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.     

Isoelectric focusing in immobilized pH gradients with carrier ampholytes added for high-resolution phenotyping of bovine beta-lactoglobulins: characterization of a new genetic variant

The genetic variants of bovine beta-lactoglobulin (beta-lg) from the "Murnau-Werdenfelser" breed were analyzed in three different isoelectric focusing (IEF) systems. While carrier ampholyte IEF with a pH gradient of 0.2 pH/cm did not resolve the new variant W from the B variant and IEF in immobobilized pH gradients (IPG) with 0.1 pH/cm only partially resolved it, adequate separation was achieved with IPG-IEF in a pH 5.25-pH 5.7 gradient, in presence of 0.8 % w/v carrier ampholytes, both over a 10 and 17 cm separation distance. Apparent isoelectric points (pI's) and genetic frequencies (f) were as follows: beta-lg A, pI = 5.370, f = 0.364; beta-lg B, pI = 5.485, f = 0.480; beta-lg W, pI = 5.492, f = 0.076; and beta-lg D, pI = 5.610, f = 0.080. The small difference of delta pI = 0.007 between beta-lg B and beta-lg W respectively, seems to originate from a "silent" substitution of neutral amino acid residues as compared to the larger delta pI's of the other genetic variants of beta-lg, which result from substitution of charged amino acids.     

Evaluation of protein loading techniques and improved separation in OFFGEL isoelectric focusing

2-DE proved to be a key technology in protein science since the two orthogonal separation dimensions are capable of protein isoform separation. Recently, Agilent introduced the OFFGEL 3100 fractionator for in solution IEF (off-gel) of proteins with the help of a 12- or 24-well frame. With this instrument also conventional focusing in IPG strips after passive in-tray rehydration can be performed. In this study, two novel IEF applications using the OFFGEL electrophoresis were developed. First, a sample cup was built and a cup-loading method for the OFFGEL device was implemented. Applying proteins via cup resulted in higher reproducibility and less protein loss compared with conventional in-tray rehydration loading. Especially, the recovery of basic and high-molecular-mass proteins seems to be favored by cup loading. These effects are more pronounced with low microgram sample amounts. Second, a 48-well OFFGEL frame was developed, which doubles the resolution of the commercially available 24-well frame. It is capable of separating proteins with small pI differences and shows potential for isoform/PTM separation.     

Efficient separation and analysis of peroxisomal membrane proteins using free-flow isoelectric focusing

We have elaborated a protocol for the fractionation of both hydrophilic and hydrophobic proteins using as a model the matrix and membrane compartments of highly purified rat liver peroxisomes because of their distinct proteomes and characteristic composition with a high quota of basic proteins. To keep highly hydrophobic proteins in solution, an urea/thiourea/detergent mixture, as used in traditional gel-based isoelectric focusing (IEF), was added to the electrophoresis buffer. Electrophoresis was conducted in the ProTeam free-flow electrophoresis (FFE) apparatus of TECAN separating proteins into 96 fractions on a pH 3-12 gradient. Consecutive sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis demonstrated that both matrix and the integral membrane proteins of peroxisomes could be successfully fractionated and then identified by mass spectrometry. This is documented by the detection of PMP22, which is the most hydrophobic and basic protein of the peroxisomal membrane with a pI > 10. The identification of 96 prominent spots corresponding to polypeptides with different physical and chemical properties, e.g., the most abundant integral membrane polypeptides of peroxisomes and specific ones of the mitochondrial and microsomal membrane, reflects the fractionation potential of free-flow (FF)-IEF, accentuating its value in proteomic research as an alternative perhaps superior to gel-based IEF.     

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.     

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.     

Toward an integrated microchip sized 2-D polyacrylamide slab gel electrophoresis device for proteomic analysis

We describe a miniaturized instrument capable of performing 2-DE. Our miniaturized device is able to perform IEF and polyacrylamide slab gel electrophoresis (PASGE) in the same unit. It consists of a compartment for a first-dimensional IEF gel, which is connected to a second-dimensional PASGE gel. The focused samples are automatically transferred from the IEF gel to the PASGE gel by electromigration. Our preliminary experiments show that the device is able to focus and separate a mixture of proteins in approximately 1 h, excluding the time required for the staining procedure. On average, the gel-to-gel retardation factor (Rf) variation was 6.2% (+/-0.9%) and pI variation was 2.5% (+/-0.6%). Separated protein spots were excised from stained gels, digested with trypsin, and further identified by MS, thus enabling direct proteomic analysis of the separated proteins.     

pI-based phosphopeptide enrichment combined with nanoESI-MS

IEF is introduced as a new principle for enrichment and separation of phosphopeptides as obtained after digestion of phosphoproteins by trypsin. Tryptic peptides and phosphopeptides exhibit pI values, which overlap in the range of about 4-6. However, after methyl esterification of all carboxyl functions, the pI values of tryptic peptides and phosphopeptides regroup in discrete clusters. In addition, mono- and diphosphorylated peptides show different but very homogeneous pI values, with variations when internal Arg, Lys, or His residues are present. Experimentally, this new concept was applied for separation of model peptides on IPG strips pH 3-10 as used in the first dimension of 2-DE. After IEF of methyl-esterified peptides, the IPG strip was cut into pieces followed by peptide extraction, desalting and MS analysis by nanoESI-MS. Phosphopeptides were found to focus in good agreement with their calculated pI values. This analytical strategy showed a resolution of about 0.2 pI units, and thus turned out to be capable of detecting minor differences in pI values, such as those occurring between pSer, pThr and pTyr residues. Using IPG strips with a pI range of 3-10, methyl esterified nonphosphorylated tryptic peptides are concentrated in the basic part of the IPG strip or even leave the strip. Thus, efficient enrichment of phosphopeptides and their subfractionation according to pI is obtained in one step. Minor hydrolytic side reactions including deamidation of Asn and partial hydrolysis of methyl esters are observed. The results show that IEF opens attractive avenues for the further advancement of analytical phosphoproteomics.     

Identification of proteins in human cerebrospinal fluid using liquid-phase isoelectric focusing as a prefractionation step followed by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionisation mass spectrometry

Cerebrospinal fluid (CSF) is in close proximity to the brain and changes in the protein composition of CSF may be indicative of altered brain protein expression in neurodegenerative disorders. Analysis of brain-specific proteins in CSF is complicated by the fact that most CSF proteins are derived from the plasma and tend to obscure less abundant proteins. By adopting a prefractionation step prior to two-dimensional gel electrophoresis (2-DE), less abundant proteins are enriched and can be detected in complex proteomes such as CSF. We have developed a method in which liquid-phase isoelectric focusing (IEF) is used to prefractionate individual CSF samples; selected IEF fractions are then analysed on SYPRO-Ruby-stained 2-D gels, with final protein identification by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS). To optimise the focusing of the protein spots on the 2-D gel, the ampholyte concentration in liquid-phase IEF was minimised and the focusing time in the first dimension was increased. When comparing 2-D gels from individual prefractionated and unfractionated CSF samples it is evident that individual protein spots are larger and contain more protein after prefractionation of CSF. Generally, more protein spots were also detected in the 2-D gels from prefractionated CSF compared with direct 2-DE separations of CSF. Several proteins, including cystatin C, IgM-kappa, hemopexin, acetyl-coenzyme A carboxylase-alpha, and alpha-1-acid glycoprotein, were identified in prefractionated CSF but not in unfractionated CSF. Low abundant forms of posttranslationally modified proteins, e.g. alpha-1-acid glycoprotein and alpha-2-HS glycoprotein, can be enriched, thus better resolved and detected on the 2-D gel. Liquid-phase IEF, as a prefractionation step prior to 2-DE, reduce sample complexity, facilitate detection of less abundant protein components, increases the protein loads and the protein amount in each gel spot for MALDI-MS analysis.     

Tailoring orthogonal proteomic routines to understand protein separation during ion exchange chromatography

Surface charge, molecular weight, and folding state are known to influence protein chromatographic behaviour onto ion exchangers. Experimentally, information related to such factors can be gathered via 2-DE methods. The application of 2-D PAGE under denaturing/reducing conditions was already shown to reveal separation trends within a large protein population from cell extracts. However, ion-exchange chromatography normally runs under native conditions. A tailored protocol consisting in a first separation based on IEF on Immobiline strips under native conditions followed by a second dimension SDS-PAGE run was adopted. The chromatographic versus electrophoretic separation behaviours of two model proteins, thaumatin (TAU) and BSA, were compared to better understand which proteomic routine would be better suited to anticipate IEX chromatographic separations. It was observed that the information contained in the pI value obtained with the adapted 2-DE protocol showed better correlation with the IEX chromatographic behaviour. On the other hand, chromatographic separations performed in the presence of urea as a denaturant have demonstrated the potential influence of hydrodynamic radius/conformation on protein separation. Moreover, the information provided by such 2-D system correlated well with the chromatographic behaviour of an additional set of pure proteins. An initial prediction of protein ion-exchange chromatographic behaviour could be possible utilizing an experimental approach based on 2-DE running under milder chemical conditions. This technique provides information that more closely resembles the separation behaviour observed with a complex biotechnological feedstock.     

Interrogation of MS/MS search data with an pI Filter algorithm to increase protein identification success

In high-throughput proteomics, the bottom-up approach has become a widely used method for the identification of proteins that is based on tryptic peptide MS/MS analysis. Separation methodologies that use IEF of tryptic peptides have recently been introduced and provide an extra dimension of peptide separation. In addition to its great fractionation capability, tryptic peptide prefractionation by IEF can also increase the protein identification success. The pI information of the peptide gained can be successfully used in a post-database search filtering step. We introduce a filtering algorithm that is based on the comparison of the experimental and theoretical pI's to validate peptide identifications by MS/MS data search engines.     

Characterization of antithrombin III from human plasma by two-dimensional gel electrophoresis and capillary electrophoretic methods

The isoforms distribution of the glycoprotein antithrombin III (ATIII) derived from human plasma was investigated by means of isoelectric focusing (IEF) in polyacrylamide gels with immobilized pH gradients (IPG) and two-dimensional gel electrophoresis (2-DE) as well as capillary electrophoretic methods. It turned out that the presence of high concentrations of chaotropics (urea, thiourea) and zwitterionic detergents (3-[(3-cholamidepropyl)dimethylammonio]-1-propanesulfonate (CHAPS)) was decisive for attaining good resolution of the protein isoforms. Resolution by IPG-IEF was obtained with excellent reproducibility and pI differences down to 0.01 pH units could be distinguished. ATIII-alpha and ATIII-beta-fractions preseparated by heparin affinity chromatography showed an analogous but shifted spot pattern consisting each of one major and three minor isoforms. The main isoforms of ATIII-alpha and ATIII-beta exhibit pI values of 5.18 and 5.32, respectively, both values determined in the presence of high concentrations of urea. The pI difference of 0.14 pH units correspond to the effect of two sialic acids absent in ATIII-beta. The formation and occurrence of ATIII dimers and trimers turned out to be dependent on the sample preparation. The results obtained by 2-DE were compared with those of capillary zone electrophoresis (CZE) and capillary IEF (CIEF). Quantitative analysis regarding the CZE separated isoforms of plasma derived ATIII yielded a content of about 70% ATIII-alpha main isoform and about 6.6% of ATIII-beta. The pI values of ATIII determined by CIEF with internal calibration were in fair agreement with the pI values of the main isoforms achieved with 2-DE.     

Fish muscle parvalbumins as marker proteins for native and urea isoelectric focusing

An isoelectric point (pI) calibration kit containing fish muscle parvalbumins was prepared and tested for its suitability for isoelectric focusing (IEF) in the presence of 8 M urea. The pattern obtained by urea CleanGel IEF consisted of nine bands covering the pI range 4.96-5.64. This range is relevant for species identification of heated fish by urea IEF. The kit may also be used for native IEF in the low pH range, as demonstrated by running an extract made from the kit together with water-soluble fish muscle proteins on Servalyt Precotes 3-6.     

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.     

Labeling effects on the isoelectric point of green fluorescent protein.

We studied the effects of fluorescent labeling on the isoelectric points (pI values) of proteins using capillary isoelectric focusing with laser-induced fluorescence detection (cIEF-LIF). Specifically, we labeled green fluorescent protein (GFP) from the jellyfish Aequorea victoria with the fluorogenic dye 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ). cIEF-LIF was used to monitor the native fluorescence of GFP and showed pI changes in GFP's FQ-labeled products. Multiple labeling of GFP with FQ produced a series of products with pI values shifted towards a low pH. We verified cIEF-LIF results with traditional slab gel IEF. Our cIEF-LIF technique can routinely detect 10(-11) M of FQ-labeled protein, whereas traditional slab gel IEF with silver stain detection gives detection limits of 10(-7) M in the same samples.     

"Early" protein synthesis of Lactobacillus delbrueckii ssp. bulgaricus in milk revealed by [35S] methionine labeling and two-dimensional gel electrophoresis

The proteomes of exponentially growing and stationary cells of Lactobacillus delbrueckii ssp. bulgaricus grown in rich medium (MRS) were separated by two-dimensional polyacrylamide gel electrophoresis (2-DE) and quantified after Coomassie staining. Stationary cells grown in MRS were inoculated in reconstituted skim milk, and "early" protein synthesis during the first 30 min of fermentation in milk was monitored by [35S]methionine labeling and 2-DE. In contrast to exponentially growing or stationary cells, the predominant "early" proteins were small (< 15 kDa) and of low pI (< 5.3). Quantification of the proteome of the "early" lag phase based on 47 "spots" revealed that only three "early" proteins accounted for more than 80% of the total label. They were identified as pI 4.7 and 4.9 isoforms of the heat-stable phosphoryl carrier protein (HPr) with 45.2 and 9.4% of total label, respectively, and an unknown protein called EPr1 ("early" protein 1) with 26.6% of total label. Although an N-terminal sequence of 19 amino acids was obtained, no homologs to EPr1 could be found. De novo synthesis of the 10 and 60 kDa heat shock proteins (GroES and GroEL) was considerably lower (0.04 and 0.9% of total label, respectively), indicating only low levels of stress. Synthesis of triosephosphate isomerase (Tpi) as marker for glycolytic enzymes reached only 0.08% of total label. Our results demonstrate that inoculation in milk, resulting in a change from glucose to lactose as carbon source, imposes only little need for synthesis of stress or glycolytic enzymes, as sufficient proteins are present in the stationary, MRS-grown cells. The high level of expression of the pI 4.7 isoform of HPr suggests a regulatory function of the presumed Ser-46 phosphorylated form of HPr.     

An optimized procedure for solubilization, reduction, and transfer of human breast cancer membrane-enriched fraction by 2-DE

The separation of integral and peripheral membrane proteins is still a challenge, although many achievements have been made in the 2-DE-based membrane proteomics. Using a human breast cancer cell line, MCF-7, we investigated the influences of Tris, reducing reagents, cup loading, and SDS on membrane protein solubilization and separation by 2-DE. The addition of Tris to the sample solution improved the solubilization of the membrane-enriched fraction, and the best-quality gel patterns were obtained at 20 mM Tris. Tributylphosphine (TBP), a reducing agent, was not optimum in the 2-DE process because it not only decreased the solubilization of hydrophobic proteins but also caused some proteins, such as hsp60, prohibitin, and actin, to be resolved to a string of spots. However, when combined with DTT, TBP could improve the resolution of 2-DE patterns. Cup loading significantly facilitated the entrance of membrane proteins into IPG strips and over 1000 protein spots with high resolution were visualized. Adopting this strategy, an ATP synthase alpha chain was resolved into two adjacent spots for the first time in 2-DE gel patterns through the adding DTT in the middle of the IEF. A high SDS concentration in the equilibration buffer enhanced the transfer and increased the staining intensity of 50% of the protein spots in the gels, but also resulted in losses of some spots.     

Quantitative evaluation of sample application methods for semipreparative separations of basic proteins by two-dimensional gel electrophoresis

The use of cup-loading for sample application has become widely used in two-dimensional electrophoresis (2-DE) for resolution of basic proteins, but no side-by-side quantitative study has been published which compares cup-loading with the alternative passive and active rehydration methods to fully promote one type of loading method over another. Replicate 2-D gels from each loading method were quantitatively evaluated for gel-to-gel reproducibility using IPG 6-11 strips and semipreparative protein loads (300 microg). Gels were stained with SYPRO Ruby and analyzed with PDQuest. An inexpensive home-made assembly for cup-loading was used with the Protean IEF Cell for separation of whole cell extracts from the archaeon, Sulfolobus solfataricus. Cup-loading was determined to be far superior for IPG 6-11 separations than active or passive rehydration methods. Cup-loading consistently produced the greatest number of detectable spots, the best spot matching efficiency (56%), lowest spot quantity variations (28% coefficient of variation, CV), and the best-looking gels qualitatively. The least satisfactory results were obtained with active rehydration, followed closely by passive rehydration in off-line tubes. Passive rehydration experiments, performed using an on-line isoelectric focusing (IEF) tray, produced comparable spot numbers to cup-loading (84%), with 55% of the spots having higher intensity but 10% more spot quantity variance than cup-loading.