From the Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark; and || Cell Signalling Unit, Childrens Medical Research Institute, Locked Bag 23, Wentworthville, NSW 2145, Australia
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Protein phosphorylation is not routinely surveyed in proteomic studies. Phosphopeptides produced by proteolysis are usually small and hydrophilic, due to the addition of the hydrophilic phosphate group and the preference for many protein kinases to target sequences rich in basic amino acids residues. Proteolytic digests examined by MS are often likely to fail to detect phosphopeptides because they are low in stoichiometry, are suppressed during the ionization (3), or do not bind to the media used to desalt the fraction or separate the peptides (e.g. 4). Recently, attempts have been made to identify phosphorylated proteins using proteomic methods combined with immobilized metal affinity chromatography (IMAC) to selectively bind phosphopeptides, spawning the sub-field of phosphoproteomics (58). After selective binding and elution of the phosphopeptides, the standard approach used in proteomics is to desalt and concentrate the eluted peptides using reversed phase (RP) chromatographic material prior to MS detection. The phosphopeptides are then eluted from the RP column with an acetonitrile gradient, either on-line or off-line with MS, or by batch-mode/isocratic elution from RP microcolumns (e.g. ZipTips) or spin-columns prior to MS.
An endemic problem in this approach is that phosphopeptides may not be retained by RP chromatography because they are too small and/or hydrophilic to bind to the C18 stationary phase (4, 9, 10). An alternative chromatographic material that is able to strongly bind hydrophilic molecules is graphite powder (11, 12). Porous graphitic carbon chromatography has been used recently to retain and resolve phosphopeptides that previously eluted with the nonbinding salts and buffers from an RP column (9). This study revealed three phosphorylated peptides from mixed-lineage kinase 3 that were not retained by RP chromatography. Recently, we introduced the use of graphite powder microcolumns packed in GELoader tips as an alternative or supplement to RP material for purification of sub-picomole amounts of peptides (10). We found that graphite powder microcolumns were able to effectively retain small and hydrophilic peptides, which could be readily eluted for MS analysis. When used in combination with RP material, we gained a significant increase in sequence coverage from tiny amounts of peptides derived from proteolytic digestion of gel-separated proteins.
In this study, we explore the use of GELoader tip microcolumns packed with graphite powder for analysis of small amounts of phosphorylated peptides. We show that a significant number of phosphorylated peptides are not retained by conventional RP chromatographic material, or even material that is slightly stronger in hydrophobicity (Poros oligo R3, which was originally designed for purification of DNA/RNA). Standard phosphoproteins and biologically relevant phosphoproteins were both analyzed after separation by gel electrophoresis. The microcolumns packed with graphite powder efficiently retained and purified phosphorylated peptides from these samples. Application of the method to phospho-dynamin I from nerve terminals revealed the previously reported singly phosphorylated peptides, but also a doubly phosphorylated peptide not previously detected by conventional methods (13). The method also revealed singly and doubly phosphorylated dynamin III, a protein not previously characterized as an in vivo phosphoprotein. The results indicate a major improvement in detection of low-abundance phosphopeptides for sequencing by tandem MS (MS/MS).
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Dynamin I
The method for purifying dynamin has been described previously (13). Briefly, dynamin I was purified from rat brain synaptosomes in a double pull-down experiment. The synaptosomes had been incubated for 1 h at 37 °C in physiological media to allow phosphorylation of proteins in the nerve terminals. Dynamin I was then affinity purified from the synaptosomes using a mixture of the glutathione S-transferase (GST)--adaptin appendage domain and GST-endophilin 1-SH3 domain bound to GSH-Sepharose for 1 h at 4 °C (13). The washed beads were heated in SDS sample buffer, and the released protein was separated by SDS-PAGE. Following zinc staining, a single dynamin I band was excised from the gel (about 2.55 µg) and submitted to in-gel tryptic digestion. The peptide mixture was applied to a microcolumn packed with IMAC beads. The eluate from the IMAC column was divided into two and vacuum dried. One aliquot was redissolved in matrix solution and analyzed by MALDI-MS. The other aliquot was applied sequentially to Poros R2 and then graphite microcolumns, from which the bound peptides were eluted onto a MALDI-MS target as described below. For MS/MS, a second dynamin I band was applied to IMAC followed by Poros R2 and graphite, as above, except that it was eluted from the graphite column using 1 µl of 0.1% formic acid in 1:1 acetonitrile:water solution. The IMAC beads were prepared by incubating a slurry of Ni2+-NTA agarose with 0.1 M EDTA, then 0.1 M acetic acid followed by 0.1 M iron (III) chloride in 0.1 M acetic acid. The beads were washed and stored in 0.1 M acetic acid. The beads were loaded into a shortened GELoader tip (Eppendorf, Hamburg, Germany) to a height of 2 mm above the neck. The sample was loaded in 20 µl of 0.1 M acetic acid. The column was washed once with 0.1 M acetic acid and then with 0.1 M acetic acid with 25% acetonitrile. The peptides were eluted with 20 µl of a 20% NH4OH solution.
SDS-PAGE
One-dimensional gel electrophoresis was performed according to Laemmli (14), using the mini Protean II gel system (Bio-Rad, Hercules, CA). The proteins were dissolved in SDS sample buffer (0.5 M Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 20 mM dithiothreitol, 0.05% bromphenol blue), boiled for 2 min, and applied to a 12% separation gel. Electrophoresis was carried out at constant 160 V. The separated proteins were visualized by zinc staining or colloidal coomassie G250 staining (15).
In-gel Digestion
In-gel digestion was performed as described (16). Briefly, the excised gel plugs were washed in digestion buffer (50 mM NH4HCO3, pH 7.8)/acetonitrile (60/40) and dried by vacuum centrifugation. Modified trypsin (10 ng/µl) or endoproteinase Glu-C (10 ng/µl) dissolved in 50 mM NH4HCO3, pH 7.8, was added to the dry gel pieces and incubated on ice for 1 h. After removing the supernatant, additional digestion buffer was added and the digestion was continued at 37 °C for 418 h. The supernatant from the digestion was used for analysis by matrix-assisted laser desorption/ionization (MALDI)-MS without peptide extraction.
Peptide Desalting
Custom-made chromatographic microcolumns used for desalting and concentration of the peptide mixture prior to mass spectrometric analysis were prepared using GELoader micropipette tips, as described in detail earlier (10, 17). The flow-through from the Poros R2 and/or Poros oligo R3 were desalted and concentrated on a microcolumn packed with graphite powder. The columns were washed with 20 µl of 0.1% trifluoroacetic acid (TFA). The peptides retained on the columns were eluted using 4HCCA in 70% acetonitrile/0.1% TFA (10 mg/ml).
Alkaline Phosphatase Treatment
Alkaline phosphatase treatment was performed directly on the previously analyzed samples after initial MALDI-MS, as described previously (18). The matrix (4HCCA or dihydroxybenzoic acid) was redissolved using 1.5 µl of 50 mM NH4HCO3, pH 7.8, containing alkaline phosphatase (0.05 unit/µl). The MALDI target was placed in a closed plastic box containing a wet tissue to prevent the samples from drying. The box was placed at 37 °C for 20 min. After incubation, the sample was acidified with 0.5 µl of 5% TFA, and the matrix was allowed to recrystallize. In cases where the previously analyzed sample was desalted and concentrated on microcolumns, additional matrix solution (0.2 µl) was added before recrystallization. Prior to MALDI-MS analysis, the surface of the sample was washed gently with 10 µl of 0.1% TFA. When very low amounts of peptides were analyzed, the dephosphorylated peptide sample was redissolved on the target, transferred to a microcolumn containing Poros R2, Poros R3, or graphite powder, desalted, and returned to the target prior to analysis. For the dynamin 1 sample, one half of the flow-through from the R2 column was dried down and resuspended in 2.5 µl of 50 mM ammonium bicarbonate with 1 mM magnesium sulfate and 0.25 units/µl alkaline phosphatase. The sample was incubated for 3 h before graphite microcolumn purification and MALD-MS analysis.
Mass Spectrometry
MALDI-MS was performed using a Voyager STR (PerSeptive Biosystems, Framingham, MA) equipped with delayed extraction. Spectra were obtained in positive reflector mode and positive linear mode using an accelerating voltage of 20 kV. MALDI-MS data analysis was performed using the MoverZ software (www.proteometrics.com). Electrospray ionization hybrid quadrupole time-of-flight mass spectrometry (ESI-MS/MS) was performed using a QSTAR XL (Applied Biosystems/MDS Sciex, Ontario, Canada). The dynamin I sample eluted from the graphite column was sprayed using a boro-silicate nanospray capillary (Proxeon Biosystems, Odense, Denmark) with 1 kV applied. Parent ions were selected using the unit resolution setting. The product ion spectra were acquired using the LINAC pulsar enhancement feature.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
We have taken a sample of in vivo phosphorylated dynamin I from nerve terminals, subjected it to in-gel tryptic digestion, and applied it to IMAC, followed by Poros R2 and graphite microcolumn separation. Fig. 4A shows a region of the MALDI-MS spectrum of the peptides eluted from the IMAC column. A monoisotopic peak matching the singly phosphorylated peptide dynamin I 774783 (SPTSSPTPQR +80 Da, theoretical monoisotopic m/z 1137.49) is present at m/z 1137.52. There are also peaks at m/z 1293.64 and 1373.54 matching singly and doubly phosphorylated dynamin I 773783 or 774784, respectively (RSPTSSPTPQR or SPTSSPTPQRR +80 and +160 Da, should appear at 1293.60 and 1373.56, respectively), overlapping the sequence of 774783 with a missed trypsin cleavage site. This situation arises because the peptide is flanked on both sides by two Arg residues, producing alternative tryptic cleavage sites. For simplicity, dynamin I 774784 is not referred to again, although the peaks in the spectra could be either peptide. The doubly phosphorylated peptide was not observed in a previous study when a similar sample was analyzed by direct MALDI-MS of IMAC beads (13). However, the singly phosphorylated peptides were easily detected in that study. In the present study, similar samples were used, but eluted from the IMAC beads. We found all of the phosphopeptides were extremely low in abundance, having signal-to-noise ratios ranging from six for the peak at m/z 1137.52 to three for the peak at m/z 1373.54. The ionization of these phosphopeptides was probably suppressed by neighboring peptides, most of which can be matched to nonphosphorylated tryptic peptides of dynamin I.
|
The lack of retention of the doubly phosphorylated peptides and poor retention of the singly phosphorylated peptides demonstrates that these phosphopeptides are too hydrophilic to bind to the Poros R2. Of course, it is difficult to rule out that their ionization was suppressed by the other peptides that were retained. Ion suppression is the most likely reason for the low-abundance detection of phosphopeptides in the IMAC eluate (Fig. 4A). The peak at m/z 1373.52 in the graphite eluate (Fig. 4D) has a 10 times higher signal-to-noise ratio than the corresponding peak in the IMAC eluate. This improved signal could be due to the separation of this phosphopeptide from the nonphosphopeptides that bound to the IMAC column (e.g. the peptide at m/z 1375.70 matching dynamin I 584594, see insets in Fig. 4, A and C) or due to the removal of low molecular mass contaminants from the IMAC eluate (e.g. salt). Nevertheless, the improved signal, demonstrates that a second major benefit of using graphite microcolumns is improved sensitivity.
Experiments were next performed to determine the phosphorylation sites within the singly and doubly phosphorylated dynamin I peptides. Previous studies reported phosphorylation of this singly phosphorylated peptide by cdk5 on either Ser-774 or Ser-778 (13), but Thr-780 resides in an excellent context to be predicted as a cdk5 substrate. In a somewhat conflicting report, Tomizawa et al. (20) suggested that the recombinant proline-rich domain of dynamin I is not phosphorylated by recombinant cdk5 in vitro when Thr-780 is mutated to Ala, suggesting Ser-774 or Ser-778 are not in vitro substrates. However, re-evaluation of their published MS/MS data (in Fig 3B of that article) suggests the presence of the phosphate group on Ser-778 rather than Thr-780. A dynamin I sample from rat brain nerve terminals was purified using graphite, following IMAC and Poros R2, as above, then analyzed by ESI-MS/MS. A peptide at m/z 569.28, corresponding to doubly charged, singly phosphorylated dynamin I 774783 was fragmented to produce the spectrum shown in Fig. 5A. Analysis of the spectrum revealed two possible interpretations. A highly abundant series of ions described dynamin I 774773, where only Ser-774 was phosphorylated. The b2 ion is helpful in placing the phosphorylation site at the N terminus, because this ion corresponds to the mass of a pSer-Pro N-terminal fragment. The y9 ion also places the pSer at the N-terminal position. A second interpretation of the spectrum places the phosphate group at Ser-778 for the same dynamin I peptide. Some of the ions overlap, i.e. they could have originated from either sequence. All ions marked with a ' (except y8', which coincides with y9-NH3) are exclusive to this second series. The transition from y5 to y6', or b4' to b5, confirms the presence of a pSer at position 778. Many of the ions of this series are very low in abundance, when compared with the pSer-774 series, but have significant signal-to-noise ratios (e.g. S/N for b4' is 20 in Fig. 5A). There was no evidence that Thr-780 was phosphorylated. The simplest interpretation is the presence of two peptides: a highly abundant peptide phosphorylated on Ser-774 and a very-low-abundance peptide phosphorylated on Ser-778.
|
As well as dynamin I singly and doubly phosphorylated peaks, four additional phosphopeptide peaks were detected in the dynamin I sample at m/z 1147.58, 1227.62, 1303.56, and 1383.57 (Fig. 4C, marked with asterisks). They also appear to be phosphopeptides because they can be dephosphorylated to peaks at m/z 1067.51 and 1223.61 (Fig. 4D, asterisks). As found for dynamin I, the dephosphorylated peaks appear to be related, because they differ only by the mass of an Arg residue. The identity of the peaks at 1147.58 and 1303.56 were previously proposed to represent a polymorphism in the sequence of dynamin I, because a Ser-Pro mutation could hypothetically account for such a series of peaks 10 units higher in m/z (13). However, this was not experimentally tested. To resolve this question, the doubly charged, singly and doubly phosphorylated peptides at m/z 574.29 and 614.26, corresponding to the peaks at m/z 1147.58 and 1227.62, respectively (Fig. 4C), were sequenced by ESI-MS/MS (Fig. 5, C and D, respectively). The sequence did not match dynamin I, but clearly matched a dynamin III tryptic peptide, which is similar in size and sequence to dynamin I. Dynamin III derives from a different gene and is known to be expressed at lower levels in the brain than dynamin I (21). The spectrum of the singly phosphorylated dynamin III peptide provided a near complete y ion series and some contributing b ions that unambiguously describe the sequence pSPPPSPTTQR of dynamin III 759768, where Ser-759 is phosphorylated. However, the ion designated b2' does not belong to this sequence and suggests an alternative sequence, where an amino acid other than Ser-759 is phosphorylated. There was insufficient information to describe this alternative sequence. The spectrum of the doubly phosphorylated peptide in Fig. 5D was found to describe the sequence pSPPPpSPTTQR. A complete y ion series enabled the sites of phosphorylation to be assigned to Ser-759 and Ser-763. There was no evidence that the two remaining potential sites, Thr-765 or Thr-766, were phosphorylated.
The positions of the two identified phosphoserines from dynamin I (Fig. 5A) are retained in analogous sequence contexts in dynamin III as Ser-759 and Ser-763. This sequence is also flanked by pairs of Arg residues, accounting for the appearance of two peptides in the MALDI spectra. Dynamin III was recently reported to be present at low abundance in presynaptic nerve terminals, but is greatly enriched in postsynaptic spines (21). The ESI-MS/MS analysis has unambiguously identified both Ser-759 and Ser-763 as in vivo phosphorylation sites, and this result is the first evidence that dynamin III is an in vivo phosphoprotein.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The results presented in this study clearly show that a large proportion of phosphopeptides derived from phosphorylated proteins by proteolytic digestion can be lost during sample purification employing RP chromatography. We have tested the ability of Poros RP resins to retain phosphopeptides, because we have previously found that this resin has a high binding capacity and consequently a high sensitivity when used in GELoader tip microcolumns. We have shown that some synthetic phosphopeptides and phosphopeptides derived by proteolytic digestion of gel-separated phosphoproteins were not retained by Poros R2 RP chromatography. However, they were efficiently retained on microcolumns packed with graphite powder. Preliminary results have shown that other C18 RP chromatographic material used for capillary high-pressure liquid chromotography on-line to MS/MS (Zorbax, SB-C18, 3.5 µm; Agilent Technologies, Wilmington, DE) also lack the ability to retain some phosphorylated peptides (data not shown). It is, however, likely that other types of RP material will have a different retention for these types of peptide, and that this may also be dependent on the mobile-phase buffer used for binding the peptides to the column.
Recently we proposed the use of sequential application of chromatographic material with increasing hydrophobic material for obtaining significantly higher sequence coverage from gel-separated proteins (10). We are currently applying peptide digests sequentially to microcolumns packed with Poros R1, R2, R3, and graphite powder, respectively, which are increasingly hydrophobic stationary phases. In addition to yielding simpler peptide mixtures to analyze by MS, the separation achieved by these stationary phases increases the probability for detection of phosphorylated peptides, due to reduced suppression effects and minimal losses. However, this procedure is an off-line method more suitable to analysis of a single protein and is therefore not easily applicable to high-throughput proteomics.
Traditional proteomic strategies employing two-dimensional gel electrophoresis (2DE) are slowly being overtaken by SDS-PAGE followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) of in-gel-derived peptides or entirely gel-free systems (LC/LC-MS/MS). The latter strategies are currently not matching the resolution obtained by 2DE for large-scale analysis of modified proteins, as the sequence coverage obtained from the LC-MS/MS analysis is relatively low, decreasing the possibility to detect protein isoforms and post-translational modifications. However, several groups have attempted to perform such analysis. The results presented in this study raise a major concern for large-scale phosphoproteomics studies that rely heavily on sample preparation by RP chromatography (e.g. 7, 22). These studies typically use a RP cleanup after elution from IMAC. In on-line experiments, they allow the salts used in IMAC elution to go to waste as they load the RP column, to prevent the salts from entering the mass spectrometer, as in LC/LC experiments using ion exchange media (23). Typically, the solvent flow is reconnected to perform LC-MS/MS of the bound material. This would result in some phosphopeptides going to waste, reducing the extent of the phosphoproteome.
Graphite powder is recommended as an aid in achieving a significant improvement in sequence coverage of proteins, thereby increasing the probability to detect phosphopeptides derived from phosphorylated proteins. We suggest that the waste from RP cleanup should be applied to a graphite column, and that the eluate from the RP and graphite columns should be analyzed in turn. Graphite powder (capillary) columns are commercially available or can be prepared from graphite material (e.g. Hypercarb; Thermo Hypersil-Keystone, Bellefonte, PA). Preliminary results using such capillary graphite columns in our laboratory have shown recovery of hydrophilic peptides in LC-MS/MS experiments using normal gradient runs. However, these experiments show lower sensitivity than if the elution was performed using MALDI matrix solution, as previously demonstrated for the GELoader tip graphite microcolumns (10). Applied as a second column after the C18 capillary column, the sequence coverage could be increased in LC-MS/MS experiments and allow detection of more peptides. In addition, the use of a C18 column prior to the graphite capillary column could extend the lifetime of such columns.
The improved detection of hydrophilic phosphopeptides using graphite has resulted in significant advances in understanding the regulation of dynamin and synaptic vesicle endocytosis. In a previous study, the phosphorylation sites in dynamin I both in vitro and in vivo were found to be Ser-774 and Ser-778. However, because only a singly phosphorylated peptide was observed with IMAC alone in that study, it was not possible to determine which amino acid was the specific phospho-acceptor. Ser-774 and Ser-778 were revealed by production of specific phosphorylation-state antibodies, while Thr-780 was not specifically investigated because phospho-amino acid analysis revealed low abundance of the pThr in vivo and in vitro. It was proposed that dynamin I might be phosphorylated on either site in a mutually exclusive fashion (13). Using the graphite column here, we have now revealed the presence of doubly phosphorylated dynamin peptides and directly confirmed phosphorylation of the two serines in dynamin I in vivo. Because blocking cdk5 activity in neurons also inhibits endocytosis after repetitive cycles, it can now be proposed that both phosphorylation sites might play a central role in synaptic vesicle endocytosis. These sites are adjacent to a number of short proline-rich motifs that may regulate protein-protein or protein-lipid interactions with dynamin. It is possible that dual phosphorylation of dynamin I regulates its interactions with specific proteins involved in endocytosis.
In addition to revealing doubly phosphorylated dynamin I, the new techniques revealed phosphorylation sites within the homologous sequence of the C-terminal tail of dynamin III. This form of dynamin is known to be expressed at low levels in nerve terminals, but is enriched in postsynaptic spines in hippocampal neurons (21). The discovery of the presence of dynamin III in pull-downs from nerve terminals is not unexpected and is consistent with the likelihood that dynamin III interacts with similar endocytic proteins as dynamin I or that dynamin III forms heterocomplexes with dynamin I. Little is known about the function or regulation of dynamin III. Its enrichment in postsynaptic spines is strongly indicative of an endocytic function in mediating postsynaptic receptor signaling. Our discovery of singly and doubly phosphorylated dynamin III provides direct evidence that it is an in vivo phosphoprotein. This raises the possibility that it may be regulated in a similar manner to dynamin I. However, it remains to directly demonstrate whether dynamin III might be a substrate for cdk5. Additionally, the phosphorylation sites at Ser-759 and Ser-763 are equally spaced and appear in a very similar context with respect to the dynamin I sites. It is interesting that these two serines, as in dynamin I, immediately precede a proline residue, suggesting they would be targeted by proline-directed protein kinases such as cdk5. We found no evidence for phosphorylation of Thr-780 in dynamin I despite that it also precedes a proline, although this cannot be definitively ruled out. In contrast, dynamin III diverges from dynamin I in this sequence and the proline is absent. This reduces the probability that it represents a phosphorylation site and raises the interesting possibility that there may be no major differences in its phosphorylation from dynamin I.
A further observation is that for both the dynamin I and dynamin III singly phosphorylated peptides, the sequence with the phosphorylation site at the N-terminal Ser produced much more abundant ions (Ser-774 and Ser-759, respectively). Care must be taken when using MS data for quantitative purposes because small changes to a sequence can affect the efficiency of ionization (3). However, the much larger relative abundance, particularly for singly phosphorylated dynamin III, which is almost exclusively Ser-759 phosphorylated, suggests that there may be a preference for phosphorylation of the N-terminal Ser to precede phosphorylation of the second site. This raises the possibility of hierarchical phosphorylation of dynamin I and III (24), which will require further investigation.
In conclusion, the use of graphite microcolumns clearly provides a significantly more complete coverage of phosphoproteomes. The method is simple, inexpensive, and readily amenable to automation. The method also greatly improves the signal-to-noise ratio for many peptides, thus providing a large increase in sensitivity. As demonstrated for dynamin, the use of graphite microcolumns following IMAC enrichment of phosphopeptides and then R2 hydrophobic columns greatly improves the coverage of phosphopeptides that can be detected within a single protein. With this simple approach it will now be possible to greatly increase the number of identified phosphorylation sites within a protein.
![]() |
FOOTNOTES |
---|
Published, MCP Papers in Press, February 2, 2004, DOI 10.1074/mcp.M300105-MCP200
1 The abbreviations used are: MS, mass spectrometry; MALDI, matrix-assisted laser desorption/ionization; RP, reversed phase; IMAC, immobilized metal affinity chromatography; GST, glutathione S-transferase; TFA, trifluoroacetic acid; 4HCCA, -cyano-4-hydroxycinnamic acid; LC, liquid chromatography; ESI, electrospray ionization; 2DE, two-dimensional gel electrophoresis.
* This work was part of the activities associated with the Danish Biotechnology Instrument Center, supported by the Danish Research Councils (to M. R. L. and P. R.) and supported by the Australian National Health and Medical Research Council (to P. J. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indi-cate this fact.
M. R. L. and M. E. G. contributed equally to this work.
¶ To whom correspondence should be addressed: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark. Tel.: 45-6550-2342, Fax: 45-6593-2661; E-mail: mrl{at}bmb.sdu.dk
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|