A combination of immobilised pH gradients improves membrane proteomics.

Membrane protein analyses have been notoriously difficult due to hydrophobicity and the general low abundance of these proteins compared to their soluble cytosolic counterparts. Shotgun proteomics has become the preferred method for analyses of membrane proteins, in particular the recent development of peptide immobilized pH gradient isoelectric focusing (IPG-IEF) as the first dimension of two-dimensional shotgun proteomics. Recently, peptide IPG-IEF has been shown to be a valuable shotgun proteomics technique through the use of acidic narrow range IPG strips, which demonstrated that small acidic p I increments are rich in peptides. In this study, we assess the utility of both broad range (BR) (p I 3-10) and narrow range (NR) (p I 3.4-4.9) IPG strips for rat liver membrane protein analyses. Furthermore, the use of these IPG strips was evaluated using label-free quantitation to demonstrate that the identification of a subset of proteins can be improved using NR IPG strips. NR IPG strips provided 2603 protein assignments on average (with 826 integral membrane proteins (IMPs)) compared to BR IPG strips, which provided 2021 protein assignments on average (with 712 IMPs). Nonredundant protein analysis demonstrated that in total from all experiments, 4195 proteins (with 1301 IMPs) could be identified with 1428 of these proteins unique to NR IPG strips with only 636 from BR IPG strips. With the use of label-free quantitation methods, 1659 proteins were used for quantitative comparison of which 319 demonstrated statistically significant increases in normalized spectral abundance factors (NSAF) in NR IPG strips compared to 364 in BR IPG strips. In particular, a selection of six highly hydrophobic transmembrane proteins was observed to increase in NSAF using NR IPG strips. These results provide evidence for the use of alternative pH gradients in combination to improve the shotgun proteomic analysis of the membrane proteome.

Characterization of the rat liver membrane proteome using peptide immobilized pH gradient isoelectric focusing.

Membrane proteins are of particular interest in proteomics because of their potential therapeutic utility. Past proteomic approaches used to investigate membrane proteins have only been partially successful at providing a comprehensive analysis due to the inherently hydrophobic nature and low abundance for some of these proteins. Recently, these difficulties have been improved by analyzing membrane protein enriched samples using shotgun proteomics. In addition, the recent application of methanol-assisted trypsin digestion of membrane proteins has been shown to be a method to improve membrane protein identifications. In this study, a comparison of different concentrations of methanol was assessed for assisting membrane protein digestion with trypsin prior to analysis using a gel-based shotgun proteomics approach called peptide immobilized pH gradient isoelectric focusing (IPG-IEF). We demonstrate the use of peptide IEF on pH 3-10 IPG strips as the first dimension of two-dimensional shotgun proteomics for protein identifications from the membrane fraction of rat liver. Tryptic digestion of proteins was carried out in varying concentrations of methanol in 10 mM ammonium bicarbonate: 0% (v/v), 40% (v/v), and 60% (v/v). A total of 800 proteins were identified from 60% (v/v) methanol, which increased the protein identifications by 17% and 14% compared to 0% (v/v) methanol and 40% (v/v) methanol assisted digestion, respectively. In total, 1549 nonredundant proteins were identified from all three concentrations of methanol including 690 (42%) integral membrane proteins of which 626 of these proteins contained at least one transmembrane domain. Peptide IPG-IEF separation of peptides was successful as the peptides were separated into discrete pI regions with high resolution. The results from this study prove utility of 60% (v/v) methanol assisted digestion in conjunction with peptide IPG-IEF as an optimal shotgun proteomics technique for the separation and identification of previously unreported membrane proteins.

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.

An immobiline DryStrip application method enabling high-capacity two-dimensional gel electrophoresis.

In the field of proteomics the need to detect low-abundance cellular components, such as regulatory proteins, is of critical importance. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is one of the most commonly used separation tools for these biological investigations. In this paper we report an alternative micropreparative 2-D PAGE sample application method, called the "paper bridge loading" method. This method makes it possible to apply a larger sample volume to commercially available immobilized pH gradient (IPG) strips. The Vh products required for focusing are only marginally longer than those used in analytical experiments. The method was compared to traditional cup loading and in-gel rehydration. With 18 cm long narrow-range Immobiline DryStrip pH 4.5-5.5, the "paper bridge" method allowed the application of 10 mg human plasma proteins compared to 3 mg with traditional loading methods. The corresponding figures using Escherichia coli sample was found to be 6 mg and less than 2 mg, respectively. The paper bridge method also showed the best results in terms of spot resolution and separation of high molecular weight proteins.

Proteomic 'contigs' of Ochrobactrum anthropi, application of extensive pH gradients.

The most extensive linear pH gradients yet employed in combination with two-dimensional gel electrophoresis are described, along with their application in proteome analysis. A significant proportion of the protein compliment of bacterial species is believed to be accessible using an extended linear pH gradient of 2.3 to 11.0. Protein standards with predicted isoelectric points (pI) ranging from 3.24 to 9.56 were used to confirm focusing positions with respect to the immobilised pH gradients (IPG) prior to mapping studies of Ochrobactrum anthropi. Multiple gel images were used to construct contiguous windows of protein expression ('proteomic contigs') within 18 cm pH gradients 2.3-5, 4-7, and 6-11 in conjunction with 15% T and 7.5% T acrylamide gels, the latter being used to resolve higher molecular weight (M(r)) proteins. Each IPG had a 5 cm region of similar pH gradient overlap at pH 4-5 and pH 6-7 that was used to construct an image of protein expression characteristic of whole cell lysates. This is reminiscent of genomic sequencing initiatives whereby portions are combined to form a contiguous picture of the whole. The protein maps obtained demonstrated a means of resolving the many tens of thousands of cellular proteins likely to occur in eukaryotic systems, but also highlighted the need to further optimise protein extraction, equilibration buffers, and separation conditions of higher M(r) proteins occurring at extreme pI. Theoretical 2-D protein maps were constructed for five organisms for which the total DNA sequence is now available. In all cases, higher M(r) acidic and basic proteins were shown to be common.

'Proteomic contigs' of Mycobacterium tuberculosis and Mycobacterium bovis (BCG) using novel immobilised pH gradients.

Tuberculosis remains a major health problem throughout the world and the failure of the existing bacille Calmette-Guérin (BCG) vaccine in recent trials has prompted a search for potential replacements. Recent advances in molecular and cell biology have cast doubts on the ability of genetic analysis alone to predict polygenic human diseases and other complex phenotypes and have therefore redirected our attention to proteome studies to complement information obtained from DNA sequencing initiatives. Novel acidic (pH 2.3-5) and basic (pH 6-11) IPG gel gradients were employed in conjunction with commercially available pH 4-7 gradients to significantly increase (fourfold) the number of protein spots previously resolved on two-dimensional (2-D) gels of Mycobacterium species. A total of 772 and 638 protein spots were observed for M. bovis BCG and M. tuberculosis H37Rv, respectively, the latter corresponding to only the pH regions 4-7 and 6-11. Of interest was the bimodal distribution observed for proteins separated from M. bovis BCG across both M(r) and pH ranges. Some differences in protein expression were observed between these two organisms, contrary to what may have been expected considering the high degree of conservation in gene order and sequence similarity between homologous genes. Further work will be directed towards a more detailed analysis of these differences, so as to allow more accurate diagnosis between vaccination and active tuberculosis. The latter is of major importance to epidemiological studies and for patient management.

Characterisation of basic proteins from Spiroplasma melliferum using novel immobilised pH gradients.

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) has become the method of choice for efficient separation of complex protein mixtures. Previously, analysis of the Spiroplasma melliferum proteome (protein complement of a genome) has been performed with pH 3-10 and narrow range pH 4-7 IPG gel strips. We report here on the use of novel 18 cm basic (pH 6-11) immobilised pH gradients (IPG) to increase the resolution of protein spots visible within 2-D gels. These gradients were synthesised to emulate the gradient of commercially available IPG gel strips in a 5 cm region of overlap so as to attempt construction of a more complete map of cellular protein expression. Approximately 50 additional gene products were detected from S. melliferum that were not previously well-resolved or visible using wide-range pH 3-10 IPG gel strips. Twenty-seven of these were electrotransferred to polyvinylidene difluoride (PVDF) membrane and analysed by N-terminal protein microsequencing. Protein spots with an initial peak yield of as little as 100 femtomoles (fm) were sequenced to 5-10 amino acid residues, demonstrating the importance of improved sample handling procedures and analytical technologies. Many essential metabolic enzymes were shown to have basic pI, including: glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, carbamate kinase and lactate dehydrogenase. A very basic protein (pI approximately 11.0) was identified as uridylate kinase, an enzyme indirectly associated with pyrimidine biosynthesis and thought be absent in some members of the bacterial class Mollicutes. The advent of novel basic (pH 6-11) IPGs has allowed the visualisation of a significantly greater percentage of the 'functional proteome', that portion of the total protein complement of a genome actively translated within a specific time frame, on 2-D electrophoresis gels. This will aid in the characterisation of translated gene products in conjunction with genome sequencing initiatives.

Reference points for comparisons of two-dimensional maps of proteins from different human cell types defined in a pH scale where isoelectric points correlate with polypeptide compositions.

A highly reproducible, commercial and nonlinear, wide-range immobilized pH gradient (IPG) was used to generate two-dimensional (2-D) gel maps of [35S]methionine-labeled proteins from noncultured, unfractionated normal human epidermal keratinocytes. Forty one proteins, common to most human cell types and recorded in the human keratinocyte 2-D gel protein database were identified in the 2-D gel maps and their isoelectric points (pI) were determined using narrow-range IPGs. The latter established a pH scale that allowed comparisons between 2-D gel maps generated either with other IPGs in the first dimension or with different human protein samples. Of the 41 proteins identified, a subset of 18 was defined as suitable to evaluate the correlation between calculated and experimental pI values for polypeptides with known composition. The variance calculated for the discrepancies between calculated and experimental pI values for these proteins was 0.001 pH units. Comparison of the values by the t-test for dependent samples (paired test) gave a p-level of 0.49, indicating that there is no significant difference between the calculated and experimental pI values. The precision of the calculated values depended on the buffer capacity of the proteins, and on average, it improved with increased buffer capacity. As shown here, the widely available information on protein sequences cannot, a priori, be assumed to be sufficient for calculating pI values because post-translational modifications, in particular N-terminal blockage, pose a major problem. Of the 36 proteins analyzed in this study, 18-20 were found to be N-terminally blocked and of these only 6 were indicated as such in databases. The probability of N-terminal blockage depended on the nature of the N-terminal group. Twenty six of the proteins had either M, S or A as N-terminal amino acids and of these 17-19 were blocked. Only 1 in 10 proteins containing other N-terminal groups were blocked.

A nonlinear wide-range immobilized pH gradient for two-dimensional electrophoresis and its definition in a relevant pH scale.

A new nonlinear immobilized pH gradient (IPG) is proposed as the first dimension for two-dimensional electrophoresis. In comparison to conventional carrier ampholyte techniques, it offers better resolution and greater reproducibility whilst allowing application of higher protein loads. Furthermore, we have checked and supplemented existing data on pK values for the immobilized groups in the presence of 8M urea. This is necessary for pH gradients to be defined in a pH scale relevant to the focusing conditions such that spot positions can be related to amino acid compositions. The data will allow definition of pH scales for the temperature range 10-25 degrees C and for a pH range covering the major part of the nonlinear pH gradient. With the latter, focusing positions are neither influenced by urea concentration nor by the choice or the concentration of detergent or carrier ampholyte. Temperature is the only parameter affecting focusing reproducibility and here any changes in focusing positions can be related to the amino acid compositions of peptides.

Micropreparative two-dimensional electrophoresis allowing the separation of samples containing milligram amounts of proteins.

We describe some simple modifications to the micropreparative two-dimensional (2-D) electrophoresis procedure using immobilized pH gradients in the first dimension and sodium dodecyl sulfate-electrophoresis in the second dimension. The geometry of the immobilized pH gradient strips has been changed to allow the use of large sample application cups that can accommodate greater sample volumes. The use of narrow range immobilized pH gradients with a large sample loading volume allows an efficient resolubilization of polypeptides after the first dimension. As a result, the vertical streaking caused by too high a protein concentration is eliminated in the second dimension. Protein identification by N-terminal sequencing is facilitated by the large protein load (1-15 mg) which can be employed using this modification. Spots not normally detectable on conventional analytical 2-D maps, even with sensitive silver staining, are observed. Results for plasma and liver proteins are shown.

The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences.

The focusing positions in narrow range immobilized pH gradients of 29 polypeptides of known amino acid sequence were determined under denaturing conditions. The isoelectric points of the proteins calculated from their amino acid sequences matched with good accuracy the experimentally determined pI values. We show the advantages of being able to predict the position of a protein of known structure within a two-dimensional gel.

Human liver protein map: a reference database established by microsequencing and gel comparison.

This publication establishes a reference human liver protein map obtained with immobilized pH gradients. By microsequencing, 57 spots or 42 polypeptide chains were identified. By protein map comparison and matching (liver, red blood cell and plasma sample maps), 8 additional proteins were identified. The new polypeptides and previously known proteins are listed in a table and/or labeled on the protein map, thus providing a human liver two-dimensional gel database. This reference map can be used to identify protein spots on other samples such as rectal cancer biopsies.

Improving the detection of proteins after transfer to polyvinylidene difluoride membranes.

N-Terminal sequence analysis of proteins separated by two-dimensional polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes has become the method for molecular characterization of proteins contained in biological samples. However, the proteins of lower abundance cannot be sequenced directly, without improving the technique. We have studied a drying method on several PVDF membranes including Trans-Blott, Immobilon P and Problott. Using Amido Black, Coomassie Brilliant Blue R-250 and Ponceau S, we have obtained, in comparison with the non-dried membranes, an enormous increase in the number of detectable proteins.

Plasma protein map: an update by microsequencing.

The reference plasma protein map, obtained with immobilized pH gradients in the first dimension of two-dimensional electrophoresis, is presented. By microsequencing, more than 40 polypeptide chains were identified. The new polypeptides and previously known proteins are listed in a table and labeled on the protein map, thus providing an update of the human plasma two-dimensional gel database.

Red blood cell protein map: a comparison between carrier-ampholyte pH gradient and immobilized pH gradient, and identification of four red blood cell enzymes.

The aim of this study was (a) to establish a red blood cell (RBC) protein map with immobilized pH gradient for the first dimension (b) to compare the pattern with previously published RBC protein map obtained with carrier-ampholyte pH gradients and (c) to localize four new enzymes on the map (i.e. 6-phosphogluconic dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, glutathione peroxidase and superoxide dismutase). This publication provides the most updated RBC polypeptide pattern with twelve proteins or enzymes localized on the map.

Moving and stationary boundaries in immobilized pH gradients.

The relations describing the concentration changes at moving boundaries in a medium containing bound, buffering group are derived for a system which, except for hydrogen and hydroxyl ions, contains one anionic and one cationic mobile constituent. The relations found have been used to calculate concentrations and conductivities in zones developing in immobilized pH gradients. Assumptions used in the calculations as well as conductivity ratios between zones have been experimentally controlled and were found to reasonably agree with expectations. It is also shown how difference in transference numbers between sample droplet and gel will cause concentration and pH changes at the gel-sample droplet interfaces and it is explained how these changes are related to ionic concentrations in the gel. The high concentration zone generated at one of the interfaces will be transported into the gel. This transport has been numerically simulated and experimentally verified. The low concentration generated at the opposite interface will cause titration impeding sample entrance in the gel through this interface even when the gel contains ions other than H+ or OH- transported towards the interface. The described phenomena explain the dependence of lateral spreading, precipitation at the application site as well as streaking and smearing along sample lanes, on the type and concentration of low molecular weight ions originally present in the gel.

Isoelectric focusing of basic proteins: the problem of oxidation of cysteines.

Isoelectric focusing of human globin chains in polyacrylamide gels dried in the ambient atmosphere and rehydrated in the presence of 8 mol/L urea produces artefactual doublets of zones as a result of oxidation by the gel. This oxidation can be avoided in separations of short duration by adding a reducing agent (e.g. 2-mercaptoethanol or dithiothreitol to the rehydration solution (Altland, K. and Rossmann, U., Electrophoresis 1985, 6, 314-325). We now demonstrate that the observed zone doublets can be explained by assuming neutralization of the contribution of dissociated sulfhydryl group of cysteine to pI by partial and reversible formation of globin dimers held together by disulfide bridges. Long time separations, requiring e.g. more than 4 h at greater than or equal to 500 V/cm, in pH gradients exceeding pH 7.5, are accompanied by artefactual oxidation from both the atmosphere and the gel matrix. Oxidation from the atmosphere as well as the effect of carbon dioxide can be eliminated by overlayering the gel with paraffin oil. Oxidation from the gel matrix can only partially be inhibited by rehydration of gels in the presence of 2-mercaptoethanol or dithiothreitol. Nearly complete protection against oxidation by the gel matrix was achieved by adding a permanent supply of 2-ME to the gel or by adding DTT to the cathodic wick towards the end of the experiment. Alkylation with iodoacetamide or iodoacetic acid resulted in stable globin patterns, which, however, displayed additional artefactual zones. Our experimental data indicate that the polyacrylamide gels function as an electron acceptor for dissociated sulfhydryl groups in proteins, even after pretreatment with strong reducing agents for proteins.

Microheterogeneity of human plasma lecithin: cholesterol acyltransferase examined by isoelectric focusing in immobilized pH gradients.

The microheterogeneity of highly purified human plasma lecithin:cholesterol acyltransferase (LCAT) has been examined by electrophoresis in immobilized pH gradients in Immobiline-polyacrylamide gels of the pH ranges 4-7 and 4.2-4.9. Seven isoforms were obtained with LCAT isolated from pools of normal plasma. Using this technique the apparent pI values at 15 degrees C for the isoforms in the pH 4.2-4.9 gradient were 4.37, 4.42, 4.48, 4.53, 4.60, 4.67 and 4.74. (SD = +/- 0.03 for all). The most intensely stained band in the isoform pattern corresponded to the isoform with a pI value of 4.48.

The influence of the support material used on band sharpness in Immobiline gels.

To achieve excellent isoelectrofocusing results with the use of Immobiline gels, the quality of the support is essential. Effects such as diffuse bands, missing protein fractions, streaking and trailing could originate from a support of inadequate quality. Two different treatments to eliminate these effects were evaluated. A washing procedure improves the band sharpness, and addition of carrier ampholytes eliminates streaking and trailing.

Effect of salt on the performance of immobilized pH gradient isoelectric focusing gels.

The effect of salt and buffer ions in the sample or in an immobilized pH gradient (IPG) on sample entry into the gel and on the final focused pattern are presented. During the initial phase of electrofocusing, ions present in the gel, either as counter ions to the immobilized charge groups of the IPG gel or added to the gel matrix during the rehydration process, are transported toward the electrodes. For ions present at a concentration exceeding approximately 1 mM the transport can be followed by the refractile line marking the trailing edge of an ion-containing zone. Gradual sample entry may be achieved by applying the sample at a site (near the anode or cathode) opposite to that from which the sharpest refractile line, marking the ion present in the highest concentration, approaches the sample. Additionally, lateral band spreading of the sample is avoided. Thus, sample applied at the cathode for IPG gels rehydrated with 1-2 mM Tris base, or at the anode for gels rehydrated with 1-2 mM acetic acid or sodium acetate, enters the gel matrix gradually without lateral band spreading. In contrast, sample applied at the anode, for Tris-containing gels, or at the cathode, for acetate-containing gels, enters rapidly in a sharp zone when the refractile line reaches the sample zone. This results in a high local protein concentration in the zone immediately behind the boundary with lateral band spreading.(ABSTRACT TRUNCATED AT 250 WORDS)