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J. Biol. Chem., Vol. 279, Issue 49, 50915-50922, December 3, 2004
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From the
Department für Biologie I, Ludwig-Maximilians Universität München, Menzinger Strasse 67, 80368 München, Germany and ¶Hoffmann-La Roche, Roche Centre for Medical Genomics, CH-4070 Basel, Switzerland
Received for publication, May 26, 2004 , and in revised form, September 22, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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For most proteomic approaches, gel-based two-dimensional systems have been coupled with mass spectrometry, which is one of the most powerful tools for protein identification (16–18). The combination of isoelectric focusing or blue native PAGE as the first dimension followed by SDS-PAGE as the second dimension is a widespread technique for the effective resolution of large numbers of soluble and membrane proteins (5, 19–21). Proteins with a grand average hydropathy (GRAVY)1 score (22) above 0.5 (23–26) and even membrane protein complexes (27, 28) can be separated by blue native PAGE in contrast to isoelectric focusing. Fast and unequivocal identification of the separated proteins is possible by analyzing internal peptide sequences. In contrast to conventional protein sequencing procedures, mass spectrometry allows the rapid and automated analysis of many proteins in a short time frame. Even though the advantages of this technology are a major break-through for the high throughput analysis of proteomic compositions, the technology is also limited to a certain extent. For example, identification of proteins of the same size but different abundance raises difficulties. Furthermore, the procedure of peptide production by protease in gel digestion can reveal major losses, and therefore, vanishing of the signal will be observed. A problem always present is the detectability of certain classes of peptides. One set of these is the class of transmembrane segments. The inability of detection was even used to analyze the topology of certain protein classes (29). It was determined that only 10% of all peptides from transmembrane proteins analyzed represent at least portions of the transmembrane segments (29). Interestingly, some groups recently succeeded in detecting and sequencing transmembrane regions from GalP (30) and bacteriorhodopsin (31, 32) in model studies. In contrast to previous reports, high amounts of protein were used in these experiments, and samples were digested in solution without electrophoretic separation of the proteins. However, highly purified membrane fractions are often not as concentrated as in such model studies. Since major interest has been drawn to the identification of protein networks and protein complexes, which have to be isolated and concentrated beforehand, the proteomic analysis of membranes and membrane complexes suffers from the reduced ability to detect membrane proteins due to the reduced amount of analyzable peptides. Furthermore, not all expected proteins will be detected in one biological sample since the expression of some membrane proteins is specific for certain tissues or developmental stages of the individual.
We have now investigated several physicochemical parameters of peptides containing transmembrane segments by experimental and bioinformatic tools. For this, we have analyzed the thylakoid membrane by mass spectrometry and the Arabidopsis thaliana proteome by bioinformatic analysis. We specifically address the molecular forces intervening with detection of peptides from transmembrane segments by mass spectrometry. Based on the results, we propose that methionine modification by hyperoxidation or cyanogen bromide digestion, for example, could be a tool to detect an increased number of transmembrane regions by mass spectrometry.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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Isolation of Protein Complexes Present in Native Membranes and Stromata
Native membrane and stromal protein complexes were prepared directly after isolation of chloroplasts from barley (Hordeum vulgare L. var. Steffi) as described (36). Chloroplasts were lysed at 4 °C in TMK buffer (10 mM Tris-HCl (pH 6.8), 10 mM MgCl2, and 20 mM KCl). Membranes were isolated by centrifugation at 3800 x g for 2 min at 4 °C. After two wash steps in TMK buffer, thylakoid membranes were resuspended in 60 µl of 750 mM 
-aminocaproic acid, 50 mM BisTris-HCl (pH 7.0), and 0.5 mM EDTA. Complexes were solubilized by incubation with 1.2% (w/v) dodecylmaltoside (final concentration) for 10 min on ice. Non-solubilized material was removed by centrifugation at 20,000 x g for 10 min at 4 °C. 5 µl of 5% (w/v) Serva blue G in 750 mM -aminocaproic acid was applied to the supernatant. 600 µg of solubilized proteins from thylakoids was loaded on one blue native gel lane.
Two-dimensional Gel Electrophoresis
First Dimension—Sample solutions of membrane proteins were applied to a 6–12% blue native PAGE system (21).
Second Dimension—Individual lanes of the blue native polyacrylamide gel were cut and incubated for 15 min at room temperature in 2% (w/v) SDS, 66 mM sodium carbonate, and 2% (v/v) 
-mercaptoethanol at 50 ml/lane (37). Lanes were positioned on top of the second dimension gel (12.5%, containing 4 M urea) and overlaid with a low melting agarose solution containing 0.5% (w/v) agarose, 192 mM glycine, 0.1% SDS, and 25 mM Tris at 50 °C. After electrophoresis, the resolved proteins were visualized by staining with Coomassie Brilliant Blue R-250 (Serva, Heidelberg, Germany), and protein spots of interest were excised with a clean scalpel and stored in Eppendorf tubes at –20 °C. Standard protein solution (Mark 12, broad-range protein standard, Novex) was provided on a piece of filter paper next to the first dimension lane.
Mass Spectrometry
In gel digestion of proteins before subjection to mass spectrometry was carried out using the method described previously (38). Mass spectrometric analyses were carried out on a Micromass Q-TOF-I hybrid mass spectrometer equipped with an orthogonal nano-electrospray ionization source operating in the positive electrospray ionization mode. Samples were loaded into medium nano-electrospray capillaries (Protana, Odense, Denmark) and positioned in the source. For external calibration, a standard solution of 0.2% (v/v) phosphoric acid (H3PO4) in 50% methanol was used. The peak at m/z 421.7589, which resulted from self-digestion of trypsin, was used for internal calibration of mass spectrometric (MS) spectra and for verification of successful digestion.
To obtain optimized spectral data, a capillary voltage of 750–900 V and a cone voltage of 45 V were used. For scanning peptide MS spectra, the quadrupole was set to the appropriate mass range of m/z 350–1600, where peptides were expected. After data collection, 25 scans were averaged at a rate of 2.0 s/scan. Instrument operation, data acquisition, and analysis were performed using MassLynx/Biolynx 3.5 and 4.0 software. For peptide detection coverage analysis, the following thylakoid proteins were analyzed in detail: PsaK, PsaG, PsaH, PsaL, LhbC, PsaB, PsbA, PsbB, PsbC, PsbD, PsbE, and PsbF.
Calculation of Hydrophobicity
For calculation of the hydrophobicity, several scales were used, viz. the hydrophobicity scale of Kyte and Doolittle (22), the scale defined by Eisenberg et al. (39), and the scale of Black and Mould (40). To calculate the mean hydrophobicity of a pool of peptides or for a protein, all hydrophobicity values of the pool or of the protein were accumulated and finally normalized by the amount of amino acids in the pool or protein. For calculation of the central region (see Figs. 5 and 6) for comparison with the calculation performed by Eisenberg et al. (39), the hydrophobicity was calculated based on the Eisenberg scale in a window of 11 amino acids; values were normalized; and the highest value was assigned to the peptide. For the region bearing the highest hydrophobicity, the hydrophobic moment was calculated as follows (Equation 1),
 | (Eq. 1) |
is the angle of the turn between two amino acids. For classical helices as discussed here, the angle was set to 100.
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-Barrel Score-barrel score was calculated as described previously (34, 41). For calculation of the exact -barrel score of the peptide pool selected, the bilayer score (41) was used. The score for each peptide was calculated for the case that the first amino acid would face the bilayer and for the case that the first residue would face the interior. The larger value was added to the exact -score of the pool. Finally, the derived sum was divided by the number of amino acids in the pool.
Calculation of Fragment Frequency and Probability Values
For analysis of the digestion frequency, the sequences were split C-terminally after arginine and lysine, and the corresponding fragments were sorted according their amino acid length. To analyze the detection probability as shown in Fig. 7C, all peptides analyzed in this work were sorted according to their hydrophobicity or their amino acid length, and the fraction of detected peptides for each parameter in a certain window was calculated. To determine the probability for detection by the two parameters hydrophobicity and length, the probability for detection at a given peptide size (P1) and at a given hydrophobicity (P2) was used to calculate the detection probability by Equation 2.
 | (Eq. 2) |
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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While analyzing the membrane proteome of chloroplasts, we recognized that several expected membrane proteins were not detectable, although they were described to be associated with thylakoid membrane complexes. Although low molecular mass proteins such as PsbF (4.4 kDa) were identified, proteins of photosystems I and II <10 kDa are often hardly detected by analysis of internal peptides. Because membrane proteins may be detected by means of their hydrophilic loop regions and because "short" peptides are detected more frequently than "long" peptides, extreme hydrophobicity and the lack of internal tryptic cleavage sites may have prevented these proteins from being detected.
Transmembrane Segments from Thylakoid Membrane Proteins Escape Detection by Electrospray Ionization MS—A detailed analysis of the thylakoid proteome revealed that especially membrane-inserted fragments could not be detected by mass spectrometry. To gain an understanding of this phenomenon, we analyzed the observed fragments of helical membrane proteins of the thylakoid membrane and -barrel membrane proteins of the outer envelope of chloroplasts (33, 34).
Tryptic digestion of proteins leads to peptides of different lengths corresponding to the distribution of tryptic cleavage sites (arginine and lysine). A number of these peptides could be detected by electrospray ionization MS (MS spectra). The quality of a spectrum depends on the amount of peptide extracted from the gel, which could be partially influenced by increasing the time of measurement. However, a relevant part of peptides of thylakoid proteins predicted from in silico analysis was not detected when spectra from tryptic digests were analyzed. In our MS spectra, peptides usually emerged between m/z 
300 and 1600. Therefore, large peptides >1.4 kDa have to be multiply charged to fit in this m/z range. However, peptides found by electrospray ionization MS normally carry charges from +2 to +4 anyway. As described previously (30, 42), peptides >4 kDa were not detected and sequenced. To decide whether a peptide signal was present or not, peak intensity had to be >50 counts/25 averaged spectra.
Doubly charged peptides <0.6 kDa (m/z 300, approximately five amino acids) were never detected in our MS spectra. A possible explanation could be charge repulsion within these peptides. For peptides longer than 15 amino acids, detectability was decreased with increased amino acid number and hydrophobicity. However, especially these long and hydrophobic peptides were of interest because tryptic digestions of 
-helical membrane proteins yields peptides containing at least 20 amino acids.
About 35% of all peptides should contain helical transmembrane segments (Fig. 1A, bar 3). However, when the theoretical statistical occurrence of helical transmembrane segments (Fig. 1A, bar 3) was compared with the observed occurrence of such peptides of 4% (bar 4), it became obvious that the detection of helical transmembrane regions is a problem. In contrast, peptides with transmembrane segments from -barrel proteins were detected in the proposed statistical frequency (Fig. 1A, bars 1 and 2). We can therefore conclude that not the membrane insertion per se results in the loss of detection of such fragments.
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50% of all peptides when no dissection regarding the localization of the peptide was performed (Fig. 1B, bar 1). In contrast, when only peptides comprising soluble regions of the proteins were analyzed, 60% of all peptides predicted were experimentally observed as well (Fig. 1B, bar 2). For peptides containing membrane regions, the overall detection rate was 35% (Fig. 1B, bar 3) and therefore significantly smaller than the detection rate for soluble peptides. We next analyzed all peptides longer than 20 amino acids. Interestingly, the overall detection rate for such peptides was decreased to 15% (Fig. 1B, bar 4), and the soluble phase peptides (bar 5) were significantly reduced to 20% as well. However, almost no peptides of membrane proteins were detected (Fig. 1B, bar 6).
We then wanted to understand why certain peptides of transmembrane proteins were detectable and others not. One physical parameter defining a transmembrane region is hydrophobicity. Therefore, the average hydrophobicity of the pool of detected (Fig. 1C, bars 2 and 4) and non-detected (bars 1 and 3) peptides was compared. Interestingly, the pool of detected peptides showed an average hydrophobicity of 0.2 independent of the cutoff length, whereas the range of the hydrophobicity determined for the detected peptides covered a range from –2 to 0.5, with two exceptions where the peptides revealed hydrophobicity values of 2.0 and 1.1. In contrast, the non-detected peptides revealed a high hydrophobicity (Fig. 1C, bars 1 and 3). Furthermore, peptides not detected and longer than 21 amino acids exceeded the hydrophobicity index of shorter peptides of the same pool by a factor of 4. These two observations point to the interpretation that the hydrophobicity and the length of peptides are critical determinants for detection by experimental means.
Comparison of the Thylakoid and the A. thaliana Proteome— From the analysis of the experimental data obtained from sequencing of the thylakoid membrane, it was determined that longer and more hydrophobic peptides are not as frequently detected compared with shorter and more hydrophilic peptides (Fig. 1B). We therefore addressed the specific role of peptide length in the increase in the hydrophobicity index. To answer this question, the proteome of the thylakoids was digested in silico at trypsin-specific cleavage sites, and the hydrophobicity and the number of fragments were calculated for each pool of peptides of a defined length (Fig. 2). Strikingly, the pools of peptides shorter than 20 amino acids showed a hydrophilic behavior in general, whereas the pools of longer peptides revealed a general hydrophobic character. To exclude that this observation is limited to the proteins of the thylakoid membrane, we analyzed the entire A. thaliana proteome. As expected, the number of peptides in a certain pool size decreased exponentially with the length of the peptides (Fig. 3A). Next, we analyzed the -barrel probability and the hydrophobicity of each peptide pool (Fig. 3, B and C). The probability for a membrane-inserted -sheet was found to be very low. Only the pools with a peptide size above 50 amino acids in length revealed a score above zero. For comparison, a cutoff score of 2 was chosen for selection as a membrane-inserted -sheet (34, 41). However, it is worthwhile to mention that the exact -strand score of the pools increased almost linearly with the peptide size of the pools (Fig. 3B). This might suggest that the portion of peptides derived from membrane-inserted -barrels increases in the pools of peptide of increasing size. As seen before for the thylakoid proteins, the average hydrophobicity of the A. thaliana proteome fragments increased almost linearly in the peptide size range of up to 30 amino acids (Fig. 3C). The pools of peptides longer than 20 amino acids showed a mean hydrophobic character, whereas the pools of peptides shorter than 21 amino acids revealed an overall hydrophilic character. Therefore, the observation made for the thylakoid proteins can be generalized for the whole proteome. Peptides longer than 20 amino acids are more hydrophobic in general, indicating that hydrophobicity is a major determinant for detection by mass spectrometry.
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-barrel score above 1, proteins with a mean hydrophobicity above 0.25 (according to the Kyte and Doolittle scale (22)), and all proteins with at least one transmembrane region identified or predicted by TMHMM (Fig. 4). The first pool was chosen as a control. The second pool contains proteins with a putative membrane -barrel fold (34, 41). This pool was analyzed to compare the transmembrane -barrel and -helical proteins (fourth pool) since analysis of the experimental data revealed a drastic difference in the coverage rate (Fig. 1A). The third pool was chosen since a mean hydrophobicity of 0.25 and larger accounts for very hydrophobic proteins for the following reasons.
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We then analyzed the peptide frequency after in silico digestion of the four pools of sequences. The distribution of peptides derived from putative -barrel proteins did not significantly differ compared with complete proteome (Fig. 4, A and B). In contrast, for the third and fourth pools, we observed a significant enrichment of peptides of 20–35 amino acids (Fig. 4, A and B). Such a result might have been expected for the pool of hydrophobic sequences since arginine and lysine (representing the cleavage sites for digestion by trypsin) are the most hydrophilic amino acids and are possibly less abundant in proteins with a high average hydrophobicity. Strikingly, however, is the result with proteins containing putative transmembrane regions. 50% of all protein sequences in the pool have only a single transmembrane domain, and only 35% have more than two predicted transmembrane regions as determined by TMHMM (data not shown). The average hydrophobicity of the complete protein pool containing putative transmembrane domains was below zero (according to the Kyte and Doolittle scale (22)), suggesting the presence of large hydrophilic domains. In addition, the arginine and lysine frequency in thylakoid membrane proteins was not significantly lower compared with the complete A. thaliana protein pool (data not shown). Still, a similar cluster of longer peptides as for the hydrophobic proteins was observed. This suggests that those transmembrane domains are usually covered by long peptides. Furthermore, the maximum peptide length was found to be in the range of 28 amino acids, but the peak expanded from 20 to 40 amino acids. Therefore, the length of the transmembrane segments derived by tryptic digestion could be one reason for the low abundance in the MS experiment as discussed above (Fig. 1B). This result can be further extended to other proteomes. While analyzing all sequences from either Escherichia coli (Fig. 4C) or Homo sapiens (Fig. 4D), we observed a similar profile as for A. thaliana (Fig. 4B). We are therefore convinced that the observations for A. thaliana can be generalized.
How Can the Detection Problem Be Solved?—We have shown that two phenomena account for the lower detectability of transmembrane segments. An increased peptide size reduced the probability for detection by experimental means (Fig. 1B), and the frequency of enlarged peptides was increased in proteins with putative membrane localization (Fig. 4, A and B). However, not only the length but also the hydrophobicity of the segment (Fig. 1C) accounted for the low detectability of the transmembrane regions since the longer peptides from soluble proteins remained detectable by mass spectrometry to a higher extent (Fig. 1B). In general, the increase in the peptide length was shown to correlate with an increase in the mean hydrophobicity of the peptides (Figs. 2, 3C, and 6). Therefore, we concluded that a reduction of the hydrophobicity and/or length of the transmembrane segment-containing peptides would allow their detection.
To analyze and prove the last idea, we went back to the experimentally derived data set. We analyzed the length of the peptides as well as their mean hydrophobicity and hydrophobic moment. To do so, we used the Eisenberg scale to compare the features of the peptides with the previously reported characteristics of transmembrane segments (39). As discussed above, most peptides not detected were of high hydrophobicity (Figs. 1C and 5C). The maximum of the gaussian distribution of the hydrophobicity for peptides not detected was 0.59 (Fig. 5C, dashed line), whereas the center of the hydrophobicity of the detected peptides was 0.15 (solid line). This clearly supports the idea that strong hydrophobicity contradicts detectability. It is worthwhile mentioning that a small cluster of highly hydrophilic peptides were not detected as well. (15% of all non-detected peptides had a peak of hydrophobicity of about –0.33.) Analyzing the peptide length supported the observation out-lined in Fig. 1B since mainly short sequences were detected, with a size distribution peaking at 10 amino acids (Fig. 5B). For peptides not detected by MS analysis, two peaks were obtained. Whereas the first peak (at approximately seven amino acids) covered only 10% of the complete area, the main peak had a wide distribution with a center at 30 amino acids, confirming the above-mentioned idea that length is a major determinant for detectability.
The physical features of the analyzed peptides can now be compared with the prediction rules for transmembrane segments as discussed previously (39). About 55% of the non-detected peptides fall into the region typical for transmembrane helices (Fig. 5A) as defined by Eisenberg et al. (39), and only 20% of all peptides are clearly assigned as soluble (Fig. 5A, to the left of the dashed line). The gaussian distribution of the hydrophobic moment of the non-detected peptides had a maximum at 0.03 (Fig. 5A, dashed line), meaning that hydrophobicity is equally distributed throughout the sequence, as the hydrophobic moment (Equation 1) is near zero. From this, it becomes obvious that a combined reduction of the hydrophobicity and an increase in the hydrophobic moment would change the physical properties of a peptide, where detection becomes more likely. But how can such a shift of the physical features be achieved?
One solution to this problem could be an oxidation of the hydrophobic transmembrane peptides. As Black and Mould (40) observed, oxidation of the amino acid methionine results in a decrease in hydrophobicity. When compared with the Eisenberg scale (39), a single oxidation of methionine would reduce the hydrophobicity from 0.64 to –0.76 (Fig. 6A). A double oxidation would decrease the hydrophobicity of the peptide to –2.0 (Fig. 6A). Such a drastic shift of one single amino acid may contribute to the physical properties of the hydrophobic transmembrane peptide in a way that the peptide becomes detectable.
To investigate this idea, the peptides were oxidized before MS analysis. This procedure revealed the detection of two previously unidentified sequences (Fig. 6, B and C). The hydrophobicity and hydrophobic moment of the two peptides were clearly shifted by oxidation (Fig. 6B, open circles). Furthermore, the oxidation placed the hydrophobicity value in a range where 80% of all analyzed peptides were detectable disregarding their length (Fig. 6B, dashed line). In contrast, before oxidation, the hydrophobicity of the two peptides was in a range where, under our experimental conditions, only 15–20% of all peptides could be found by MS analysis.
A second solution would be a reduction of the size of the fragments. One possibility is the subsequent cyanogen bromide cleavage of the peptides. Since both methods would target methionines in transmembrane regions, we analyzed the methionine content in different proteomes (Fig. 7A). We observed that the overall methionine content in eukaryotes was reduced in comparison with the analyzed prokaryotic proteome (Fig. 7A, compare bars 1, 4, and 7). However, when only proteins with putative transmembrane regions were analyzed, the methionine content was increased in sequences from E. coli and H. sapiens (Fig. 7A, bars 2 and 5), but not from the plant proteome (bar 8), compared with the corresponding content of the complete proteome. Analysis of the transmembrane segments then showed a significant increase in the methionine distribution in all three cases (Fig. 7A, bars 3, 7, and 9). Therefore, methionine is an obligate target to further optimize transmembrane peptide analysis. Supporting this notion, almost 60% of all predicted transmembrane segments in E. coli and almost 50% in the eukaryotic proteomes contain a methionine (Fig. 7B). We conclude that targeting methionine by chemical modification or cleavage should increase the detectability of transmembrane segments by mass spectrometry.
Indeed, when the transmembrane peptides not detected by mass spectrometry in our approach (Fig. 7C, shaded triangles) were subsequently cleaved in silico by cyanogen bromide (open circles), we determined that most of the peptides fall into the region with respect to amino acid length and hydrophobicity of the proteolytic fragment where detection of peptides becomes most likely according to our experience (0, 25, 50, and 75% detection probability boundaries) (Fig. 7C). In a subsequent experimental preliminary approach, we were able to detect 14 new peptides (Fig. 7C, open squares) as part of predicted transmembrane regions. This demonstrated that cyanogen bromide modification of tryptic membrane proteins increases the probability of their detection. Hence, methionines should be seen as the major target to identify peptides from transmembrane regions.
Conclusion—It is well known that membrane proteins are detected by means of their soluble regions and that short peptides are detected more frequently than long peptides (Fig. 1B) (44). However, we have clearly demonstrated that transmembrane segment-containing fragments escape detection by mass spectrometry because of their size and their hydrophobicity (Fig. 5). In addition, longer fragments were found to be more hydrophobic than shorter fragments (Fig. 3); and in turn, transmembrane region-containing proteins revealed a higher yield of fragments longer than 20 amino acids compared with soluble proteins (Fig. 4). Therefore, the low abundance of peptides containing membrane segments is an intrinsic experimental problem since those regions are present in long and hydrophobic peptides. In contrast to peptides representing soluble regions, the decrease in the peptide size cannot be proposed as a tool for an increase in peptide coverage since many short peptides containing transmembrane segments still escape detection (Fig. 5B). However, alteration of the hydrophobicity would clearly increase the probability of detection. One way to modify the hydrophobicity is the oxidation of such peptides (Fig. 6A) since methionines are highly enriched in transmembrane segments (Fig. 7, A and B). As demonstrated, such oxidation revealed the detection of two peptides not detected before (Fig. 6, B and C) since the hydrophobicity value was drastically altered. Furthermore, analysis of the second method of targeting methionines, viz. subsequent cyanogen bromide digestion, revealed that the shorter and sometimes less hydrophobic peptides became detectable (Fig. 7C). We therefore propose that the development of techniques quantitatively targeting the methionines of peptides will reveal the detectability of transmembrane segment-containing fragments by mass spectrometry.
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FOOTNOTES |
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The on-line version of this article (available at http://www.jbc.org) contains Supplemental Fig. 1.
To whom correspondence may be addressed. Tel.: 0049-89-17861-272; Fax: 0049-89-12861-206; E-mail: eichacker{at}lrz.uni-muenchen.de. || To whom correspondence may be addressed. Tel.: 0049-89-17861-182; Fax: 0049-89-17861-185; E-mail: schleiff{at}lrz.uni-muenchen.de.
1 The abbreviations used are: GRAVY, grand average hydropathy; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; MS, mass spectrometric.
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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HE11
aa) was identified for each peptide using the scale developed by Eisenberg et al. (
