Development and Application of a Two-Phase, On-Membrane Digestion Method in the Analysis of Membrane Proteome Jian Zhou, Yong Lin, Xingcan Deng, Jianying Shen, Quanyuan He, Ping Chen, Xianchun Wang,* and Songping Liang* College of Life Sciences, Hunan Normal University, Changsha 410081, P. R. China Received August 11, 2007
Abstract: Analysis of membrane proteins, particularly integral membrane proteins, still presents a great challenge due to their poor water solubility and low abundance though much effort has been devoted to the solubilization and enrichment of the protein class. In this paper, a two-phase, on-membrane digestion method was developed and applied in the analysis of rat liver membrane proteome. The two-phase system was constituted by mixing n-butanol and 25 mM NH4HCO3. Comparative experiments indicated that the proteins on membranes could be digested in the two-phase system more efficiently than in both 60% methanol and 25 mM NH4HCO3 solutions under the same conditions, thereby improving the identification of the membrane proteins. When the established two-phase system and CapLC-MS/MS was used to analyze rat liver membrane proteome, a total of 411 membrane proteins were identified, more than 80% of which were transmembrane proteins with 1–12 mapped transmembrane domains (TMDs). Because of its extraction and dissolution actions, the two-phase on-membrane digestion system we developed could efficiently improve the digestion and removal of adsorbed nonmembrane proteins, and remarkably increase the number and coverage of identified membrane proteins, particularly the transmembrane proteins. Using our procedure to identify a complementary protein set from all fractions of the twophase system could achieve a higher coverage of the membrane proteome. Keywords: two-phase system • on-membrane digestion • membrane protein • proteome • n-butanol
Introduction Membranes are critical components of cellular structure and function involving the partitioning of organelles, protecting the integrity of genome and proteome, and providing defense against foreign molecules and external conditions that may damage or destroy the cell.1The functions of cells are closely related to the proteins present in the membranes because the membrane proteins are main components of the membranes and carry out many essential biological membrane functions. * To whom correspondence should be addressed. Fax, 86-731-886-1304; e-mails, (S.L.)
[email protected], (X.W.)
[email protected].
1778 Journal of Proteome Research 2008, 7, 1778–1783 Published on Web 02/29/2008
A better knowledge of the membrane proteins would greatly help to understand the highly diverse structure and functions of the cell. However, although the protein chemistry and proteomics have been greatly developed in recent years, the analysis of membrane proteins still presents an analytical challenge, because most membrane proteins not only are lowabundance, but also are not readily soluble in pure aqueous buffers due to their hydrophobicity and tendency of aggregation. These properties of the membrane proteins could lead to the low number and coverage of proteins identified. In recent years, many refinements, such as subcellular prefractionation2,4 and use of detergents,5,7 have been directed at the enrichment and solubilization of membrane fraction to improve the identification of membrane proteins. Among the subcellular prefractionation methods, the most commonly used approach to isolate plasma membrane and intercellular membranes is the use of differential centrifugation followed by density gradient centrifugation3 or the aqueous two-phase partitioning.8,9 Nevertheless, although the membrane proteins in the membrane fraction obtained with the subcellular prefractionation methods were enriched compared with those in whole-cell lysate, the identification efficiency of membrane proteins, in particular integral membrane proteins, is still far from satisfactory. A high percentage of the identified proteins are usually contaminating proteins and membrane-associated proteins such as cytoskeletal proteins. The real membrane proteins, in particular low-abundance integral membrane proteins, including regulatory proteins and ion channels, are mostly out of the scope of standard proteomic techniques. One of the main reasons is that the proteins absorbed on the membrane are generally in ample abundance and override the relatively small amounts of membrane proteins. For improvement of representation of membrane proteins in membrane fraction, many efforts have been made to remove undesirable proteins, such as rinsing the membranes with strong ionic and high pH solution, and applying protein depolymerising conditions. For example, Marmagne et al. used chloroform/methanol, NaOH, and nonionic detergents to extract and treat the membrane fraction purified from Arabidopsis thaliana cells by differential centrifugation and aqueous two-phase partitioning.10 Bartee et al. employed sodium bicarbonate (pH 11.5) and ammonium bicarbonate (pH 8.5) to sequentially rinse the membranes purified from Hela-Tet Off cell using discontinuous sucrose gradient ultracentrifugation in order to remove adsorbed undesirable proteins.11 With these measures, the identification effectiveness of membrane proteins was enhanced and the 10.1021/pr070526j CCC: $40.75
2008 American Chemical Society
Two-Phase On-Membrane Digestion Method identified membrane proteins achieved to 50–60% of the total proteins identified. In addition, for simplification of the treatment process and improvement of membrane protein identification, on-membrane digestion with protease in the presence or the absence of organic solvent was also introduced into the removal of undesirable proteins and the cleavage of peripheral membrane proteins and exposed domains of integral membrane proteins from the membranes.1,12 The results demonstrated that this method had special advantages in membrane protein enrichment and could facilitate the identification of the protein set. In this paper, a two-phase, on-membrane digestion system, which was prepared by mixing n-butanol and 25 mM NH4HCO3, was developed and applied to the on-membrane digestion of rat liver membrane proteins with the aim to improve the identification of membrane proteins. The results show that the method we used has the advantages of simplicity, low cost, and high efficiency, and could remarkably increase the number and coverage of identified membrane proteins, particularly the transmembrane proteins.
Materials and Methods Materials. Trypsin (proteomics sequencing grade), dithiothreitol (DTT), iodoacetamide (IAA), trifluoroacetic acid (TFA), 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), and sucrose were obtained from Sigma-Aldrich (St. Louis, MO). Acrylamide, bisacrylamide, urea, glycine, Tris, and sodium dodecyl sulfate (SDS) were from Amresco (Solon, OH). BioRad DC protein assay kit was from Bio-Rad Laboratories (Hercules, CA). HPLC-grade acetonitrile, acetone, and nbutanol were purchased from Shanghai Chemical Reagent Company of National Medicine Group of China (Shanghai, China). Water was obtained with a Milli-Q Plus purification system (Millipore, Bedford, MA). All other reagents were domestic products of highest grade available. Sprague–Dawley rats (weighting 150–200 g) were from Hunan Academy of Traditional Chinese Medicine (Changsha, China). Preparation of Rat Liver Membrane. Rats were killed after being starved for 24 h, and their liver was excised. After a wash with 0.9% NaCl and removal of the gall bladder and blood vessels, the livers were minced into pieces and homogenized in an ice-cold solution containing 50 mM HEPES (pH 7.4), 1 mM CaCl2, and 0.1 mM phenylmethylsulfonyl fluoride (PMSF). Unbroken cells and debris were removed by centrifugation at 3200g for 15 min at 4 °C. The resulting supernatant was centrifuged at 100 000g for 30 min at 4 °C, and the pellet was collected and washed with the above buffer three times.1,13 Protein concentration of the membrane fraction was determined by Bradford assay. Comparison of Three On-Membrane Digestion Methods. Before digestion, the rat liver membrane fraction was reduced with 10 mM DTT in 25 mM NH4HCO3 at 37 °C for 1 h and alkylated by 55 mM IAA in 25 mM NH4HCO3 in the dark at room temperature for 45 min. For comparison, three aliquots of the treated membrane fraction (150 µg of protein each) were taken and used for on-membrane digestion in a 300-µL twophase system, 150 µL of 60% methanol1,13 and 150 µL of 25 mM NH4HCO314 solution, respectively. The two-phase system was prepared by mixing n-butanol and 25 mM NH4HCO3 at a ratio of 1:1 (v/v). Trypsin was added at a 100:1 protein/enzyme ratio. All the Eppendorf tubes containing reaction mixture were incubated at 37 °C for 24 h, with intermittent gentle vortexing.
technical notes After the digestion was completed, the digested membranes in all the three digestion systems as well as the proteins in the top phase (TP) of the two-phase system were dissolved in sample lysis solution containing 10 mM Tris-HCl, pH 6.8, 20% glycerol, 4% SDS, and 100 mM DTT and analyzed using 12% SDS-PAGE to compare the effectiveness of on-membrane digestion. For comparison of membrane protein identification efficiencies, the 60% methanol, NH4HCO3, bottom- and top-phases of the two-phase system were centrifuged at 10 000g for 10 min and, the supernatants were separately collected for CapLC-MS/ MS analysis. Analysis of Rat Liver Membrane Proteome Using the Two-Phase System. The two-phase, on-membrane digestion together with CapLC-MS/MS was applied to the analysis of rat liver membrane proteome, and experiments were repeated three times. After the digestion in the two-phase system was completed, the tryptic digests were distributed in three regions: top phase (TP), bottom phase (BP), and the residual membranes at phase interface (IP). The three parts were separately collected. The BP fraction was acidified with diluted acetic acid and centrifuged at 10 000g for 15 min, and the supernatant was analyzed by CapLC ion trap mass spectrometry (Bruker Daltonics) without further digestion. Because both of IP and TP contained some remainder proteins with relatively larger molecular weight, the proteins in the two fractions were lysed and prefractionated before being used for CapLC-MS/MS analysis in order to decrease the complexity and increase the efficiency of protein identification. The protein mixtures from IP and TP were, respectively, separated on a nonporous (NPS) C18 reversed-phase column (Beckman Coulter, Fullerton, CA). A 30-min elution gradient with linear increase of 3.33% B/min was used in which solvent A was 0.1% aqueous TFA and solvent B was 0.08% TFA in acetonitrile at a flow rate of 0.75 mL/min. The eluted 10 fractions were collected and then concentrated using a SpeedVac concentrator to 5–10 µL. The proteins were reduced with 10 mM DTT in 25 mM NH4HCO3 at 56 °C for 1 h and alkylated by 55 mM IAA in 25 mM NH4HCO3 in the dark at room temperature for 45 min. Trypsin (0.2 µg) was added to each tube and incubated overnight at 37 °C. After the digestion was stopped by acidification, the tryptic peptide mixture was centrifuged at 10 000g for 15 min and the supernatant was collected for CapLC-MS/MS analysis. Mass Spectrometric Analysis of Tryptic Digests. Tryptic digests prepared as described above were analyzed by CapLC ion trap mass spectrometry (Bruker Daltonics) coupled with an automated Agilent 1200 LC system equipped with an autosampler and a C18 reverse-phase column (PepMap, 180 µm i.d., 15 cm long, LC-Packings). Before separation on the reversephase capillary column, the sample was preconcentrated on a C18 precolumn (500 µm i.d., 3.5 cm long, Bruker). When the sample was separated on the C18 PepMap column, the flow rate was 3 µL/min and the column temperature was set to 25 °C. For the chromatography, the following solvents were used: solvent A (98% H2O, 1.9% acetonitrile, and 0.1% formic acid), solvent B (95% acetonitrile, 4.9% H2O, and 0.1% formic acid). The online LC separation used a gradient from 3% to 30% B in 13 min and then 30% to 80% B in 80 min, followed by 80% B for 10 min, then by 3% B for 8 min. The peptides eluted from the column were online directed into the mass spectrometry. The LC-MS system was controlled using Chemstation B01 (Agilent) and EsquireControlTM 6.1 (Bruker Daltonics) softJournal of Proteome Research • Vol. 7, No. 4, 2008 1779
technical notes wares. The nebulizer pressure was 10 psi. Drying gas flow rate was 5 µL/min. Drying gas temperature was 300 °C. Capillary voltage was 4000 V. The full MS scan mode was standardenhanced (m/z 350-1600 Da). Peptide ions were detected in MS scan, and seven most abundant in each MS scan were selected for collision-induced dissociation (MS/MS), using datadependent MS/MS mode over the m/z range of 100–2000 Da. Data Processing and Bioinformatics. Raw mass spectrometry data were processed, and Mascot compatible mgf files were created using Data AnalysisTM 3.3 software (Bruker Daltonics) with the following parameters: compounds (autoMS) threshold 10 000, number of compounds 300, retention time windows 1.0 min. Searches were performed using Mascot software 2.0 (Matrixscience, London, U.K.), and the international protein index (IPI) rat database was used for protein identification. Search parameters were set as follows: enzyme, trypsin; allowance of up to one missed cleavage; mass tolerance, 1.5 Da and MS/MS mass tolerance, 0.5 Da; fixed modification, carbamoylmethylation (C); variable modification, oxidation (at Met); auto hits allowed (only significant hits were reported); results format as peptide summary report. The theoretical molecular weights of identified proteins were retrieved from Mascot output files. The grand average hydropathy (GRAVY) values for identified proteins and peptides were analyzed using the ProtParam program (http://tw.expasy.org/tools/protparam. html). Mapping of transmembrane (TM) regions for the identified proteins was conducted using the TMHMM 2.0 program based on transmembrane hidden Markov model (http:// www.cbs.dtu.dk/services/TMHMM) by submitting the FASTA files.15 The subcellular localization and function of identified proteins were retrieved by gene ontology (GO) prediction and function annotations, respectively. Text-based annotation files were available for download from GO database ftp site at ftp:// ftp.geneontology.org/pub/go.16
Zhou et al.
Figure 1. Comparison of membrane proteins identified using the solution fractions of the three on-membrane digestion systems. (A) Total number of membrane proteins and transmembrane proteins identified in solution fractions of each digestion method; (B and C) membrane protein distribution as a function of predicted TMDs and GRAVY indexes.
Results Comparison of Three On-Membrane Digestion Methods. To evaluate the effects of membrane protein digestion in the three on-membrane digestion systems, we detected the molecular size of tryptic digests in digested membranes as well as in TP by using 12% SDS/PAGE. The image (Supplementary Figure 1 in Supporting Information) shows that, after onmembrane digestion for 24 h, the IP and TP contained few proteins of greater than about 15 kDa, whereas the membranes that were on-membrane-digested in 60% methanol and 25 mM NH4HCO3 contained much more high-molecular-weight proteins, suggesting that the effectiveness of two-phase, onmembrane digestion is better than that of the other two onmembrane digestion systems under the same experimental conditions. Since when methanol and NH4HCO3 were reportedly used for on-membrane digestion of membrane proteins only the solution fractions were used to identify proteins,1,14 in our experiments, for further comparison of the effects of three onmembrane digestion methods on identification of membrane proteins, the 60% methanol, 25 mM NH4HCO3, TP and BP of the on-membrane digestion systems were centrifuged and the supernatants were analyzed using tandem mass spectrometry coupled with reverse-phase capillary column. The results of membrane protein identification (Figure 1) show that, after the membranes were on-membrane-digested in the two-phase system (n-butanol), methanol, or NH4HCO3, 143, 132, and 77 membrane proteins were identified, including 114 (79.7%), 102 1780
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(77.3%), and 56 (72.7%) transmembrane proteins, respectively (Figure 1A). Figure 1B shows the transmembrane protein distribution as a function of the predicted TMDs. The general protein distribution profiles of the three methods are similar. Most identified transmembrane proteins have 1–2 TMDs. There are transmembrane proteins identified based on the two-phase system in every bin. Compared with the other two methods, two-phase system could lead to identifying more transmembrane proteins with 1–2 and 5–10 TMDs. In addition, the twophase system is also superior to the other two methods in terms of the GRAVY values of identified membrane proteins (Figure 1C). Analysis of Rat Liver Membrane Proteome Using the Two-Phase System. Because the remainder proteins in IP and TP fractions had a few of relatively large molecular weight proteins and these proteins could not be efficiently separated by gel electrophoresis as shown in Supplementary Figure 1 in Supporting Information, we used detergent-assisted method to lyse and extract these proteins. The obtained proteins were separated by NPS-RP-HPLC before being used for in-solution digestion and CapLC-MS/MS analysis. Multidimensional separation at the protein and peptide level prior to LC-MS/MS was helpful to the decrease in the complexity of sample and the increase in efficiency of protein identification. However, the MALDI-TOF mass spectrometric analysis17 of BP fraction indicated that there were not significant amounts of proteins and peptides with molecular weight greater than 4.5 kDa. A
technical notes
Two-Phase On-Membrane Digestion Method
Figure 2. Distribution and overlap of the identified membrane proteins and their matching peptides in three different fractions of the two-phase system. (A) Membrane proteins; (B) peptides from membrane proteins; TP, top phase; BP, bottom phase; IP, residual membranes at phase interface.
majority of the tryptic digests in the fraction were distributed in the range of 1.0–3.0 kDa (Supplementary Figure 2 in Supporting Information), which is compatible with the effective analysis range of tandem mass spectrometry, and can be directly analyzed by the instrument without further digestion. When mass spectrometry data was used to search against the rat IPI protein sequence database, a total of 411 nonredundant membrane proteins were identified in this study (Supplementary Table in Supporting Information). Of these, 103 (25.1%) were identified in TP, BP, and IP; 69 (16.8%), 62 (15.1%), and 52 (12.7%) were identified only in TP, BP, and IP, respectively; the rest were identified in two of the three fractions (Figure 2A and Supplementary Table in Supporting Information). The results demonstrate that, when the membrane proteins were digested in the two-phase, on-membrane digestion system, only about one-fourth of the tryptic digests of membrane proteins was distributed in all the three fractions, and there still were more than 10% of membrane proteins that could be identified only in IP after digestion. To probe the distribution profile of tryptic digests in the twophase, on-membrane digestion system, we made a statistics on the distribution of peptides identified in the three different fractions. In this study, a total of 1203 unique peptides corresponding to 411 membrane proteins were identified. Of these peptides, only 56 (4.7%) were identified in all the three fractions; 123 (10.2%), 408 (33.9%), and 376 (31.3%) were identified only in TP, BP, and IP, respectively. The rest were identified in two of the three fractions (Figure 2B). The lower overlap of peptides identified in these fractions indicates that the peptide distribution in these fractions has complementarity. Therefore, analysis of complete membrane proteome needs comprehensive utilization of the tryptic digests in all fractions of the digestion system. Several physicochemical characteristics of the identified membrane proteins were defined, including theoretical molecular weight, hydrophobicity (expressed as hydropathy (GRAVY) value), and the number of transmembrane domains (TMDs). The molecular weights of the identified membrane proteins are distributed in the range of 6-540 kDa. Of these, 375 (91.2%) are in the range of 10–80 kDa (Figure 3A). The GRAVY values of 376 (91.5%) membrane proteins are in the range of -0.6 to 0.3, and more membrane proteins with GRAVY value of >0.3 were identified from IP and TP than from BP (Figure 3B), suggesting that the proteins remaining in the IP and entering the TP are relatively of high hydrophobicity. TMHMM predication showed that, among the identified 411 membrane proteins, 330 (80.3%) were transmembrane proteins with 1–12 transmembrane domains (TMDs); 208 (63.0%) transmembrane proteins have only one TMD, and the number of transmembrane proteins with one TMD identified from BP was
greater than those from TP and IP. However, more transmembrane proteins with more than three TMDs were identified from TP and IP than from BP, suggesting that utilizing the tryptic digests in TP and IP could identify more hydrophobic proteins with multitransmembrane domain (Figure 3C). As regards the GRAVY values of the peptides, in the range of -2.0 to 0.5, the number of peptides identified from BP was greater than that from IP and TP. In contrast, in the range of >0.5, more peptides were identified from IP and TP than from BP (Figure 3D). These results indicate that IP and TP are more hydrophobic peptides-rich fractions. The identified membrane proteins were categorized on the basis of their GO function annotations (Table 1), though this classification is not strict due to the multiple functions of a protein. The data in Table 1 show that the membrane proteins identified involve a broad spectrum of biological membrane functions, including substance transport, signal transduction, binding, recognition and metabolism, and so on. Of the identified 411 membrane proteins, 211 (51.3%) involve binding and transport; 34 (8.3%) are receptors, channels, and signaling proteins, which are generally considered as low-abundance membrane proteins; 54 (13.1%) are metabolism-related enzymes. Besides, there are 27 (6.6%) proteins being classified into “others”, and 33 (8%) are proteins of unknown function. It is interesting to note that more unique membrane proteins with binding functions could be identified from IP than from TP and BP, but no antigen was identified from IP in the present experiment, which reflected the difference in the resistance of these two kinds of proteins to the on-membrane digestion.
Discussion Effectiveness of the Two-Phase System. To evaluate the effectiveness of the two-phase system in the on-membrane digestion of membrane proteins, we made a comparison of the system with 60% methanol1,13 and 25 mM NH4HCO314 that were reportedly used for on-membrane digestion of membrane proteins. By comprehensive analysis of experimental results from SDS-PAGE and membrane protein identification, we can retrieve some important information. First, during the digestion in the on-membrane digestion systems, a variety of membrane and membrane-associated, as well as membrane-adsorbed, proteins were removed from the membranes or reduced to smaller polypeptides. The digestion efficiency of the two-phase system is superior to those of the other two systems, because the digested membranes from the two-phase system contained much less and smaller proteins and peptides than those from the other two systems under the same experimental conditions. The superiority of the two-phase system was further supported by the fact that the method had led to more membrane and transmembrane proteins to be identified. Second, most remainder proteins in the IP of the two-phase system were concentrated around 5 kDa (Supplementary Figure 1 in Supporting Information, lane IP). In our experiments, we discovered that prolongation of the incubation time could not achieve the complete digestion. These indicated that there was a limit for the on-membrane digestion of membrane proteins under the experimental conditions, which presumably reflected the presence of post-translational modifications such as glycosylation, too tight binding of membrane-associated proteins, and incomplete denaturation of the proteins. The high Journal of Proteome Research • Vol. 7, No. 4, 2008 1781
technical notes
Zhou et al.
Figure 3. Distribution of identified membrane proteins and their matching peptides in the three different fractions of two-phase system as a function of (A) molecular masses, (B) protein hydrophobicity gauged by the GRAVY indexes, (C) predicted TMDs, and (D) the hydrophobicity of peptides detected in LC-MS/MS. Table 1. Functional Classification of the Unique Membrane Proteins Identified from the Three Fractions of Two-Phase System parameter
TPa
BPb
IPc
total
Binding Transporter Channel Receptor Signal transduction Metabolism Antigen Others Unknown Total
78 46 9 7 4 39 34 14 15 246
84 42 8 9 7 37 28 22 14 251
101 49 9 3 2 43 0 18 20 245
133 78 10 13 11 54 52 27 33 411
a
Top phase.
b
Bottom phase. c Interface membrane.
degree of glycosylation and the globular nature of extracellular domains may mask many susceptible sites for cleavage by trypsin.18 Third, there were a certain number of remainder proteins having entered the TP of the two-phase system (Supplementary Figure 1 in Supporting Information, lane TP). These proteins had molecular weight slightly higher than that of the remainder proteins in IP, which indicted that the proteins entering the TP had also undergone the digestion, though this digestion was not complete. n-Butanol, like other organic solvents such as methanol,1,14 could not completely solubilize the membranes, but could destabilize the membranes and extract proteins from the affected membranes. Furthermore, trypsin could digest the extracted membrane proteins in the water and NH4HCO3containing n-butanol, although the enzymatic activity in organic solvents was generally in the range of 20-40% of that in aqueous solutions.19 In addition, the tryptic peptides that entered the BP had a size smaller than 4.5 kDa, with the most peptides in the range of 1.0–3.0 kDa, which was compatible with the effective analysis range of mass spectrometry and can be directly analyzed by 1782
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the instrument (Supplementary Figure 2 in Supporting Information) without further digestion. This suggests that, containing small amounts of n-butanol, the water-rich BP fraction is favorable to the further digestion of proteins and larger peptides entering the phase. Comprehensive Analysis of the Membrane Proteome. Membrane proteins carry out many essential cellular functions. Many efforts have been made by researchers to obtain a complete profile of membrane proteome, including the use of on-membrane digestion, to improve the analysis of membrane proteins, in particular integral membrane proteins, Blonder et al.1 developed a membrane sample preparation method combining carbonate extraction, surfactant-free organic solventassisted solubilization, and on-membrane proteolysis in the presence of 60% methanol. Niesen et al.12 depleted nonmembrane molecules from entire tissue homogenate by high-salt, carbonate, and urea washes, followed by treatment of the membranes with sublytic concentrations of digitonin and protein digested on-membrane by endoproteinase Lys-C. Fischer et al.14 described a modified membrane and integral membrane proteins enrichment and cleavage procedure in their attempt toward the complete membrane proteome. The results demonstrated that the membrane sample preparation methods mentioned above indeed improved the enrichment and identification of membrane proteins including integral membrane proteins. However, their techniques involved multiple rinsing and/or predigestion steps, which unavoidably led to the loss of some membrane proteins and peptides. Furthermore, after extraction with organic solvent and on-membrane digestion, the remainder membrane fragments were removed and discarded, leading to the loss of another part of membrane proteins. Our results indicated that, after on-membrane digestion, there were more than 10% membrane proteins that could be identified only in IP, and the distribution profiles of tryptic digests in the three fractions were complementary to one another. Therefore, for insight into the complete membrane
technical notes
Two-Phase On-Membrane Digestion Method proteome, the comprehensive analysis of tryptic digests in all the fractions was necessary. Besides a certain number of cytosol or membrane-associated proteins, the analysis of combined data using our protocol yielded 411 membrane proteins, 80.3% of which were classified as integral membrane proteins with 1–12 transmembrane domains (TMDs). However, because some integral membrane proteins lacking putative R-helices can be predicted to be anchored to the membranes as a result of post-translational modification such as myristoylation and prenylation, the percentage of integral membrane proteins obtained using our procedure should be greater than 80.3%. The superiority of our protocol is mainly due to the effectiveness of our newly developed two-phase, on-membrane digestion method. The extraction and dispersion actions exerted by the n-butanolrich TP and the water-rich BP efficiently improved the digestion and removal of the proteins absorbed on the membranes. n-Butanol was able to solubilize some hydrophobic proteins and induce depolymerization of microtubules in vivo and in vitro.20,21 On the other hand, during the two-phase, onmembrane digestion, the larger hydrophobic proteins removed from the membranes predominantly entered the n-butanolrich TP containing small mounts of water and NH4HCO3, where they were further digested by trypsin. The smaller peptides produced in TP and IP were continuously extracted into the water-rich BP due to their increased hydrophilicity, where the peptides were, if necessary, further digested. Although there were certain amounts of reminder proteins in IP and TP that had relatively high molecular weight, we used detergentcontaining lysis buffer to solubilize these proteins, followed by prefractionation and in-solution digestion before LC-MS/MS analysis, thereby decreasing the loss of membrane proteins to the smallest extent.
Conclusions After equilibrium, the on-membrane digestion system consisting of n-butanol and 25 mM NH4HCO3 was separated into two phases: n-butanol-rich TP containing small amounts of water and NH4HCO3, water-rich BP containing small amounts of n-butanol and NH4HCO3. The remainder membranes were distributed at phase interface. During the digestion, the proteins, including the absorbed nonmembrane proteins, membrane-associated proteins, and bona fide membrane proteins, as well as their tryptic digests, were affected not only by proteolysis, but also by extraction and dissolution of the two phases, which in turn improved the digestion further. The multiple actions of the two-phase, on-membrane digestion system made its digestion effectiveness better than that of the reported aqueous- and organic-aqueous solvent systems. After digestion, the tryptic digests were not evenly distributed, and therefore, only a comprehensive analysis of the digests in all fractions could achieve a higher coverage of the membrane proteome. The protein identification results support the high effectiveness of our method in removing undesirable proteins and enriching membrane proteins, particularly integral membrane proteins. The method we newly developed has the advantages of simplicity, low cost, and high efficiency and is
suitable for the analysis of complete membrane proteome of various sources.
Acknowledgment. This work was supported by grants from Special Program for Key Basic Research of the Ministry of Science (2004CCA00300), Technology of China and National 973 Projects of China (2001CB5102), and National Natural Science Foundation of China (30430170, 90408017). Supporting Information Available: Supplementary Figure 1 shows the evaluation and comparison of the onmembrane digestion effectiveness by SDS-PAGE of remainder proteins in membranes after digestion in two-phase system, 60% methanol, and 25 mM NH4HCO3, respectively. Supplementary Figure 2 shows the MALDI-TOF mass spectrum of the bottom phase fraction (BP) of the two-phase, on-membrane digestion system. Supplementary Table shows the information on the membrane proteins identified based on the two-phase system. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Blonder, J.; Goshe, M. B.; Moore, R. J.; Pasa-Tolic, L.; Masselon, C. D.; Lipton, M. S.; Smith, R. D. J. Proteome Res. 2002, 1, 351– 360. (2) Josic, D.; Brown, M. K.; Huang, F.; Callanan, H.; Rucevic, M.; Nicoletti, A.; Clifton, J.; Hixson, D. C. Electrophoresis 2005, 26, 2809–2822. (3) Dreger, M. Mass Spectrom. Rev. 2003, 22, 27–56. (4) Peltier, J. B.; Ytterberg, A. J.; Su, Q.; van Wijk, K. J. J. Biol. Chem. 2004, 279, 49367–49383. (5) Fountoulakis, M.; Gasser, R. Amino Acids 2003, 24, 19–41. (6) Navarre, C.; Degand, H.; Bennett, K. L.; Crawford, J. S.; Mortz, E.; Boutry, M. Proteomics 2002, 2, 1706–1714. (7) McCarthy, F. M.; Burgess, S. C.; van den Berg, B. H. J.; Koter, M. D.; Pharr, G. T. J. Proteome Res. 2005, 4, 316–324. (8) Morré, D. M.; Morre, D. J. J. Chromatogr., B 2000, 743, 377–387. (9) Schindler, J.; Lewandrowski, U.; Sickmann, A.; Friauf, E.; Nothwang, H. G. Mol. Cell. Proteomics 2006, 5, 390–400. (10) Marmagne, A.; Rouet, M. A.; Ferro, M.; Rolland, N.; Alconm, C.; Joyard, J.; Garin, J.; Barbier-Brygoo, H.; Ephritikhine, G. Mol. Cell. Proteomics 2004, 3, 675–691. (11) Bartee, E.; McCormack, A.; Früh, K. PLoS Pathogens 2006, 2, 975– 988. (12) Niesen, P. A.; Olsen, J. V.; Podtelejnikov, A. V.; Andersen, J. R.; Mann, M.; Wiœniewski, J. R. Mol. Cell. Proteomics 2005, 4, 402– 408. (13) Ruth, M. C.; Old, W. M.; Emrick, M. A.; Meyer-Arendt, K.; AvelineWolf, L. D.; Pierce, K. G.; Mendoza, A. M.; Sevinsky, J. R.; Hamady, M.; Knight, R. D.; Resing, K. A.; Ahn, N. G. J. Proteome Res. 2006, 5, 709–719. (14) Fischer, F.; Wolters, D.; Rögner, M.; Poetsch, A. Mol. Cell. Proteomics 2006, 5, 444–453. (15) Krogh, A.; Larsson, B.; von Heijne, G.; Sonnhammer, E. L. J. Mol. Biol. 2001, 305, 567–580. (16) Ashburner, M.; Ball, C. A.; Blake, J. A.; Botstein, D.; Butler, H.; Cherry, J. M.; Davis, A. P.; Dolinski, K.; Dwight, S. S.; Eppig, J. T. Nat. Genet. 2000, 25, 25–29. (17) Zhang, L. J.; Wang, X. E.; Peng, X.; Wei, Y. J.; Cao, R.; Liu, Z.; Xiong, J. X.; Yin, X. F.; Ping, C.; Liang, S. Cell. Mol. Life Sci. 2006, 63, 1790– 1804. (18) Bledi, Y.; Inberg, A.; Linial, M. Briefings Funct. Genomics Proteomics 2003, 2, 254–265. (19) Zaks, A.; Klibanov, A. M. J. Biol. Chem. 1988, 263, 8017–8021. (20) Bai, H. X.; Yang, F.; Yang, X. R. J. Proteome Res. 2006, 5, 840–845. (21) Hirase, A.; Hamada, T.; Itoh, T. J.; Shimmen, T.; Sonobe, S. Plant Cell Physiol. 2006, 47, 1004–1009.
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