIGE substantially enhances analysis depth of complex proteomes such as mammalian cell and tissue extracts

Current gel-based protein profiling methods such as 2-DE and fluorescent 2-D difference in gel electrophoresis (DIGE) evaluate small portions of complex proteomes. Hence, sample prefractionation is essential for more comprehensive proteome coverage and detection of low-abundant proteins. In this study, we describe the combination of DIGE labeling with microscale solution IEF (MicroSol-IEF) fractionation and subsequent analysis on slightly overlapping narrow pH range 2-D gels. By fluorescently tagging and mixing samples and controls prior to prefractionation, complications resulting from minor run-to-run variations during MicroSol-IEF separations of multiple samples are avoided. This greatly improves the reliability of quantitative comparisons. To illustrate its utility, this 3-D DIGE strategy was applied to analysis of human melanoma cells and mouse lung tissue extracts. Approximately 1000 reproducible spots can be obtained from narrow range 2-D gels of individual MicroSol-IEF fractions, and approximately 6000 spots can be obtained from entire proteomes. Quantitative changes in closely related samples could be more reliably detected and the method has a greatly increased capacity to distinguish between closely related protein isoforms. Thus the 3-D DIGE strategy produces a powerful method for more comprehensive and more reliable quantitative comparisons of protein profiles of very complex proteomes.     

Iron-regulated proteins in Phanerochaete chrysosporium and Lentinula edodes: differential analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional polyacrylamide gel electrophoresis profiles

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis (2-DE) were used to identify iron-responsive proteins in the white-rot species (Phanerochaete chrysosporium and Lentinula edodes), by comparing the differential patterns of cellular and membrane proteins obtained from iron-sufficient and iron-deficient mycelia. Six cellular proteins induced by iron restriction have been observed in SDS-PAGE for P. chrysosporium and twelve for L. edodes. In 2-DE, the numbers of iron-restricted induced proteins were 12 and 9, respectively, in a resolution range of 15-60 kDa and pI 4.5-8.1. SDS-PAGE for the plasma membrane protein did not show differences, whereas the outer-membrane protein profile showed 6 and 5 proteins induced by iron depletion in P. chrysosporium and L. edodes, respectively. The results presented here are important data to unravel mechanisms of biosynthesis and/or transport of the iron-complexing agents in ligninolytic fungi and to further correlate them to the ligninolytic processes.     

Subproteomics based upon protein cellular location and relative solubilities in conjunction with composite two-dimensional electrophoresis gels

Progress in the field of proteomics is dependent upon an ability to visualise close to an entire protein complement via a given array technology. These efforts have previously centred upon two-dimensional gel electrophoresis in association with immobilised pH gradients in the first dimension. However, limitations in this technology, including the inability to separate hydrophobic, basic, and low copy number proteins have hindered the analysis of complete proteomes. The challenge is now to overcome these limitations through access to new technology and improvements in existing methodologies. Proteomics can no longer be equated with a single two-dimensional electrophoresis gel. Greater information can be obtained using targeted biological approaches based upon sample prefractionation into specific cellular compartments to determine protein location, while novel immobilised pH gradients spanning single pH units can be used to display poorly abundant proteins due to their increased resolving power and loading capacity. In this study, we show the effectiveness of a combined use of two differential subproteomes (as defined by relative solubilities, cellular location and narrow-range immobilised pH gradients) to increase the resolution of proteins contained on two-dimensional gels. We also present new results confirming that this method is capable of displaying up to a further 45% of a given microbial proteome. Subproteomics, utilising up to 40 two-dimensional gels per sample will become a powerful tool for near-to-total proteome analysis in the postgenome era. Furthermore, this new approach can direct biological focus towards molecules of specific interest within complex cells and thus simplify efforts in discovery-based proteome research.     

An insight into the abundant proteome of 46BR.1G1 fibroblasts deficient of DNA ligase I

This work presents the proteome profile of cultured human skin fibroblasts established from a patient affected by DNA ligase I (Lig I) deficiency syndrome, a rare disorder characterized by immunodeficiency, growth retardation and sun sensitivity. 2-DE (in the 3-10 and 4-7 pH ranges) was the separation technique used for the production of maps. MALDI-TOF/MS and LC-MS/MS were the mass spectrometry platforms applied for the identification of proteins in gel spots. A total of 154 proteins, including 41 never detected before in skin fibroblasts with this approach, were identified in gel spots analyzed. This newly generated extensive database provides for the first time a global picture of abundant proteins in 46BR.1G1 skin fibroblasts. While being relevant to the particular disorder considered, these results may be regarded as an intriguing starting point on the way to achieve a reference map of the proteins highly expressed in an inherited syndrome with defect in DNA replication and repair pathways.