Proteomic profiling combining solution-phase isoelectric fractionation with two-dimensional gel electrophoresis using narrow-pH-range immobilized pH gradient gels with slightly overlapping pH ranges

This paper describes a simple new approach toward improving resolution of two-dimensional (2-D) protein gels used to explore the mammalian proteome. The method employs sample prefractionation using solution-phase isoelectric focusing (IEF) to split the mammalian proteome into well-resolved pools. As crude samples are thus prefractionated by pI range, very-narrow-pH-range 2-D gels can be subsequently employed for protein separation. Using custom pH partition membranes and commercially available immobilized pH gradient (IPG) strips, we maximized the total separation distance and throughput of seven samples obtained by prefractionation. Both protein loading capacity and separation quality were higher than the values obtained by separation of fractionated samples on narrow-pH-range 2-D gels; the total effective IEF separation distance was ~82 cm over the pH range pH 3–10. This improved method for analyzing prefractionated samples on narrow-pH-range 2-D gels allows high protein resolution without the use of large gels, resulting in decreased costs and run times.      

Quantitative evaluation of protein recoveries in two-dimensional electrophoresis with immobilized pH gradients

In this study, metabolically radiolabeled Escherichia coli cell extracts were used to systematically evaluate protein recoveries at each step of two-dimensional (2-D) electrophoresis and using different sample application methods. Sample application using sample cups resulted in better protein recovery compared with sample loading by rehydration when the Multiphor system was used. At least 50% or more of an E. coli extract was lost when high protein amounts (500 microg) were loaded by rehydration using this system, which employs separate holders for rehydration and isoelectric focusing (IEF). In contrast, when the IPGphor system was used, rehydration sample loading consistently yielded the highest overall protein recoveries. These improved protein recoveries were due to integration of rehydration and electrophoretic separation in a single unit. Even at high protein loads (500 microg), less than 15-20% of the proteins were lost when proteins were loaded by rehydration using sample buffer containing 2% carrier ampholytes in the ceramic immobilized pH gradient (IPG) strip holders used for both rehydration and IEF. Regardless of the loading conditions used, carrier ampholytes in the sample buffer increased protein recoveries. Use of thiourea did not significantly affect protein recoveries but did improve protein resolution in 2-D gels as expected. In summary, these results show the best protein recoveries are obtained for all protein loads when samples are applied to IPG strips during rehydration using a single device for both rehydration and IEF. In contrast, the poorest recoveries are obtained when rehydration and IEF are performed in separate devices, and losses increase dramatically with increasing protein loads using this approach.     

Spot overlapping in two-dimensional polyacrylamide gel electrophoresis maps: relevance to proteomics

Proteomics requires a large-scale, simultaneous separation of proteins from a mixture, assessment of the relative abundance of these molecules, and identification and characterization of each component. In 2-D PAGE separations, the best method of choice for protein analysis, separation of all the proteins present in the sample is still far to be achieved and comigrating proteins in the same spot are in general present. A statistical estimation of the degree of spot overlapping present in a 2-D PAGE separation is here described: for different conditions of spot overcrowding in the map, the degree of overlapping can be quantified in terms of purity degree of each spot or percentage of proteins that will appear in the map as a single spot. A computer simulation approach is described: it is based on the protein separation pattern present in the experimental maps. The results thus obtained are compared to a theoretical model (statistical degree of peak overlapping model) based on random spot position. The described procedures were applied to an experimental reference map of human plasma. The severity of spot overlapping in 2-D PAGE maps is estimated and the influence of different experimental conditions (strip dimension, detector system performance, pI range) is discussed. These informations are useful to quantitatively estimate the degree of error associated with identification and quantitation of each protein and to set-up experimental conditions which will increase resolution and greatly decrease the probability of spot overlapping.     

Recent developments and contributions from Chinese scientists in multidimensional separations for proteomics and traditional Chinese medicines

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 in protein separations performed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Yet the 2-D PAGE-mass spectrometry (MS) approach has its drawbacks with regard to automation, sensitivity, and throughput. Consequently, considerable effort has been devoted to the development of non-gel-based proteome separation technologies in an effort to alleviate the shortcomings of 2-D PAGE. In addition, traditional Chinese medicines (TCMs), due to their long period of clinical testing and reliable therapeutic efficacy, are attracting increased global attention. However, hundreds or even thousands of components are usually present in TCMs, which results in great difficulties of separation. As a mainstream separation tool, multidimensional liquid separation systems have shown powerful separation ability, high peak capacity, and excellent detectability in the analysis of complex samples including biological samples and TCMs, etc. Therefore, this review emphasizes the most recent advances in multidimensional liquid chromatography and capillary electrophoresis-based separation techniques, and the corresponding applications in proteomics and TCMs. In view of the significant contributions from Chinese scientists, this review focuses mainly on the work of Chinese scientists in the above fields.     

Decoding two-dimensional polyacrylamide gel electrophoresis complex maps by autocovariance function: a simplified approach useful for proteomics

This paper describes a mathematical approach applied for decoding the complex signal of two-dimensional polyacrylamide gel electrophoresis maps of protein mixtures. The method is helpful in extracting analytical information since separation of all the proteins present in the sample is still far from being achieved and co-migrating proteins are generally present in the same spot. The simplified method described is based on the study of the 2-D autocovariance function (2D-ACVF) computed on an experimental digitized map. The first part of the 2D-ACVF allows for the estimation of the number of proteins present in the sample (2D-ACVF computed at the origin) and of the separation performance (mean spot size). Moreover, the 2D-ACVF plot is a powerful tool in identifying order in the spot position, and singling it out from the complex separation pattern. This method was validated on synthetic maps obtained by computer simulation to describe 2-D PAGE real maps and reference maps retrieved from the SWISS-2DPAGE database. The results obtained are discussed by focusing on specific information relevant in proteomics: sample complexity, separation performance, and identification of spot trains related to post-translational modifications.     

Novel moving reaction boundary-induced stacking and separation of human hemoglobins in slab polyacrylamide gel electrophoresis

We developed a novel polyacrylamide gel electrophoresis (PAGE) method to stack and separate human hemoglobins (Hbs) based on the concept of moving reaction boundary (MRB). This differs from the classic isotachophoresis (ITP)-based stacking PAGE in the aspect of buffer composition, including the electrode buffer (pH 8.62 Tris–Gly), sample buffer (pH 6.78 Tris–Gly), and separation buffer (pH 8.52 Tris–Gly). In the MRB-PAGE system, a transient MRB was formed between alkaline electrode buffer and acidic sample buffer, being designed to move toward the anode. Hbs carried partial positive charges in the sample buffer due to its pH below pI values of Hbs, resulting in electromigrating to the cathode. Hbs would carry negative charges quickly when migrated into the alkaline electrode buffer and be transported to the anode until meeting the sample buffer again. Thus, Hbs were stacked within a MRB until the transient MRB reached the separation buffer and then separated by zone electrophoresis with molecular sieve effect of the gel. The experimental results demonstrated that there were three clear and sharp protein zones of Hbs (HbA_1c, HbA₀, and HbA₂) in MRB-PAGE, in contrast to only one protein zone (HbA₀) in ITP-PAGE for large-volume loading (≥15 μl), indicating high stacking efficiency, separation resolution, and good sensitivity of MRB-PAGE. In addition, MRB-PAGE was performed in a conventional slab PAGE device, requiring no special device. Thus, it could be widely used in separation and analysis of diluted protein in a standard laboratory.

Detection and identification of human bronchoalveolar lavage proteins using narrow-range immobilized pH gradient DryStrip and the paper bridge sample application method.

The use of two-dimensional gel electrophoresis as a tool for the investigation of human bronchoalveolar lavage fluid (BALF) has been hampered by technical difficulties. In the last decade attempts have been made to establish a two-dimensional (2-D) protein map of BALF samples, resulting in the identification of a number of proteins present in BALF. In this study, we report an improved sample handling and separation protocol for investigation of human BALF proteins. The sample has been analyzed by employing a number of strategies, including the 'paper bridge' sample application method in combination with narrow range immobilized pH gradient (IPG) DryStrips, followed by comparison to the corresponding plasma map. Using peptide mass fingerprinting, we have identified 49 proteins in the narrow pH range 4.5-5.2 from an individual healthy BALF sample. Furthermore, we identified 17 BALF proteins, not detected in plasma. Twelve of these proteins have, to our knowledge, not previously been described in the BALF 2-D map. The mapping of BALF proteins with inclusion of those at low concentration increases the possibility to subsequently screen patient material for disease markers.     

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.     

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.     

Narrow-band fractionation of proteins from whole cell lysates using isoelectric membrane focusing and nonporous reversed-phase separations

Preparative isoelectric focusing (PIEF) is used to achieve narrow-band fractionation of proteins from whole cell lysates of Escherichia coli (E. coli). Isoelectric membranes create well-defined pH ranges that fractionate proteins by isoelectric point (pI) upon application of an electric potential. A commercial IsoPrime device (Amersham-Pharmacia BioTech) is modified for the PIEF separation to lessen run volumes significantly. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) analysis of chamber contents indicates that excellent pH fractionation is achieved with little overlap between chambers. PIEF pH fractions are further separated using nonporous reversed-phase high-performance liquid chromatography (NPS-RP-HPLC) and HPLC eluent is analyzed on-line by electrospray ionization-time of flight-mass spectrometry (ESI-TOF-MS) for intact protein molecular weight (MW) analysis. The result is a pI versus MW map of bacterial protein content. IEF fractionation down to 0.1 pH units combined with intact protein MW values result in a highly reproducible map that can be used for comparative analysis of different E. coli strains.     

A novel droplet-tap sample-loading method for two-dimensional gel electrophoresis

A novel procedure, droplet-tap mode, has been devised for sample application for two-dimensional gel electrophoresis (2DE) expression profiles. The sample was loaded by evenly distributed tapping of droplets of the sample on to the rehydration buffer (RB) and then lowering the strip on to the solution surface. At normal loading concentrations, the number of spots obtained was increased by approximately one-third by this new approach compared with the rehydration loading procedure. The method also resulted in significantly improved resolution compared with cup loading when high concentrations of proteins were present, indicating its potential usefulness in micropreparative separation. In addition, recovery of the proteins confirmed that protein uptake was enhanced by use of this method. By enabling improved performances in 2DE, the proposed procedure has much potential for sample loading to meet the requirements of global proteome analysis.Electronic Supplementary Material Electronic Supplementary Material found in     

Fractionation and separation of human salivary proteins by pH-gradient ion exchange and reversed phase chromatography coupled to mass spectrometry

In the present work, a 2-D capillary liquid chromatography method for fractionation and separation of human salivary proteins is demonstrated. Fractionation of proteins according to their pI values was performed in the 1-D employing a strong anion exchange (SAX) column subjected to a wide-range descending pH gradient. Polystyrene-divinylbenzene (PS-DVB) RP columns were used for focusing and subsequent separation of the proteins in the 2-D. The SAX column was presaturated with a high pH buffer (A) consisting of 10 mM amine buffering species, pH 9.0, and elution was performed with a low pH elution buffer (B) having the same buffer composition and concentration as buffer A, but pH 3.5. Isoelectric point fractions eluting from the 1-D column were trapped on PS-DVB trap columns prior to back-flushed elution onto the PS-DVB analytical column for separation of the proteins. The 1-D fraction eluting at pH 9.0-8.7 was chosen for further analysis. After separation on the RP analytical column, nine RP protein fractions were collected and tryptic digested for subsequent analyses by MALDI TOF MS and column switching capillary LC coupled to ESI TOF MS and ESI QTOF MS. Eight proteins and two peptides were identified in the pH 9.0-8.7 fraction using peptide mass fingerprinting and uninterpreted MS/MS data.     

Preparative two-dimensional gel electrophoresis with agarose gels in the first dimension for high molecular mass proteins

A two-dimensional gel electrophoresis (2-DE) method that uses an agarose isoelectric focusing (IEF) gel in the first dimension (agarose 2-DE) was compared with an immobilized pH gradient 2-DE method (IPG-Dalt). The former method was shown to produce significant improvements in the 2-D electrophoretic separation of high molecular mass proteins larger than 150 kDa, up to 500 kDa, and to have a higher loading capacity, as much as 1.5 mg proteins in total for micropreparative runs. The extraction medium found best in this study for agarose 2-DE of mammal tissues was 6 M urea, 1 M thiourea, 0.5% 2-mercaptoethanol, protease inhibitor cocktail (Complete Mini EDTA-free), 1% Triton X-100 and 3% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Trichloroacetic acid (TCA) treatment of the agarose gel after IEF is to be carefully weighed beforehand, because some high molecular mass proteins were less likely to enter the second-dimensional polyacrylamide gel after TCA fixation, and proteins such as mouse skeletal muscle actin gave pseudospots in the agarose 2-DE patterns without TCA fixation. As a good compromise we suggest fixation of proteins in the agarose gel with TCA for one hour or less. The first-dimensional agarose IEF gel containing Pharmalyte as a carrier ampholyte was 180 mm in length and 2.5-4.8 mm in diameter. The gel diameter was shown to determine the loading capacity of the agarose 2-DE, and 1.5 mg liver proteins in total were successfully separated by the use of a 4.8 mm diameter agarose gel.     

Development of native two-dimensional electrophoresis methods for the separation and detection of platinum carrying serum proteins: initial steps

The initial development steps of a native and powerful two-dimensional electrophoretic (2-D) method for the separation of platinum-proteins is described. Mild conditions were selected, particularly for the second dimension, e.g., avoiding buffer systems with platinophile N- or S-donor groups. Therefore, the separation reagents were checked if and at which concentration they can be used for this purpose. In the first dimension isoelectric focusing (IEF) was performed using immobilised pH gradients (IPGs). Native polyacrylamide gel electrophoresis (PAGE) was done in the second dimension. Detection of proteins was achieved via silverstaining. For the determination of platinum in the ultra-trace range, double focusing inductively coupled plasma mass spectrometry (HR-ICP-MS) was used. Autoradiography (¹⁹¹Pt tracer) will be done additionally in the future as a fast, powerful and elegant way of detecting the platinum carrying proteins after the second dimension.     

High-resolution 2-DE for resolving proteins, protein adducts and complexes in plasma

A 2-DE system has been devised in which proteins are first separated in their native state followed by separation according to mass under denaturing conditions (Nat/SDS-PAGE). Hydrophilic properties of the gel and the presence of dihydroxybisacrylamide in the first dimension allowed a good resolution for high-molecular-weight proteins and maintained interactions. With this method 252 plasma spots have been resolved and 140 have been characterized by MS as isoforms of 60 proteins, a relevant part of which (12) were not detected by traditional 2-D gels or by other nondenaturing 2-D techniques. The list includes complement factors (C4d, C7), coagulation factors (coagulation factor II, fibrin beta), apolipoproteins (apolipoprotein B) and cell debris (vinculin, gelsolin, tropomyosin, dystrobrevin beta, fibrinectin I). Nat/SDS PAGE also allowed separation of nicked forms of albumin, Apo B100 and alpha2-macroglobulin and showed the presence of atypical albumin adducts corresponding to post-translational and oxidation products. Our system provides therefore new tools for resolving proteins, protein aggregates and complexes and amplifies the potentiality of traditional electrophoretic analysis.     

Ultrasensitive native fluorescence detection of proteins with miniaturized polyacrylamide gel electrophoresis by laser side-entry excitation

Direct detection of separated proteins inside polyacrylamide gels has many advantages compared to staining methods. Ultrasensitive native fluorescence detection of proteins with miniaturized 1-D and 2-D PAGE was achieved with laser side-entry excitation. The detection limit for R-phycoerythrin protein spots in 1-D SDS-PAGE with 532 nm excitation was as low as 15 fg, which corresponds to only 40,000 molecules. The average detection limit of six standard native proteins was 5 pg per band with 275 nm excitation. The dynamic range spanned more than three orders of magnitude. By using the same detection setup, approximately 150 protein spots from 30 ng of total Escherichia coli extraction were detected on a 0.8 cm x 1 cm gel in 2-D separation. The significant improvement in sensitivity for laser side-entry excitation comes from higher excitation power and lower background level compared with other excitation modes.     

On-line coupling of high performance gel filtration chromatography with imaged capillary isoelectric focusing using a membrane interface

A high performance liquid chromatography system, a sample preparation device, and an imaged capillary IEF (CIEF) instrument are integrated and multiplexed on-line. The system is equivalent to two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), by transferring the principle of 2-D separation to the capillary format. High performance liquid chromatography (HPLC) provides protein separation based on size using a gel filtration chromatography (GFC) column. Each eluted protein is sampled and directed to a novel microdialysis hollow fiber membrane device, where simultaneous desalting and carrier ampholyte mixing occurs. The sample is then driven to the separation column in an on-line fashion, where CIEF takes place. The fluidic technology used by our 2-D system leads to natural automation. The coupling of the two techniques is simple. This is attributed to high speed and efficiency of the sample preparation device that acts as an interface between the two systems, as well as the speed and simplicity of our whole column absorption imaged CIEF instrument. To demonstrate the feasibility of this approach, the separation of a mixture of two model proteins is studied. Sample preparation and CIEF were complete in just 4-5 min, for each of the eluted proteins. Total analysis time is about 24 min. Three-dimensional data representations are constructed. Challenges and methods to further improve our instrument are discussed, and the design of an improved horseshoe-shaped sample preparation sample loop membrane interface is presented and characterized.     

The application of two-dimensional polyacrylamide gel electrophoresis to medical microbiology: molecular epidemiology of viruses and bacteria

A variety of molecular methods can be used to identify protein and nucleic acid markers with which to investigate the epidemiology of viruses and bacteria. This paper reviews the application of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) for studying microbial molecular epidemiology. A small format 2-D PAGE system is described for locating protein markers in group B coxsackie viruses (CVB) and Haemophilus influenzae isolates. Representative isolates of CVB serotypes 2, 4, and 5 were compared by analysing the intracellular proteins present in CVB-infected HEp-2 cells by 2-D PAGE protein gels. Although some of the virus-induced proteins had similar electrophoretic mobilities, the three serotypes could be distinguished from each other on the basis of a major virus-induced protein of molecular weight between 39,000 and 43,000. Protein differences were demonstrated among six serotype 2 CVB (CVB-2) isolates. Four clinical CVB-2 isolates collected over a period of four months had indistinguishable two-dimensional protein profiles. Comparison of the two-dimensional protein profiles of cloned virus stocks prepared from a single clinical CVB isolate demonstrated that it was a heterogeneous virus population. The proteins of nontypable and type-b H. influenzae isolates were compared. Up to 160 proteins, detected by staining with Coomassie Brilliant Blue R, were resolved by 2-D PAGE. Although protein differences between individual bacterial isolates were detected, comparable two-dimensional protein profiles were found for the two groups of H. influenzae isolates. There was no similarity in the two-dimensional protein profiles of H. influenzae and Aeromonas. Potential protein markers were identified that may be useful in long-term studies of H. influenzae epidemiology.     

Revisiting electroblotting of immobilized pH gradient gels: a new protocol for studying post-translational modification of proteins

Currently, one of the most important techniques in proteome analysis is two-dimensional electrophoresis that is widely used for separation of thousands of different protein spots. Nevertheless, characterization of special aspects in protein patterns, e.g., separation of protein isoforms generated by post-translational modifications, requires individual detection methods, e.g., immunoblotting. Blotting of proteins after fractionation in immobilized pH gradients has always caused some problems. In this paper we present an optimized protocol for immunoblotting after isoelectric focusing using immobilized pH gradient (IPG) strips cast on Net-Fix as an internal support that is permeable to electric current. The focusing procedure can be carried out in commonly used IPG systems, e.g., the IPGphor by Amersham Biosciences, where electrically assisted rehydration can be performed. This may be of interest for many laboratories, because the same system as used for the first dimension of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is involved. As an example, we describe separation and detection of up to seven isoforms of recombinant erythropoietin beta using semidry blotting of IPG strips and visualization by chemiluminescence detection.     

Rapid and sensitive separation of trace level protein digests using microfabricated devices coupled to a quadrupole--time-of-flight mass spectrometer

The application of microfabricated devices coupled to a quadrupole time-of-flight mass spectrometer (Qq-TOF-MS) is presented for the analysis of trace level digests of gel-isolated proteins. In order to enhance the sample loading for proteomics analyses, two different on-chip sample preconcentration techniques were evaluated. First, a sample stacking procedure that used polarity switching to remove the sample buffer prior to zone electrophoresis was easily integrated on the microfabricated devices. With the present chip design, this preconcentration technique provided up to 70 nL sample injection with sub-nM detection limits for most peptide standards. For applications requiring larger sample loading, a disposable adsorption preconcentrator using a C18 membrane is incorporated outside the chip. This preconcentration method yielded lower peptide recoveries than that obtainable with sample stacking, and provided a convenient means of injecting several microL of sample with detection limits of typically 2.5 nM for hydrophobic peptides. The analytical merits of both sample enrichment approaches are described for the identification of bands isolated from two-dimensional (2-D) gel separation of protein extracts from Haemophilus influenzae. Accurate molecular mass measurements (< 5 ppm) in peptide mapping experiments is obtained by introducing an internal standard via a post-separation channel. Rapid identification of trace level peptides is also demonstrated using on-line tandem mass spectrometry and database searching with peptide sequence tags.