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Originally published In Press as doi:10.1074/mcp.M500181-MCP200 on October 13, 2005.
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Molecular & Cellular Proteomics 5:265-273, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

A Novel Two-dimensional Electrophoresis Technique for the Identification of Intrinsically Unstructured Proteins*,S

Veronika Csizmók, Edit Szollosi, Peter Friedrich and Peter Tompa{ddagger}

From the Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, H-1518 Budapest, Hungary


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Intrinsically unstructured proteins (IUPs) lack a well defined three-dimensional structure under physiological conditions. They constitute a significant fraction of various proteomes, but only a handful of them have so far been identified. Here we report the development of a two-dimensional electrophoresis technique for their de novo recognition and characterization. This technique consists of the combination of native and 8 M urea electrophoresis of heat-treated proteins where IUPs are expected to run into the diagonal, whereas globular proteins either precipitate upon heat treatment or unfold and run off the diagonal in the second dimension. This behavior was born out by a collection of 10 known IUPs and four globular proteins. By running Escherichia coli and Saccharomyces cerevisiae extracts, several novel IUPs were also identified by mass spectrometric analysis of spots at or near the diagonal. By comparing this novel method to several other techniques, such as the PONDR® predictor, hydrophobicity-net charge plot, CD analysis, and gel filtration chromatography, it was shown to provide dependable global assessment of disorder even in dubious cases. Overall the reproducibility and ease of performance of this technique may promote the proteomic scale recognition and characterization of protein disorder.

The long standing dogma that tied protein function to a well defined three-dimensional structure has been increasingly challenged lately by the recognition that for many proteins/protein domains the native, functional state is intrinsically unstructured/disordered (1–4). Such proteins constitute a significant fraction of various proteomes: from studies based on their sequence attributes (2, 5) and heat stability (6) it has been ascertained that as much as 25% of all residues may fall into disordered regions in the proteomes of different species. Still only about 100 of them have so far been identified (2–4, 7). Systematic studies aimed at identifying novel IUPs are all the more compelling as IUPs1 play essential physiological and pathological roles (1, 2, 4, 8).

IUPs have so far been identified by the chance observation of the structural anomaly of proteins studied for their functional interest. We reasoned that a straightforward technique to separate IUPs from globular proteins in a cellular extract could be established by the combination of a native gel electrophoresis of heat-treated proteins followed by a second, denaturing gel containing 8 M urea. The rationale for the first dimension is that IUPs are very often heat-stable as demonstrated for Csd1 (9), MAP2 (10), NACP (11), stathmin (12), and p21Cip1 (13) for example. Heat treatment thus results in a good initial separation from globular proteins, most of which aggregate and precipitate. In the native gel, IUPs and rare heat-stable globular proteins will then be separated according to their charge/mass ratios. Combining this first dimension with an 8 M urea second step is rationalized by the usual structural indifference of IUPs to chemical denaturation by trichloroacetic acid, guanidine HCl, or urea as reported for Csd1 (9), NACP (11), ß-casein (14), stathmin (12), and p21Cip1 (13) for example. As urea is uncharged and IUPs are just as "denatured" in 8 M urea as under native conditions, they are expected to run the same distance in the second dimension and end up along the diagonal. Heat-stable globular proteins, on the other hand, will unfold in urea, slow down in the second gel, and accumulate above the diagonal. Because of this effective separation, IUPs are amenable to subsequent MS identification.

In this work we elaborated this novel separation principle. By 10 known IUPs and four globular proteins, it was shown that IUPs did occupy diagonal positions in the second gel and separate from globular proteins, and thus the gel can probe the IUP nature of a protein available in very low quantity and limited purity. By running Escherichia coli and Saccharomyces cerevisiae extracts, we demonstrated that a combination of this novel 2D technique and MS analysis can lead to the identification of novel IUPs.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Control Proteins—
IUPs and globular control proteins were selected from the literature (ERD102 and others (2–4)) and obtained from various sources. We purchased stathmin (Calbiochem, catalog number 569390); {alpha}- and ß-caseins (both from ICN Biomedicals, catalog numbers 100251 and 100321); DARPP-32 (Calbiochem, catalog number 251755); and BSA (catalog number A 7638), ovalbumin (catalog number A 2512), fetuin (catalog number F 3004), and alcohol dehydrogenase (catalog number A 7011, all four from Sigma). The GF calibration kit (catalog number 17-0442-01) was from Amersham Biosciences. Thermus thermophilus IPMDH was a gift from Prof. Peter Závodszky (Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences). Csd1 and MAP2c were prepared as described previously (15), and ERD10 was cloned by PCR from an Arabidopsis thaliana cDNA library obtained from Dr. László Szabados (Institute of Plant Biology, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary). The cDNA of NACP cloned into a pT7-7 vector was obtained from Dr. Michael J. Volles (Center for Neurological Disease, Harvard Medical School, Cambridge, MA). Mypt1-(304–511) in a pET21 vector was a gift from Prof. Ferenc Erdodi (Department of Medical Chemistry, University Medical School of Debrecen, Debrecen, Hungary) and was expressed as described previously (16). Bob-1 in a pET 32 vector was obtained from Prof. Ben Luisi (Department of Biochemistry, University of Cambridge, Cambridge, UK) and purified as described previously (17). All proteins were dialyzed into a buffer of 50 mM Tris, 150 mM NaCl, 2 mM dithioerythritol, pH 7.5 and kept at –80 °C in aliquots until use.

Novel Proteins—
The cDNA of novel proteins was amplified by PCR by primers encoding a 5' NdeI and a 3' XhoI restriction sites either from genomial DNA (for E. coli ORF1, ORF2, YhgI, DnaKs, and GccH) or from a cDNA library generated by RT-PCR (for S. cerevisiae tropoM, Ubi6, sRib, and calmodulin). The products were ligated into pET-22b vector (Novagen) and transformed into E. coli BL21(DE3) strain. Expression of these proteins and S. cerevisiae transcription factor IIA (TFIIA) (the expression vector of which was a gift from Dr S. Hahn, The Fred Hutchinson Cancer Research Center, Seattle, WA) was induced by 0.5 mM isopropyl 1-thio-ß-D-galactopyranoside at 30 °C for 3 h. The proteins were purified to homogeneity by heat-treating (10 min x 100 °C) cell extracts following DEAE-cellulose ion exchange chromatography of the supernatants.

Extracts of E. coli and S. cerevisiae
E. coli cells were grown overnight in NZYM medium (NZ-amine, NaCl, yeast, MgSO2) and S. cerevisiae cells for 2 days in YP medium (yeast extract, pecton) (both 500 ml). The cells were centrifuged at 4,000 rpm at 4 °C for 20 min, washed in 10 ml of 50 mM Tris/HCl, pH 7.5, and centrifuged again. E. coli cells were then suspended in 2 ml of 50 mM Tris/HCl, 150 mM NaCl, 2 mM dithioerythritol, pH 7.5 and disrupted by sonication (10 x 10 s, 24 mµ at 0 °C on an MSE sonicator with 1-min breaks between runs). S. cerevisiae cells were disrupted by three freezing/thawing cycles in liquid nitrogen, homogenizing in a Potter-Elvehjem tissue grinder, and subsequent sonication as with E. coli. In both cases, disrupted cells were centrifuged for 100,000 x g for 30 min at 4 °C, and the supernatant was heated to 100 °C for 10 min and centrifuged again for 100,000 x g for 30 min at 4 °C. The final supernatant usually contained 2 mg/ml protein and was kept at –80 °C in small aliquots for 2D runs.

2D Electrophoresis—
The 2D electrophoresis combined a native gel and an 8 M urea gel, both prepared according to Laemmli (18) but without SDS. To the heat-treated samples (1 µg each of control proteins or 100 µg of E. coli or S. cerevisiae extract) Formula volume of native sample buffer was added, and the sample was run on a native gel of 0.7-mm thickness either in small format (8 x 6 cm, Mini-Protean II) or large format (20 x 16 cm, Protean II XL, both from Bio-Rad). The gel was briefly stained with BioSafeTM Coomassie G250 (Bio-Rad), and the lane containing protein bands was cut out. This 10–15-mm-wide strip was then soaked into the upper gel buffer plus 8 M urea for 45 min and placed on top of a second gel prepared with 8 M urea. This second dimension had no upper (stacking) gel, and it was cast between 1-mm spacers to accommodate the strip swollen in urea. The second gel was run 2.5 times longer than the first at the same voltage and stained in colloidal Coomassie (19) overnight.

Mass Spectrometry—
Potential IUPs to be identified were cut out of the 2D gel and analyzed at the Proteomics Research Group of the Biological Research Center of the Hungarian Academy of Sciences (Szeged, Hungary). Briefly spots were in-gel digested by trypsin and analyzed by MALDI-TOF MS first. The resulting monoisotopic peptide masses (peptide mass fingerprint) were then subjected to a database search to identify the protein(s) present. Protein identities were confirmed by postsource decay spectra, providing sequence information on selected peptides.

Gel Filtration Chromatography—
The folded/unfolded nature of proteins was characterized by GF chromatography. 100 µl of each of the proteins was run on an Amersham Biosciences Superdex 200 (1 x 30-cm) column at 0.5 ml/min in 50 mM Na2HPO4, 150 mM NaCl (or occasionally 1 M NaCl to exclude aggregation), pH 7.0 buffer on an Amersham Biosciences FPLC system. Proteins were recorded at 280 nm. The column was calibrated by the following globular proteins (molecular mass): ribonuclease A (13.7 kDa), chymotrypsinogen A (25.0 kDa), ovalbumin (43.0 kDa), BSA (67.0 kDa), and alcohol dehydrogenase (146.8 kDa). The Mw of novel proteins was determined from a calibration curve constructed by plotting log Mw values versus elution volume.

Characterization of Protein Disorder—
Sequence-specific assessment of protein disorder was obtained by the PONDR® (Predictor of Natural Disordered Regions) algorithm (2, 5) available at www.pondr.com. Access to PONDR was provided by Molecular Kinetics (6201 La Pas Trail, Suite 160, Indianapolis, IN 46268; E-mail: main{at}molecularkinetics.com) under license from the Washington State University Research Foundation. PONDR is copyright ©1999 by the Washington State University Research Foundation, all rights reserved. The percentage of disorder is calculated as the ratio of residues with a PONDR score above 0.5.

The global tendency of proteins for disorder was also assessed by calculating their mean net charge (R) and mean hydrophobicity (H) (see Ref. 7). R was calculated as (Glu + Asp) – (Lys + Arg) divided by the length of the protein. H was calculated with the ProtScale server (us.expasy.org/tools/protscale.html) with settings as defined in Ref. 7. IUPs and globular proteins in the hydrophobicity-charge space thus defined (the CH plot) are usually separated by a line defined by the equation r = 2.785H – 1.151 (7). The tendency of a given protein for disorder was characterized by its distance from this line (set as positive for IUPs and negative for ordered proteins).

Structural disorder can also be characterized by the distribution of secondary structural elements determined by CD spectroscopy. To this end, CD spectra were decomposed into {alpha}-helix, ß-sheet, turn, and coil by the CDpro software (lamar.colostate.edu/~sreeram/CDPro/main.html), and coil was considered to correspond to disordered regions of the proteins.

The hydrodynamic dimension, a very sensitive measure of globularity, was characterized by the ratio of the apparent Mw determined by GF chromatography and the absolute Mw calculated from the amino acid sequence and also by the ratio of the apparent and real Stokes radius derived from Mw,app and Mw by the equation: log(RS) = 0.369 log(Mw) – 0.254 (20). Because the column was calibrated by globular proteins, these ratios are close to 1 for folded proteins. The value of Mw,app/Mw = 2–3 and the value of RS,app/RS = 1.4–1.5 are typical of the proteins in a molten globule (MG) state and the value of Mw,app/Mw = 4–5 and the value of RS,app/Rs = 1.7–1.9 are typical of the extended, premolten globule (PMG) or random coil-like (U) proteins (3).

Other Methods—
CD spectra were recorded in a Jasco J-720 spectropolarimeter in a buffer of 10 mM NaH2PO4, 150 mM NaCl, pH 7.0. In the case of gels run for MS analysis, all solutions were prepared from Elix/Synergy purified water (Millipore) and filtered to sterility through a 0.2-µm filter (Whatman). All the equipment was washed and rinsed in Millipore water. The gels were handled with great care to avoid contamination. Spots cut out were transferred to 100 µl of sterile water and sealed for transport to the MS laboratory. Protein concentration was determined according to Bradford (21).


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Separation of IUPs from Globular Proteins—
To provide evidence that the combination of a native gel and an 8 M urea gel results in the appropriate separation of IUPs from globular proteins, we ran a panel of purified IUPs and globular proteins (Fig. 1A). This time the sample was not boiled to keep all globular proteins in solution and demonstrate the full resolving power of the 2D technique. As expected, IUPs ran to, or very near, the diagonal of the second gel, whereas globular proteins remained way above the diagonal, mostly along a second line of much smaller slope. It should be noted, however, that one IUP ({alpha}-casein) ran a little above the diagonal, and one globular protein (fetuin) ran closer to IUPs. These deviations probably result from a significant residual structure in caseins (22) and the resistance to denaturation of fetuin (6). Nevertheless this run overall demonstrated that the 2D electrophoresis separates IUPs and globular proteins as predicted.


Figure 1
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  		FIG. 1. The native/8 M urea 2D electrophoresis of IUPs and globular proteins. A, a mixture of proteins (1 µg of each) was run on a 7.5% native gel in the first dimension (without heat treatment) and on a 7.5% gel containing 8 M urea in the second dimension in the large format (20 x 16 cm). The second gel was visualized by colloidal Coomassie staining. Individual proteins marked are as follows: IUPs: 1, stathmin; 2, MAP2c; 3, Mypt1-(304–511); 4, ERD10; 5, ß-casein; 6, NACP; 7, Csd1; 8, Bob-1; 9, DARPP32; and 10, {alpha}-casein; globular proteins: 11, fetuin; 12, IPMDH; 13, BSA; and 14, ovalbumin. A continuous line marks the diagonal of the gel to where IUPs run. A dashed line marks the position of globular proteins. For further details, see "Experimental Procedures." B, 1 µg each of four globular proteins (11, fetuin; 12, IPMDH; 13, BSA; and 14, ovalbumin) and two IUPs (4, ERD10; and 7, Csd1) mixed in a total volume of 30 µl were boiled for 10 min. The precipitated protein was pelleted by centrifugation for 10 min at 10,000 x g, and the supernatant was run on a small format (8 x 6-cm) 7.5% native/8 M urea 2D gel. The two IUPs define the diagonal. Three of the globular proteins (BSA, ovalbumin, and IPMDH despite its thermophilic origin) precipitate upon heat treatment and are absent from the second gel: their expected positions are marked. Serum fetuin resists heat treatment but runs above the diagonal in the second dimension.

 
Probing the IUP Nature of Structurally Uncharacterized Proteins—
The 2D format provides a simple means of probing the IUP nature of a structurally uncharacterized protein even if it is available in microgram quantities and/or is of limited purity. This point was demonstrated by mixing only two IUPs (ERD10 and Csd1) and the four globular control proteins together and running them on a small format 2D gel after heat treatment (Fig. 1B). The two IUPs mark the diagonal, and they are clearly distinguished from all the globular proteins either because the latter precipitate upon heat treatment (IPMDH, BSA, and ovalbumin) or run off the diagonal (fetuin). Essentially the same behavior was observed if either of the globular proteins alone was mixed with the two IUPs. Thus, this simple approach of running two known IUPs with an uncharacterized protein obtained in a cloning/purification experiment can establish the order/disorder status of a protein available in a minute quantity only.

Identification of E. coli and S. cerevisiae IUPs by Combining the 2D Technique with MS—
The major asset of the 2D technique is that it can recognize novel IUPs not yet described as such. This point is demonstrated by running heat-treated extracts of E. coli and S. cerevisiae in the large format gel (Figs. 2 and 3) and identifying spots at, and above, the diagonal by MS (Tables I and II). It is of note that the procedure is highly reproducible as parallel runs resulted in very similar gel patterns (not shown).


Figure 2
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  		FIG. 2. Separation and identification of IUPs from E. coli. A large format 7.5–15% gradient 2D gel was run with a heat-treated extract of E. coli strain BL21 (see "Experimental Procedures"). The gel was visualized by colloidal Coomassie, and dots marked were cut out and sent for MS identification. The results are summarized in Table I.

 

Figure 3
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  		FIG. 3. Separation and identification of IUPs from S. cerevisiae. A large format 7.5–15% gradient 2D gel was run with a heat-treated extract of S. cerevisiae (see "Experimental Procedures"). The gel was visualized by colloidal Coomassie, and dots marked were cut out and sent for MS identification. The results are summarized in Table II (please note that spots 8a and 8b correspond to the same protein, probably as a result of post-translational modification).

 

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  		TABLE I Novel IUPs identified in E. coli

The proteins identified on the diagonal (1–8) or above the diagonal (9–15) of the 2D gel of the E. coli extract (Fig. 2) are listed with their GenBankTM (gi) accession number. The sequences submitted to disorder prediction by PONDR (VL-XT, default setting); the percentage of structural disorder thus estimated is also shown. The net charge/molecular mass (the latter in kDa) ratio was also calculated. PTS, phosphoenolypyruvate phosphotransferase system; FKBP, FK506-binding protein.

 

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  		TABLE II Novel IUPs identified in S. cerevisiae

The proteins identified on the diagonal (1–10) or above the diagonal (11–13) of the 2D gel of S. cerevisiae extract (Fig. 3) are listed with their GenBankTM (gi) accession number. The percentage of structural disorder estimated by PONDR prediction (VL-XT, default setting) is also shown. The net charge/molecular mass (the latter in kDa) ratio was also calculated.

 
As estimated by their PONDR score (Tables I and II), the amount of disorder in proteins of diagonal positions is very high (52.1 ± 14.1%), exceeding that of certain typical IUPs (NACP,: 37.1%; ß-casein, 41.15%). Although many of these proteins have never been structurally characterized, data from the literature confirm the structural disorder of some. For example, ribosomal proteins in general are held to lack a well defined structure when separated from rRNA (2, 7). The structural disorder of yeast ribosomal acidic proteins P1{alpha} and P2ß has been reported (23), whereas E. coli ribosomal L7/L12 is a heat-stable, molten globule-like protein that acquires structure only upon dimerization (24). A similar behavior was reported for GroES (25). E. coli acyl carrier protein may also be a molten globule as it is stabilized by Mg2+ binding (Protein Data Bank code 1acp), but it is extremely flexible in solution (26), and its Vibrio harveyi homologue is fully unfolded under physiological conditions (27). Some of the proteins have separate disordered and ordered regions. For example, the structure of region 80–156 of the acetyl-CoA carboxylase BCCP subunit is known (Protein Data Bank code 1a6x), but the protein has a highly flexible N-terminal linker region (28). Proteins above the diagonal are heat-stable enzymes (e.g. superoxide dismutase), which typically require a well defined structure for function. Nevertheless the high flexibility of calmodulin is necessary for its regulatory function (29).

Benchmarking the 2D Technique against Other Methods—
Our current base of knowledge on protein disorder relies on a few experimental techniques and derived bioinformatic predictors. The different techniques (mostly x-ray crystallography, CD, and NMR (2, 3, 7)), however, probe distinct aspects of protein disorder (4), which results in a certain level of uncertainty in the output of disorder predictors (see Ref. 30). Thus, benchmarking the 2D technique against other methods is of both theoretical and practical importance.

To this end, we generated the PONDR pattern and the CH plot for all control and newly identified proteins (Supplemental Figs. S1–S6). Besides clear cut cases, there were several contradictory proteins that would be difficult to correctly classify by any single method (see also Fig. 4). For example, Bob-1 was largely disordered by PONDR (73.44%) but appeared ordered on the CH plot. Caseins and NACP (Fig. 4A) would be held mostly ordered by both approaches, although they are classical examples for disorder (11, 31), also underscored by the CD spectra here (Supplemental Fig. S1). On the other extreme, IPMDH is a thermostable globular enzyme (Protein Data Bank code 1ipd), yet its level of disorder predicted by PONDR exceeded these three IUPs, whereas its structural status was correctly identified by the CH plot (Fig. 4B). Similar inconsistencies were apparent with some of the novel proteins identified as disordered by the 2D technique. For example, DnaKs (acronyms of protein names are given in Tables I and II) had a lower level of disorder by PONDR (39.07%) than GccH (53.49%), but it was disordered by the CH plot, whereas GccH (Fig. 4C) appeared ordered. GroES and L7/L12 (Fig. 4D) had a rather high level of disorder by PONDR (43.3 and 57.02%) and are thought to be disordered in the monomer state (24, 25), but they were predicted as ordered by the CH plot. By both techniques, YhgI appeared balanced between order and disorder without a clear clue as to where it belongs.


Figure 4
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  		FIG. 4. PONDR score and hydrophobicity-net charge plot of selected disordered proteins. The PONDR score and CH plot is shown for NACP (A), IPMDH (B), GccH (C), and L7/L12 (D) for which the assessment of global order/disorder by these techniques is questionable. Further such cases, referred to under "Results," are shown in Supplemental Figs. S1–S6.

 
To clarify this ambiguity, we selected those cases where the domination of disorder is dubious. These proteins and some clear cut cases were cloned and expressed, and their disorder was assessed by various independent means, such as their position on the 2D gel (on/above diagonal), PONDR pattern, location on the CH plot, coil content by CD, and apparent Mw and RS by GF chromatography (Table III). These comparisons show that the 2D technique is most suitable for assessing global disorder, i.e. an unfolded state permitting various levels of residual structure. For proteins that are probably fully disordered (Csd1, ORF1, and TFIIA) or fully ordered (ORF2), all approaches agreed. These have an extended, PMG or random coil-like (U) structure. The methods mostly disagreed on proteins for which PONDR predicted a roughly equivalent amount of order and disorder, making it almost impossible to decide whether they are basically ordered with disordered loops or mostly disordered with a significant residual structure. The misclassified control proteins (caseins and NACP) were correctly placed by the 2D technique alongside CD and GF chromatography. With the novel proteins, such as YhgI, DnaKs, and GccH from E. coli and Ubi6 from S. cerevisiae, which have a relatively high coil content compatible with either a folded or unfolded state, the 2D technique agreed with GF chromatography in marking them as IUPs. With tropomyosin, a coiled-coil protein disordered in the monomer state (32), PONDR is inconclusive, and the CH plot puts it as very disordered, whereas by CD it is predominantly {alpha}-helical. GF chromatography agreed with the 2D technique in ascertaining its overall disorder. An interesting example is sRib, which showed little disorder by PONDR® but appeared to exist in an extended conformation by GF and occupied a diagonal position on the 2D gel.


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  		TABLE III Comparison of various measures of disorder for selected proteins

Various measures of disorder are compared for control proteins and newly identified E. coli and S. cerevisiae (yeast) proteins. For each protein, its position relative to the diagonal on the 2D gel (on/above), percentage of disorder predicted by PONDR®, distance on the CH plot from the line separating disordered (+) and ordered (–) proteins (multiplied by 100), percentage of residues in coil (disordered) conformation by CD measurements, the ratio of the apparent and real Mw, and the ratio of the apparent and calculated Stokes radius (apparent values derived from GF chromatography experiments) are given. Details of the measurements and calculations are given under "Results" and "Experimental Procedures." CaM, calmodulin.

 
In all, the 2D technique correctly identified the global folded/unfolded character of proteins, most closely agreed with the hydrodynamic technique GF, and identified both extended (U or PMG type) and more compact (MG type) disordered proteins correctly. Thus, it may be a useful complement of other techniques more related to local structural preferences of the protein.


   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
A systematic search for IUPs has not been possible until now because of the lack of an appropriate technique for their separation from globular proteins and each other. In this study we proposed a novel 2D electrophoresis format conceived with this goal in mind. As shown by known IUPs and globular proteins and by identifying novel IUPs from cell extracts, the combination of a native gel of heat-treated samples and a denaturing gel of 8 M urea resulted in the appropriate separation. The technique is reproducible, is easy to perform, and is readily adaptable to high throughput studies. Its comparison with other experimental and bioinformatic techniques showed that it provides dependable assessment of global structural disorder even in contradictory cases. Although its resolving power does not match that of the conventional 2D technique, it enables specific applications in the rapidly advancing field of IUPs in two different but related directions.

Its first practical application is the rapid characterization of a single protein in terms of its IUP status. As demonstrated, a simple run of the 2D format can tell with high certainty if the protein is structured or unstructured. Given that the technique can provide information on a protein of very small quantity and limited purity, it will be a useful complement to other techniques that are more demanding on protein quantity and quality (2, 4). Actually with a good antibody at hand, it may even be performed on cell/tissue extracts without prior purification.

The other application is in the analysis of cellular extracts. This point was demonstrated by the large format gel with E. coli and S. cerevisiae extracts and the identification of a range of novel, mostly disordered proteins. The list of proteins actually identified (Tables I and II) raises intriguing points. The majority of proteins have never been described as IUPs, which is consistent with the fact that we know only a small fraction of the "disorderome" (2, 4). This is also underscored by the lack of an overlap between identified E. coli and S. cerevisiae proteins despite the evolutionary conservation of protein disorder in some protein families (2, 33). Furthermore when we compared the 2D pattern of the extracts from E. coli and S. cerevisiae, more proteins were seen in the diagonal of the latter in agreement with predictions that the frequency of protein disorder increases with increasing complexity of the organisms (2, 5).

Whereas the 2D technique offers unique applications, it has some limitations as well. Because of the application of a native gel in the first dimension, its resolving power does not match that of the conventional 2D electrophoresis. This can be partially overcome by applying mild, non-charged detergents, and this will certainly be one future direction of our studies. A related point is that some IUPs occur at very low levels not detected by conventional staining. Improving the sensitivity of staining/detection, increasing sample load in the presence of detergents, or prefractionation of samples may be of help here. An important further limitation is that under the standard conditions of native electrophoresis, proteins of net positive charge are lost. As roughly half of the IUPs have a basic isoelectric point (7), the buffer system needs to be changed to extend the analysis over these as well.

Notwithstanding these limitations, this technique has already yielded significant information on the identity of novel IUPs. With the conceived improvements implemented, its potential is expected to increase in the future. Given the ubiquity and functional importance of IUPs, this novel 2D electrophoresis technique may represent an important step toward developing a high throughput proteomic tool for these proteins.


   ACKNOWLEDGMENTS
 
We are indebted to Dr. Katalin F. Medzihradszky and Dr. Éva Klement for the MS analysis of 2D proteins. Thanks are also due to Prof. Peter Závodszky, Prof. Ben Luisi, Prof. Ferenc Erdodi, Dr. László Szabados, Dr. Steven Hahn, and Dr. Michael J. Volles for various proteins and constructs.


  FOOTNOTES
 
Received, June 14, 2005, and in revised form, October 12, 2005.

Published, MCP Papers in Press, October 13, 2005, DOI 10.1074/mcp.M500181-MCP200

1 The abbreviations used are: IUP, intrinsically unstructured protein; Bob-1, B cell-specific transcription co-activator; Csd1, calpastatin domain 1; DARPP32, dopamine- and cAMP-regulated phosphoprotein; GF, gel filtration; CH plot, mean hydrophobicity-mean net charge plot; IPMDH, 3-isopropylmalate dehydrogenase; MAP2c, microtubule-associated protein 2c; MG, molten globule; Mypt1-(304–511), myosin phosphatase target subunit 1 (fragment 304–511); NACP ({alpha}-synuclein), non-Aß component of Alzheimer’s amyloid plaque, precursor; PMG, premolten globule; PONDR®, Predictor of Natural Disordered Regions; RS, Stokes radius; 2D, two-dimensional; TFIIA, transcription factor IIA; GccH, glycine cleavage complex H; DnaKs, DnaK suppressor; BCCP, biotin carboxyl comer protein of acetyl-CoA carboxylase. Back

2 V. Csizmók, E. Szollosi, P. Friedrich, and P. Tompa, unpublished observations. Back

* This work was supported in part by International Senior Research Fellowship GR067595 from the Wellcome Trust. Back

S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material. Back

{ddagger} Supported by a Bolyai János Scholarship. To whom correspondence should be addressed: Inst. of Enzymology, Biological Research Center, Hungarian Academy of Sciences, P. O. Box 7, H-1518 Budapest, Hungary. Tel.: 361-279-3143; Fax: 361-466-5465; E-mail: tompa{at}enzim.hu


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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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