Narrow Range Immobilized pH Gradient Research Articles

A Novel Prefractionation Method Combining Protein and Peptide Isoelectric Focusing in Immobilized pH Gradient Strips

To increase sensitivity and analytical depth in shotgun proteomics, prefractionation of complex samples is often used. Here we describe a novel prefractionation method, Sandwich high resolution isoelectric focusing, which combines both protein and peptide isoelectric focusing. In the first step, intact proteins are separated on the basis of isoelectric point (pI) using traditional immobilized pH gradient (IPG) strips. Segments in the IPG-strip containing proteins of interest are subsequently cut out and applied to in-strip digestion, without subsequent peptide elution. In the second peptide isoelectric focusing step, the strip segments are used as loading bridges. The peptides are thereby directly applied to the peptide isoelectric focusing, without an intermediate elution step, and separated on narrow range IPG strips to reduce the complexity on the peptide level. In the final step, the peptides are eluted into 96-well plates and analyzed with mass spectrometry. In a proof of principle experiment, using this method to zoom in on pI regions of interest in human plasma, we identify over 800 proteins, with concentrations spanning over 6 orders of magnitude.

Comparative proteome analysis of Bacillus anthracis with pXO1 plasmid content

Bacillus anthracis the causative agent of anthrax, is an important pathogen among the Bacillus cereus group of species because of its physiological characteristics and its importance as a biological warfare agent. Tripartite anthrax toxin proteins and a poly-D-glutamic acid capsule are produced by B. anthracis vegetative cells during mammalian hosts infection and when cultured in conditions that are thought to mimic the host environment. To identify the factors regulating virulence in B. anthracis the whole cell proteins were extracted from two B. anthracis strains and separated by narrow range immobilized pH gradient (IPG) strips (pH 4-7), followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins that were differentially expressed were identified by the peptide fingerprinting using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). A total of 23 proteins were identified as being either upregulated or downregulated in the presence or absence of the virulence plasmid pXO1. Two plasmid encoded proteins and 12 cellular proteins were identified and documented as potential virulence factors.

Observed peptide pI and retention time shifts as a result of post-translational modifications in multidimensional separations using narrow-range IPG-IEF

Modified peptides constitute a sub-population among the tryptic peptides analyzed in LC-MS based shotgun proteomics experiments. For larger proteomes including the human proteome, the tryptic peptide pool is very large, which necessitates some form of sample fractionation. By carefully choosing the sample fractionation and separation methods applied as shown here for the combination of narrow-range immobilized pH gradient isoelectric focusing (IPG-IEF) and nanoUPLC-MS, significantly increased information content can be achieved. Relatively low standard deviations were obtained for such multidimensional separations in terms of peptide pI (<0.05 pI units) and retention time (<0.3 min for a 350 min gradient) for a selection of highly complex proteomics samples. Using narrow-range IPG-IEF, experimental and predicted pI were in relative good agreement. However, based on our data, retention time prediction algorithms need further improvements in accuracy to match state-of-the-art reversed-phase chromatography performance. General trends of peptide pI shifts induced by common modifications including deamidations and N-terminal modifications are described. Deamidations of glutamine and asparagines shift peptide pI by approximately 1.5 pI units, making the peptides more acidic. Additionally, a novel pI shift (+~0.4 pI units) was found associated with dethiomethyl Met modifications. Further, the effects of these modifications as well as methionine oxidation were investigated in terms of experimentally observed retention time shifts in the chromatographic separation step. Clearly, post-translational modification-induced influences on peptide pI and retention time can be accurately and reproducibly measured using narrow-range IPG-IEF and high-performance nanoLC-MS. Even at modest mass accuracy (±50 ppm), the inclusion of peptide pI (±0.2 pI units) and/or retention time (±20 min) criteria are highly informative for human proteome analyses. The applications of using this information to identify post-translationally modified peptides and improve data analysis workflows are discussed.

Multipotent Adult Germline Stem Cells and Embryonic Stem Cells: Comparative Proteomic Approach

Spermatogonial stem cells isolated from the adult mouse testis acquire under certain culture conditions pluripotency and become so-called multipotent adult germline stem cells (maGSCs). They can be differentiated into somatic cells of the three germ layers. We investigated a subset of the maGSCs and ESCs proteomes using cell lines derived from two different mouse strains, narrow range immobilized pH gradients to favor the detection of less abundant proteins, and DIGE to ensure confident comparison between the two cell types. 2-D reference maps of maGSCs and ESCs in the pI ranges 3-6 and 5-8 were created, and protein entities were further processed for protein identification. By peptide mass fingerprinting and tandem mass spectrometry combined with searches of protein sequence databases, a set of 409 proteins was identified, corresponding to a library of 166 nonredundant stem cell-associated proteins. The identified proteins were classified according to their main known/postulated functions using bioinformatics. Furthermore, we used DIGE to highlight the ESC-like nature of maGSCs on the proteome scale. We concluded that the proteome of maGSCs is highly similar to that of ESCs as we could identify only a small subset of 18 proteins to be differentially expressed between the two cell types. Moreover, comparative analysis of the cell line proteomes from two different mouse strains showed that the interindividual differences in maGSCs proteomes are minimal. With our study, we created for the first time a proteomic map for maGSCs and compared it to the ESCs proteome from the same mouse. We confirmed on the proteome level the ESC-like nature of maGSCs.

In this issue: Proteomics 15/2008

AbstractIn this issue of Proteomics you will find the following highlighted articles:An old dog refines new tricksOld dogs are reputed to be slow learners but they can be subtle manipulators, able to induce younger dogs and gullible owners to share the food dish in their favor or choose the path they prefer. Two‐dimensional gel electrophoresis has been around for more than 25 years but “new and improved” versions continue to appear. Ericsson et al. scramble the order of several steps to get more information out of the combination of IPG/IEF and “shotgun” peptide analysis. Developed for studying mechanisms of drug resistance in small cell lung cancer, the modified protocol fractionates sonically disrupted cells into microsomes and soluble fractions before tryptic digestion and iTRAQ labeling for later quantitation. Digested samples were fractionated on narrow range immobilized pH gradient strips from which they were eluted for MALDI TOF or LC‐MS/MS analysis. Detection and identification of transmembrane proteins were dramatically improved.Ericsson, H. et al., Proteomics 2008, 8, 3008–3018.An evanescent view of a lectin micro‐array: through a glass faintlyLectins have the ability to distinguish closely‐related carbohydrate moieties attached (or not) to other molecules such as proteins, peptides, lipids, cells, etc. In some respects, they are much like antibodies, just not quite as specific. Using an array of 45 different lectins, the glycan portion of glycoproteins can be identified by its binding profile. Here, Uchiyama et al. report the improvements they have made to the reproducibility and sensitivity of the system. The binding of rhodamine‐labeled probes was detected in an evanescent field fluorescence‐based instrument that was capable of reaching, 10pM levels without having to wash off unbound probe. Depositing lectin spots with a non‐contact type printer, at the right humidity, and blocking with a non‐proteinaceous material greatly improved sensitivity.Uchiyama, N. et al., Proteomics 2008, 8, 3042–3050.The innate defense: multiplex proteomic probingThe body's first line of defense against pathogen infection is the innate response. In the case of bacterial infection, it is initiated by the sensing of the universal Gram‐positive cell wall lipopolysaccharide (LPS) component by macrophages primarily (but not solely) through the Toll‐like receptor 4 (TLR4). To understand the regulation of the LPS response, Gu et al. developed a multiplex quantitative proteomic analysis procedure to follow the response of TLR4+ and TLR4− cell lines. The method of choice was amino acid‐coded mass tagging (AACT, also referred to as SILAC). It showed high efficiency of labeling (95%) which eliminated interference with quantitation by the unlabeled fraction. Using triplex labeling of lysine (13C, 15N), the authors confirmed that TLR4− cells did show a response to LPS: 25 proteins were up‐regulated in TLR4+ cells, 5 in TLR4− cells. More than 500 proteins could be quantitated.Gu, S. et al., Proteomics 2008, 8, 3061–3070.

Cover Picture: Proteomics 15/2008

AbstractIn this issue of Proteomics you will find the following highlighted articles:An old dog refines new tricksOld dogs are reputed to be slow learners but they can be subtle manipulators, able to induce younger dogs and gullible owners to share the food dish in their favor or choose the path they prefer. Two‐dimensional gel electrophoresis has been around for more than 25 years but “new and improved” versions continue to appear. Ericsson et al. scramble the order of several steps to get more information out of the combination of IPG/IEF and “shotgun” peptide analysis. Developed for studying mechanisms of drug resistance in small cell lung cancer, the modified protocol fractionates sonically disrupted cells into microsomes and soluble fractions before tryptic digestion and iTRAQ labeling for later quantitation. Digested samples were fractionated on narrow range immobilized pH gradient strips from which they were eluted for MALDI TOF or LC‐MS/MS analysis. Detection and identification of transmembrane proteins were dramatically improved.Ericsson, H. et al., Proteomics 2008, 8, 3008–3018.An evanescent view of a lectin micro‐array: through a glass faintlyLectins have the ability to distinguish closely‐related carbohydrate moieties attached (or not) to other molecules such as proteins, peptides, lipids, cells, etc. In some respects, they are much like antibodies, just not quite as specific. Using an array of 45 different lectins, the glycan portion of glycoproteins can be identified by its binding profile. Here, Uchiyama et al. report the improvements they have made to the reproducibility and sensitivity of the system. The binding of rhodamine‐labeled probes was detected in an evanescent field fluorescence‐based instrument that was capable of reaching, 10pM levels without having to wash off unbound probe. Depositing lectin spots with a non‐contact type printer, at the right humidity, and blocking with a non‐proteinaceous material greatly improved sensitivity.Uchiyama, N. et al., Proteomics 2008, 8, 3042–3050.The innate defense: multiplex proteomic probingThe body's first line of defense against pathogen infection is the innate response. In the case of bacterial infection, it is initiated by the sensing of the universal Gram‐positive cell wall lipopolysaccharide (LPS) component by macrophages primarily (but not solely) through the Toll‐like receptor 4 (TLR4). To understand the regulation of the LPS response, Gu et al. developed a multiplex quantitative proteomic analysis procedure to follow the response of TLR4+ and TLR4− cell lines. The method of choice was amino acid‐coded mass tagging (AACT, also referred to as SILAC). It showed high efficiency of labeling (95%) which eliminated interference with quantitation by the unlabeled fraction. Using triplex labeling of lysine (13C, 15N), the authors confirmed that TLR4− cells did show a response to LPS: 25 proteins were up‐regulated in TLR4+ cells, 5 in TLR4− cells. More than 500 proteins could be quantitated.Gu, S. et al., Proteomics 2008, 8, 3061–3070.

Towards the Application of Proteomics to Human Thyroid Tissue

In this paper we describe for the first time a systematic approach to proteome analysis of human thyroid tissue. We report different methods to decrease the complexity of the human thyroid tissue proteome by applying different solubilization strategies and correcting for thyroglobulin protein abundance; to increase the protein resolution by prefractionation and by the use of narrow-range pH gradients; to detect proteins using sensitive and quantitative stains; and to identify soluble and membrane-bound thyroid tissue proteins by mass spectrometry analysis. MAIN OUTCOME/RESULTS: We found that buffers containing high contents of urea and detergents allow the best solubilization of human thyroid tissue proteins; highly variable abundance of thyroglobulin is a major pitfall of human thyroid proteome analysis, which in contrast to centrifugal ultrafiltration, size-exclusion chromatography and microdissection, can be countered best by adapting the protein amount to the thyroglobulin content per sample; prefractionation leads to a significant enrichment of proteins and allows subcellar localization of thyroid proteins; application of narrow-range immobilized pH gradient (IPG) strips allows further improvement of spot detection and separation; and protein detection with the fluorescent stain ruthenium II Tris bathophenanthroline disulfonate (RuBPs) is a highly sensitive and reliable tool for quantitative proteome analysis. Finally, in a pilot study of four patients with benign nodular thyroid disease we found that the described procedures allow a highly reproducible detection and identification of alterations in protein expression between nodular and corresponding normal thyroid tissues. Application of the described methods provides the basis for a highly sensitive and reproducible proteome analysis of the human thyroid, providing an additional novel tool to elucidate complex proteins changes in human thyroid biology as well as pathophysiology of human thyroid disease.

Protein quality and identification of the storage protein subunits of tofu and null soybean genotypes, using amino acid analysis, one- and two-dimensional gel electrophoresis, and tandem mass spectrometry

The protein quality of 11 null and 2 tofu soybean genotypes were determined from their total protein content, their amino acid composition, and their glycinin and β-conglycinin contents. There were highly significant differences (P < 0.001) in their total storage proteins, and amino acid contents. Total protein among these genotypes ranged from 33 to 37%, with arginine being the third highest amino acid (7.4–10.9 g/100 g protein) followed by glutamic and aspartic acids. Methionine accounted for only 1.6–2.4 g/ 100 g of protein. All genotypes contained a good balance of essential amino acids (EAA9), ranging from 43.5 to 47.3% of the total protein, limited only in methionine and possibly threonine and valine. Two-dimensional gel electrophoretic (2-DE) reference maps, using narrow range immobilized pH gradient (IPG) strips, revealed unique differences in the proteome, and subunit expression of glycinin and β-conglycinin, among these null genotypes, which can then be correlated with their protein quality. Out of a total of 111 basic (pH 6–11), and 223 acidic (pH 4–7) protein spots separated by 2-DE, 41 soybean storage protein spots were excised, and identified by liquid chromatography on-line with electro spray LCQ DecaXP tandem quadrupole time-of-flight mass spectrometry (LC/MS/MS). These methods will enable accurate evaluation of protein quality in soybeans, based on their protein digestibility-corrected amino acid score, assessment of the genetic variability of soybean genotypes, and serve as very effective tools for assisting plant breeders in their selection of high quality soybean varieties.

Identification of Apolipoprotein A-I as a “STOP” Signal for Myopia

Good visual acuity requires that the axial length of the ocular globe is matched to the refractive power of the cornea and lens to focus the images of distant objects onto the retina. During the growth of the juvenile eye, this is achieved through the emmetropization process that adjusts the ocular axial length to compensate for the refractive changes that occur in the anterior segment. A failure of the emmetropization process can result in either excessive or insufficient axial growth, leading to myopia or hyperopia, respectively. Emmetropization is mainly regulated by the retina, which generates two opposite signals: "GO/GROW" signals to increase axial growth and "STOP" signals to block it. The presence of GO/GROW and STOP signals was investigated by a proteomics analysis of the retinas from chicken with experimental myopia and hyperopia. Of 18 differentially expressed proteins that were identified, five displayed an expression profile corresponding to GO/GROW signals, and two corresponded to STOP signals. Western blotting confirmed that apolipoprotein A-I (apoA-I) has the characteristics of a STOP signal both in the retina as well as in the fibrous sclera. In accordance with this, intraocular application of the peroxisome proliferator-activated receptor alpha agonist GW7647 resulted in up-regulation of apoA-I levels and in a significant reduction of experimental myopia. In conclusion, using a comprehensive functional proteomics analysis of chicken ocular growth models we identified targets for ocular growth control. The correlation of elevated apoA-I levels with reduced ocular axial growth points toward a functional relationship with the observed morphological changes of the eye.

Current Progress in Sample Preparation for Two-Dimensional Electrophoresis in Proteomics

Two dimensional polyacrylamide gel electrophoresis (2DE) has been a core technology of current proteomics for its high resolution and ability to detect proteins with post-translational modifications. This technology has been widely applied in numerous biomedical research projects including biomarker discovery and drug development. The application of narrow-range immobilized pH gradient strips (IPGs) and advanced detection methodologies have increased the resolution of 2DE technology. However, 2DE still suffers from its limitation in unambiguous detection of proteins of lowabundance. To improve this, sample preparation is the first and key step for successful 2DE analysis. This article summarizes current progresses in sample handling and prefractionation aiming to enrich proteins of interest according to their special physical and chemical properties so that the application of 2DE can be greatly extended. Keywords: Proteomics, sample preparation, protein prefractionation, protein separation, 2DE, two dimensional gel electrophoresis, protein profiling

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.

New Insights into the Rat Spermatogonial Proteome: Identification of 156 Additional Proteins

Despite the essential role played by spermatogonia in testicular function, little is known about these cells. To improve our understanding of their biology, our group recently identified a set of 53 spermatogonial proteins using two-dimensional (2-D) gel electrophoresis and mass spectrometry. To continue this work, we investigated a subset of the spermatogonial proteome using narrow range immobilized pH gradients to favor the detection of less abundant proteins. A 2-D reference map of spermatogonia in the pH range 4-9 was created, and protein entities fractionated in a pH 5-6 2-D gel were further processed for protein identification. A new set of 156 polypeptides was identified by peptide mass fingerprinting and tandem mass spectrometry. These polypeptides corresponded to 102 different proteins, which reflect the complexity of post-translational modifications. Seventy-nine of these proteins were identified for the first time in spermatogonia. All identified proteins were classified into functional groups. This work represents a first step toward the establishment of a systematic spermatogonia protein database.

Proteomic analysis of the rat liver

Rat is a useful, widely used animal model for biological and toxicity studies. We analyzed total and cytosolic rat liver proteins by applying proteomics technologies. The proteins were separated by two-dimensional electrophoresis employing broad and narrow range immobilized pH gradient strips, followed by MALDI-MS analysis of the tryptic digests. Two hundred and seventy-three different gene products were identified, of which approximately 60% were enzymes with a broad spectrum of catalytic activities. Most of the identified proteins were detected in other rat protein samples as well, which were analyzed in our laboratory. Fifteen gene products were detected for the first time. These were represented by one spot each, whereas most of the frequently detected proteins were represented by multiple spots. In average, approximately five to 10 spots corresponded to one gene product. The database includes a large number of proteins known to be involved in toxicology-relevant pathways and may be useful in toxicity prediction studies.

Global analysis of the Brucella melitensis proteome: Identification of proteins expressed in laboratory-grown culture.

Brucella melitensis is a facultative intracellular bacterial pathogen that causes brucellosis, a zoonotic disease primarily infecting sheep and goats, characterized by undulant fever, arthritic pain and other neurological disorders in humans. A comprehensive proteomic study of strain 16M was conducted to identify and characterize the proteins expressed in laboratory-grown culture. Using overlapping narrow range immobilized pH gradient strips for two-dimensional gel electrophoresis, 883 protein spots were detected between pH 3.5 and 11. The average isoelectric point and molecular weight values of the detected spots were 5.22 and 46.5 kDa, respectively. Of the 883 observed protein spots, 440 have been identified by matrix-assisted laser desorption/ionization-mass spectrometry. These proteins represent 187 discrete open reading frames (ORFs) or 6% of the predicted 3197 ORFs contained in the genome. The corresponding ORFs of the identified proteins are distributed evenly between each of the two circular B. melitensis chromosomes, indicating that both replicons are functionally active. The presented proteome map lists those protein spots identified to date in this study. This map may serve as a baseline reference for future proteomic studies aimed at the definition of biochemical pathways associated with stress responses, host specificity, pathogenicity and virulence. It will also assist in characterization of global proteomic effects in gene-knockout mutants. Ultimately, it may aid in our overall understanding of the cell biology of B. melitensis, an important bacterial pathogen.

Preparative two-dimensional gel electrophoresis at alkaline pH using narrow range immobilized pH gradients.

A reproducible high-resolution protein separation method is the basis for a successful differential proteome analysis. Of the techniques currently available, two-dimensional gel electrophoresis is most widely used, because of its robustness under various experimental conditions. With the introduction of narrow range immobilized pH gradient (IPG) strips (also referred to as ultra-zoom gels) in the first dimension, the depth of analysis, i.e. the number of proteins that can be resolved, has increased substantially. However, for poorly understood reasons isoelectric focusing on ultra-zoom gels in the alkaline region above pH 7 has suffered from problems with resolution and reproducibility. To tackle these difficulties we have optimized the separation of semipreparative amounts of proteins on alkaline IPG strips by focusing on two important phenomena: counteracting water transport during isoelectric focusing and migration of dithiothreitol (DTT) in alkaline pH gradients. The first problem was alleviated by the addition of glycerol and isopropanol to the focusing medium, leading to a significant improvement in the resolution above pH 7. Even better results were obtained by the introduction of excess of the reducing agent DTT at the cathode. With these adaptations together with an optimized composition of the IPG strip, separation efficiency in the pH 6.2-8.2 range is now comparable to the widely used acidic ultra-zoom gels. We further demonstrated the usefulness of these modifications up to pH 9.5, although further improvements are still needed in that range. Thus, by extending the range covered by conventional ultra-zoom gels, the depth of analysis of two-dimensional gel electrophoresis can be significantly increased, underlining the importance of this method in differential proteomics.

The rat liver mitochondrial proteins.

Subcellular fractionation increases the probability of detection of low-abundance proteins. We prepared a fraction highly enriched in mitochondrial proteins from rat liver. The proteins were analyzed by two-dimensional (2-D) electrophoresis using broad-and narrow-range immobilized pH gradient strips, and identified by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS). 192 different gene products were detected, of which approximately 70% were enzymes with a broad spectrum of catalytic activities. Most of the identified proteins were detected in other rat protein samples as well, which were analyzed in our laboratory. Eight gene products were detected for the first time. These were represented by one spot each, whereas most of the frequently detected proteins were represented by multiple spots. In average, approximately 10-15 spots corresponded to one gene product.

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.

Mass spectrometry and proteomics.

the 15th asilomar conference on Mass Spectrometry this October was devoted to the role of mass spectrometry (MS) in proteomics. The Asilomar Conference site is in a picturesque national park in Pacific Grove, CA, overlooking the Pacific Ocean. The conference aims to bring together scientists from a

'98 Escherichia coli SWISS-2DPAGE database update.

The combination of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), computer image analysis and several protein identification techniques allowed the Escherichia coli SWISS-2DPAGE database to be established. This is part of the ExPASy molecular biology server accessible through the WWW at the URL address http://www.expasy.ch/ch2d/ch2d-top.html . Here we report recent progress in the development of the E. coli SWISS-2DPAGE database. Proteins were separated with immobilized pH gradients in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. To increase the resolution of the separation and thus the number of identified proteins, a variety of wide and narrow range immobilized pH gradients were used in the first dimension. Micropreparative gels were electroblotted onto polyvinylidene difluoride membranes and spots were visualized by amido black staining. Protein identification techniques such as amino acid composition analysis, gel comparison and microsequencing were used, as well as a recently described Edman "sequence tag" approach. Some of the above identification techniques were coupled with database searching tools. Currently 231 polypeptides are identified on the E. coli SWISS-2DPAGE map: 64 have been identified by N-terminal microsequencing, 39 by amino acid composition, and 82 by sequence tag. Of 153 proteins putatively identified by gel comparison, 65 have been confirmed. Many proteins have been identified using more than one technique. Faster progress in the E. coli proteome project will now be possible with advances in biochemical methodology and with the completion of the entire E. coli genome.

Characterisation of wool intermediate filament proteins separated by micropreparative two-dimensional electrophoresis.

Wool intermediate filament proteins (IFP) are a subclass of the cytokeratins, a group of structural proteins which form intermediate filaments in many cell types. Post-translational modifications, such as phosphorylation, play an important role in the control of intermediate filament assembly. Two-dimensional electrophoresis has previously been used to study the IFP distribution in wools with different physical characteristics. Charge heterogeneity has been observed in Type I and Type II IFP. In a previous study, two-dimensional electrophoresis of alkaline phosphatase-treated wool protein extracts was used to show that Type II IFP are phosphorylated. To facilitate post-separation analysis, micropreparative two-dimensional electrophoresis was used to separate milligram quantities of wool protein. Direct phosphoamino acid analysis has confirmed the presence of phosphorylation on serine residues on Type II IFP, whose identity was confirmed by amino acid compositional analysis. The isoelectric points of Type I IFP are very similar and they do not separate completely on the commercially available pH 4-7 immobilized pH gradients (IPG) used in this study. In situ tryptic digestion followed by automated Edman sequencing of the high performance liquid chromatography (HPLC)-separated peptides was used to confirm the identity of this group as Type I IFP. To improve the separation of the Type I IFP it will be necessary to use narrow range IPGs such as Immobiline DryPlates which are available from Pharmacia Biotech, in the pH ranges 4.2-4.9, 4.5-5.4, 5.0-6.0 and 5.6-6.6.