HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Supplemental Data All Versions of this Article: T500036-MCP200v1 5/12/2392    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Glossary Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by McDonald, T. Articles by Van Eyk, J. E. Search for Related Content PubMed PubMed Citation Articles by McDonald, T. Articles by Van Eyk, J. E. Social Bookmarking                      What's this?

Molecular & Cellular Proteomics 5:2392-2411, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

---

Technology

Expanding the Subproteome of the Inner Mitochondria Using Protein Separation Technologies

One- and Two-dimensional Liquid Chromatography and Two-dimensional Gel Electrophoresis^*,S

Todd McDonald^,,¶, Simon Sheng^,¶, Brian Stanley^,,¶, Dawn Chen^||,**, Young Ko, Robert N. Cole^||,**, Peter Pedersen^|| and Jennifer E. Van Eyk^,,||,,

From the Departments of Medicine, ^|| Biological Chemistry, and Biomedical Engineering and ^** The Technical Implementation and Coordination Core of The Johns Hopkins NHLBI Proteomics Center, The Johns Hopkins University, Baltimore, Maryland 21224 and Department of Physiology, Queen’s University, Kingston, Ontario K7L 3N6, Canada

	ABSTRACT

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Currently no single proteomics technology has sufficient analytical power to allow for the detection of an entire proteome of an organelle, cell, or tissue. One approach that can be used to expand proteome coverage is the use of multiple separation technologies especially if there is minimal overlap in the proteins observed by the different methods. Using the inner mitochondrial membrane subproteome as a model proteome, we compared for the first time the ability of three protein separation methods (two-dimensional liquid chromatography using the ProteomeLab^TM PF 2D Protein Fractionation System from Beckman Coulter, one-dimensional reversed phase high performance liquid chromatography, and two-dimensional gel electrophoresis) to determine the relative overlap in protein separation for these technologies. Data from these different methods indicated that a strikingly low number of proteins overlapped with less than 24% of proteins common between any two technologies and only 7% common among all three methods. Utilizing the three technologies allowed the creation of a composite database totaling 348 non-redundant proteins. 82% of these proteins had not been observed previously in proteomics studies of this subproteome, whereas 44% had not been identified in proteomics studies of intact mitochondria. Each protein separation method was found to successfully resolve a unique subset of proteins with the liquid chromatography methods being more suited for the analysis of transmembrane domain proteins and novel protein discovery. We also demonstrated that both the one- and two-dimensional LC allowed for the separation of the -subunit of F₁F₀ ATP synthase that differed due to a change in pI or hydrophobicity.

2-DE, a classical proteomics technology that separates proteins based on their pI and molecular weight, has a practical dynamic range of 10⁴ orders of magnitude (for reviews, see Refs. 3 and 4). This restricts the analysis of a proteome to the most abundant proteins, and so it often underrepresents proteins with extreme hydrophobicity, mass, or isoelectric point. Another common separation method is 1-DLC, which separates proteins based on hydrophobicity. In proteomics, 1-DLC has primarily been used for peptide separation prior to MS, but it can be used for protein separation prior to enzymatic digestion and analysis by MS (for a review, see Ref. 5; e.g. see Refs. 6 and 7). 2-DLC traditionally couples a charge-based method (e.g. isoelectric focusing or strong cation exchange) as a first dimension with RP-HPLC as the second dimension thereby increasing the extent of protein fractionation compared with 1-DLC. As with 1-DLC, this method has been used primarily in proteomics for peptide separation; however, it is increasingly being applied to the separation of complex intact protein mixtures (for a review, see Ref. 5; e.g. see Refs. 8–10). This increased use is (in part) due to the commercialization of 2-DLC systems, including the PF2D (Beckman Coulter), which is based upon the system developed by Lubman and colleagues (e.g. Refs. 11–14). The PF2D uses chromatofocusing in the first dimension (separating proteins based on their pI) and reversed phase chromatography in the second dimension. Except for a single report utilizing chromatographic isoelectric focusing (first dimension) to separate peptides prior to a multidimensional protein identification technology experiment (15) the PF2D has been used exclusively for protein separation. To date, the only comparison of the PF2D with any other protein separation technology examined the rice proteome through a limited comparison between the PF2D and 2-DE (16). Thus, both the scope of proteome coverage by PF2D alone and its synergy with other proteins separation method is not clear.

Mitochondria generate the majority of ATP in the cell, and their dysfunction has been implicated in many different diseases. Mitochondria have both an outer and an inner membrane structure with the components of the oxidative phosphorylation pathway located in (or associated with) the inner mitochondrial membrane (IMM). To understand these diseases, a determination of the members of this subproteome (including post-translational modifications (PTMs)) is of great interest (17–19). Although intact mitochondria have been studied using different proteomics technologies (20–26, 28–30), these databases comprise only part of the estimated 697–4532 total mitochondrial proteins (31). However, because these estimations can have a false discovery rate of up to 68% (31), the absolute number of mitochondrial proteins is not currently known. A problem with the existing mitochondrial databases derived from proteomics analysis has been the bias toward proteins localized to the matrix and outer membrane and the lack of IMM-associated proteins (21, 25). To increase the coverage of the IMM subproteome, Da Cruz et al. (29, 30) used an enriched IMM preparation and demonstrated that there are novel proteins within this subproteome. Using the same well characterized IMM preparation (29–32) we tested the hypothesis that there would be a minimal overlap of observed proteins when using three different separation technologies (2-DE, 1-DLC, and 2-DLC) thereby expanding proteome coverage.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
IMM Preparation—
Mitochondria were isolated from frozen rat liver, and the IMM subproteome was isolated according to the protocol of Pedersen et al. (32). Purity was assessed as described previously (30). For 2-DE, the IMM proteins were solubilized by incubation in either 5% (w/v) CHAPS, 5% (w/v) N-decyl-N,N'-dimethyl-3-ammonio-1-propanesulfonate, or 5% (w/v) amidosulfobetaine-14 in double distilled H₂O for 15 min at room temperature prior to the addition IEF buffer (8 M urea, 2 M thiourea, 4% detergent, 1% (w/v) DTT, and 0.25% (v/v) carrier ampholytes). For 2-DLC, the IMM proteins were solubilized in 1% (w/v) SDS followed by precipitation with ice-cold acetone and resuspended in 2 ml of PF2D chromatofocusing start buffer (Beckman Coulter, Carlsbad, CA). For 1-DLC the IMM subproteome was solubilized in 0.1% (v/v) trifluoroacetic acid with 20% (v/v) acetonitrile, pH 2.4.

2-DE Analysis—
The IMM subproteome (200–750 µg) was resolved on IPG Ready Strips (17 cm, pH 4–7 or 3–10 linear gradient ( BioRad)). Strips were actively rehydrated with solubilized protein in 350 µl of IEF buffer at 50 V for 10 h, and then a rapid voltage ramping method was applied as follows: 100 V for 1 h, 500 V for 1 h, 1000 V for 1 h, linear gradient to 10,000 V over 1 h, and finally 10,000 V for 40 kV-h using a Protean IEF cell (Bio-Rad). To separate proteins with basic pI values, the sample was cup-loaded into IPG Dry Strips (18 cm, pH 6–11, GE Healthcare) in 100 µl of IEF buffer as follows: 150 V for 8 h, 500 V for 1 h, 1000 V for 1 h, linear ramping to 10,000 V for 30 min, and finally 10,000 V for 30 kV-h over 10 h. A Peltier temperature control platform maintained gels at 20 °C. Focused IPG strips were stored at –80 °C until SDS-PAGE at which time they were thawed to room temperature and incubated for 15 min in equilibration buffer (50 mM Tris-HCl, pH 8.8, 6 M urea, 30% (w/v) glycerol, 2% (w/v) SDS) with 1% (w/v) DTT followed by 15 min in equilibration buffer with 2% (w/v) iodoacetamide (reduction/alkylation). IPG strips were embedded in a 5% acrylamide stacking gel, and proteins were resolved by 8, 10, 12, or 15 SDS-PAGE (Protean II XL, 200 x 220 x 1 mm, Bio-Rad). For 1-D SDS-PAGE sample buffer containing protein was boiled for 10 min in 3x SDS Sample Buffer (New England Biolabs, Beverly, MA) prior to loading into 5% acrylamide stacking gel and resolved by 6, 12, or 15% acrylamide. 2-DE gels were silver-stained according to Shevchenko et al. (33). Stained gels were scanned with a Powerlook II scanner (UMAX Data Systems, Fremont, CA) on a Sun Ultra5 computer (Sun Microsystems, Palo Alto, CA). Gels were vacuum-dried between cellophane sheets until protein spots/bands were manually excised.

2-DLC (PF2D) Analysis—
2-DLC analysis of solubilized IMM proteins (3 mg) was carried out on a PF2D (Beckman Coulter) (12). The first dimension separated proteins on the basis of their pI using a pH gradient generated on the column (see below). Fractions from the first dimension were collected (FC/I Module) and sequentially injected onto the second dimension RP-HPLC column (see below). The fractions were collected into 96-deepwell plates for subsequent digestion with trypsin and mass spectrometry analysis (see below). With the PF2D, the first and second dimensions occur sequentially in an automatic manner.

Chromatofocusing—
Proteins were separated in the first dimension on a chromatofocusing (CF) column by mixing two buffers differing in their pH, start buffer (pH 8.5) and eluent buffer (pH 4.0), to create a linear pH gradient from pH 8.5 to 4.0 (see Fig. 2A). Initially the CF column was equilibrated for 130 min with start buffer at a flow rate of 0.2 ml/min. 3 mg of solubilized IMM protein sample was injected onto the equilibrated column. After a stable base line was established (20 min), the pH gradient was started by introducing the eluent buffer (flow rate, 0.2 ml/min) for 75 min. Finally the column was washed with 1 M NaCl. Fractions were collected at 0.3 pH intervals during the pH gradient portion of the run and every 5 min before and after.

View larger version (31K): [in this window] [in a new window]   FIG. 2. 2-DLC separation of rat liver inner mitochondrial membrane proteins. 3.0 mg of liver inner mitochondrial membrane proteins was separated in the first dimension using chromatofocusing (CF-LC) with proteins with pI values greater than pH 8.5 eluting in the void volume followed by a descending pH gradient from pH 8.5 to 4.0 and finally in a salt wash for pI values less than pH 4.0 (A, left). The dotted line represents the change in pH from 8.5 to 4.0. CF fractions with a pH range of 0.3 units from the first dimension were collected individually and subsequently separated using RP-HPLC in the second dimension (A, right). Each RP-HPLC fraction was distinct and unique (for examples, see B). The arrows indicate the fraction where the -isoform of F1F0 ATP synthase was present (retention times, 16.4 and 18.8 min). The 2-DLC composite map of the intensity of the protein profiles from the second dimension RP-HPLC elutions across all pH ranges is shown in C. Each RP-HPLC fraction was processed prior to ESI MS/MS for protein identification; many proteins were identified that were not identified using either 2-DE or RP-HPLC alone. For more details see Table I and Table S1.

View larger version (17K): [in this window] [in a new window]   FIG. 4. Reversed phase high performance liquid chromatography. 100 µg of inner mitochondrial membrane proteins solubilized in 2% TFA, 20% acetonitrile was resolved by reversed phase high performance liquid chromatography using a linear gradient of 0–100% acetonitrile over 30 min (absorbance, solid line; gradient, dotted line). 38 fractions were processed and analyzed by ESI MS/MS for protein identification; many proteins were identified that were not identified using either 2-DE or 2-DLC. For more details see Table I. The arrows indicate the fraction where the -subunit of F1F0 ATP synthase was present (retention times, 16.4 and 18.8 min).

Protein Identification to Create a Non-redundant Protein Database—
Protein identification by peptide mass fingerprinting was conducted with the database search tool MS-Fit in the program ProteinProspector (41). Mass tolerance was limited to 25 ppm after internal calibration to trypsin mass peaks. Identifications required a minimum of five mass peaks corresponding to the major peaks in the spectra, a minimum MOWSE score of 10⁵ with no other scores for different proteins within two orders and no other mass peaks above 50% intensity that could not be attributed to the identified protein or known contaminants, and greater than 40% sequence coverage. Proteins identified with amino acid sequences obtained from ESI MS/MS had a minimum of two peptide matches (see on-line Supplemental Fig. S1 for representative MS spectra) with a minimum MASCOT score of 40 for each peptide. When protein identification was made with two peptide matches, the fragments had to be unique to the protein (and not matched to another other potential identification (i.e. two non-redundant peptides). Further stringency was added by eliminating any peptide that could be assigned to more than one protein. To create a non-redundant database, protein identifications were manually examined in the database for possible redundancies including multiple names and homologies because numerous instances were found where the same protein was contained in multiple database protein identifications. Redundancy was eliminated through the use of a command line version of BLAST (blastclust; ftp.ncbi.nih.gov) that clustered accession numbers according to a 90% protein sequence similarity over 90% of their length. Those proteins that matched these criteria were considered to be the same protein. An estimate of hydrophobicity was calculated from a GRAVY. This score along with the theoretical pI and mass were obtained using the proteomics package in BioJava (biojava.org/wiki/Main_Page) on the intact (non-processed) amino acid sequence. In an attempt to estimate the effect of mitochondrial signal sequence, the theoretical pI, molecular weight, and GRAVY score for mitochondrial signal sequence from 20 well characterized mitochondrial proteins were calculated (see Supplemental Table S1). The average is reported as a footnote in Table I. The prediction of transmembrane domains was carried using the dense alignment surface transmembrane prediction server (35). The extent of amino acid sequence homologies for all unknown or similar proteins was determined by BLAST (us.expasy.org/tools/blast/), and if identity was over 95% homology at amino acid sequence it was considered to be the same protein (see Supplemental Table S1 for MS spectra characterization). If BLAST search could not find a similar protein, the protein was left as undefined or theoretical.

Western Blotting—
For 1-D analysis, 200 µl of LC fractions was evaporated to dryness in a Speed Vac, and proteins were solubilized in LDS sample buffer (Invitrogen) and resolved by 1-D SDS-PAGE using NuPAGE® Novex Bis-Tris gels with MES running buffer (Invitrogen). For 2-DE analysis, 200-µl fractions were evaporated to dryness in a Speed Vac, and proteins were solubilized in IEF buffer and resolved in a Protean IEF cell (Bio-Rad). IPG Dry Strips (7 cm, pH 3–10, GE Healthcare) were actively rehydrated with solubilized protein in 115 µl of IEF buffer at 50 V for 10 h, and then a rapid voltage ramping method was applied as follows: 100 V for 1 h, 500 V for 1 h, 1000 V for 1 h, linear gradient to 5000 V over 1 h, and finally 5000 V for 10 kV-h. The second dimension was performed using NuPAGE Novex Bis-Tris gels with MES running buffer (Invitrogen). Gels were transferred to a PVDF membrane for Western blotting using a wet transfer apparatus (Bio-Rad) in NuPAGE Transfer Buffer (Invitrogen) at 100 V for 45 h at 4 °C. Western blot analysis was carried out using a mouse monoclonal antibody specific to the -subunit of F₁F₀ ATP synthase according to the manufacturer’s protocol (clone 7H10, Molecular Probes-Invitrogen). The primary antibody was detected with rabbit anti-mouse antibody conjugated to alkaline phosphatase (Jackson Immunoresearch Laboratories) and CDP-Star chemiluminescence reagent (PerkinElmer Life Sciences-Mandel Scientific).

	RESULTS AND DISCUSSION

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
We had previously shown that preincubation with the IEF-compatible zwitterionic detergent N-decyl-N,N'-dimethyl-3-ammonio-1-propanesulfonate for 10 min at room temperature prior to the addition of standard IPG buffer (see "Experimental Procedures") improved isoelectric focusing, reduced horizontal streaking, and increased the number of distinct protein spots visualized in 2-DE from less than 30 to 145 using the same broad range conditions (pH 3–10, 12% SDS-PAGE) (Ref. 24 and Fig. 1A). Furthermore extending the pH gradient over a larger distance by using 18-cm pH 4–7 IPG (Fig. 1B), pH 6–11 IPG (Fig. 1C), and 12% SDS-PAGE (or 6 and 15% SDS-PAGE, data not shown) to collectively expand the proteome allowed for the visualization of over 200 protein spots. Of these, 115 spots were identified corresponding to 77 non-redundant proteins (Table I). 20 proteins including the -subunit of F₁F₀ ATP synthase (NCBI accession number 114523; Figs. 1 and 3) were visualized as multiple spots by 2-DE due to modifications that altered their pI (Table I, see *).

View larger version (77K): [in this window] [in a new window]   FIG. 1. 2-DE analysis of rat liver inner mitochondrial membrane separated using various pH gradients. A–C, rat inner mitochondrial membrane proteins separated using pH 3–10 IPG followed by 12% SDS-PAGE (A), pH 4–7 IPGs/12% SDS-PAGE (B), and pH 6–11 IPG/12% SDS-PAGE (C). Proteins were annotated using NCBI accession number (see Table I for protein identification). For more details see "Experimental Procedures." Insets show enlargements of two regions (1 and 2) of the gels illustrating improved spot resolution with narrow pH gradient gels. This allowed the identification of two forms of ubiquinol-cytochrome-c reductase complex core protein I (inset 1, accession number 92090651) and four forms of the -subunit of F1F0 ATP synthase (inset 2, accession number 114523).

View larger version (26K): [in this window] [in a new window]   FIG. 3. Western blotting for the -subunit of F1F0 ATP synthase reveals PTM. The -subunit of ATP synthase was identified in two RP-HPLC fractions (at retention times 18.8 and 16.4 min ('')) by ESI MS/MS. Western blotting of these fractions with monoclonal antibody specific to the -subunit revealed a 59-kDa and a 39-kDa form (A). The small GRAVY score difference between these two forms was resolved by RP-HPLC (–0.138 and –0.144 for 59 and 39 kDa, respectively). The intensity of the signal suggests the smaller form was present in the inner membrane at significant levels. B, Western blotting of inner mitochondrial membrane proteins resolved by 2-DE revealed both the smaller and larger form but also confirmed the presence of a modification to the larger form that altered its pI, a PTM that was not present in the smaller form.

A total of 146 non-redundant proteins was identified by 2-DLC (Table I) through ESI MS/MS analysis of a total of 106 fractions. 18 proteins were identified in more than two non-sequential fractions (in either dimension) suggesting that these proteins may have a potential PTM (40). The -subunit of F₁F₀ ATP synthase separated into two second dimension fractions suggesting it also has a modification that alters its hydrophobicity (Fig. 2B). These fractions were analyzed by 1-D SDS-PAGE followed by Western blot for the -subunit. This showed that the full-length -subunit from F₁F₀ ATP synthase of 59 kDa eluted in 18.8 min, whereas a 38-kDa lower molecular mass form eluted in a fraction at 16.4 min (Fig. 3). Although the 38-kDa form was not identified in the initial silver-stained 2-DE analysis (due to its low abundance), its presence was confirmed by 2-DE Western blot (Fig. 3B). Because the theoretical pI of the -subunit is greater than 8.5, the multiple pI forms observed by 2-DE eluted in the void volume (pH > 8) of the first dimension of the PF2D. The 2-DLC was also useful in separating distinct protein isoforms. For example, acetyl-coenzyme A acetyltransferase also eluted in the void volume of the first dimension but eluted from the second dimension at different retention times (16.3 and 18.4 min). ESI MS/MS analysis showed that the less hydrophobic fraction contained isoform 1 of acetyl-coenzyme A acetyltransferase based upon the identification of three unique amino acid sequences (Supplemental Fig. S2). The later eluting fractions contained both protein isoforms (1 and 2) of acetyl-coenzyme A acetyltransferase based upon the observation of unique amino acid sequences (Supplemental Fig. S1). The different retention times for isoform 1 suggest that there is a yet uncharacterized hydrophobic modification.

For 1-DLC, the IMM proteins were solubilized in 2% TFA with 20% acetonitrile (pH 2.3) and resolved by reversed phase chromatography using a linear gradient from 0 to 100% B over 30 min (Fig. 4). ESI MS/MS analysis of 38 fractions identified 230 non-redundant proteins with each fraction containing between one and 30 proteins (Table I). The -subunit of F₁F₀ ATP synthase eluted in reversed phase fractions with retention times equivalent to those observed by 2-DLC (Fig. 4, arrows).

Comparison of the Protein Separation Technologies—
The combined IMM subproteome database obtained from the three protein separation technologies consisted of a total of 348 non-redundant parent proteins (Table I). There was little overlap (7%) between the proteins observed by the three different methods and only 24% overlap between any two methods. 21% of the total proteins were identified only by 2-DLC, and 47% were identified only by 1-DLC even though pI, mass, and GRAVY score calculations were similar (Fig. 5). This in-depth comparison highlights the advantages and limitation of the three protein separation methods. Even with optimization of solubilization and running conditions, 2-DE is limited with respect to proteins with extreme mass, hydrophobicity, and pI, although it has the advantage of distinguishing multiple forms of proteins with differences in molecular mass or isoelectric point (Table I). Conversely the 2-DLC and 1-DLC have the advantage of enriching for low mass proteins below 30 kDa and basic proteins with pI values above 8.5/9.0. With the PF2D the basic proteins are concentrated in the void volume from the first dimension so individual pI information is lost for this class of protein. To date, the majority of studies using the PF2D (or similar LC system) have not analyzed this basic fraction. Because the PF2D requires an approximate 10-fold higher protein load compared with the other two methods (to track profiles by absorbance) one would expect to be able to detect lower abundance protein leading to greater proteome coverage. Also despite 2-DLC and 1-DLC sharing the same step (RP-HPLC), the overlap in protein identifications was surprisingly small (9%). This difference may be due to different solubilization buffers used in preparation for each method. For instance, 2-DLC required a pH 8.5 aqueous buffer, whereas RP-HPLC contained 2% TFA (pH 2.3) as well as an organic solvent. Membrane proteins are notoriously difficult to solubilize in aqueous buffers (27), and the addition of an organic solvent to the start buffer for 1-DLC may have increased the identification of certain classes of proteins. For instance, translocase of the inner mitochondrial membrane (TIM), solute carrier, and cytochrome P450 family members are predicted (35) to have multiple transmembrane domains (ranging from one to seven) (data not shown), which would limit their solubility. These proteins possess a range of GRAVY scores (–0.53 to 0.09), yet GRAVY scores represent an average of hydrophobicity. Highly hydrophobic or hydrophilic protein domains affecting retention may be underrepresented by presenting the data in this manner. The detection of this unique class of proteins is advantageous for experiments in which the goal is to determine which proteins are present; however, for quantitative studies 1-DLC is of limited use due to its reduced ability to resolve individual proteins compared with 2-DLC. Additionally both LC methods would most likely require additional downstream methods for quantitative analysis. In this respect, 2-DLC has the advantage because image analysis of the elution profiles can limit the number of fractions requiring further analysis.

View larger version (35K): [in this window] [in a new window]   FIG. 5. Distribution of the biochemical properties of proteins identified with 2-DE, 2-DLC, or 1-DLC. A shows the molecular mass distribution over 10-kDa increments of the proteins identified by 2-DE, 2-DLC, or 1-DLC. B shows the pI of the proteins identifications (from Table I) over increments of 0.5 units of the proteins separated by either 2-DE (black bars), 1-DLC (dashed bars), or 2-DLC (gray bars). Identifications following 2-DE revealed clustering of protein pI values between 5.0–6.5 and 7.5–9.0, whereas proteins identified following 2-DLC and 1-DLC tended to be shifted toward higher pI values. C shows the hydrophobicity distribution of identified proteins separated by 2-DE, 1-DLC, or 2-DLC. Hydrophobic proteins with positive GRAVY scores were identified using the three protein separation methods, although there is a slight increase with 1-DLC and 2-DLC. Note that the theoretical values were calculated on the intact non-processed amino acid sequence. The mitochondrial signal sequence from 20 known mitochondrial proteins would have contributed an average mass, pI, and GRAVY score of 3.4 ± 0.8 kDa, 0.85 ± 0.58 pI, and –0.03 ± 0.08 units, respectively (see "Experimental Procedures" and Supplemental Table S2 for more details). D shows the overlap of proteins identified by each technology. Only 7% of identifications were common to all three, and significant portions were observed by only a single technology.

Conclusion—
The data show a low degree of overlap between the protein separation methods described and very significantly extended our coverage of the IMM proteome by using an integrative approach. Furthermore using the PF2D, a 2-DLC system, we were able to identify a unique subset of proteins not observed by 2-DE or 1-DLC. This may be due to difference in starting protein quantity (3 mg for the PF2D to 100 µg for 1-DLC). 1-DLC was able to resolve a unique set of proteins with highly hydrophobic domains. Also the majority of the proteins not observed by previous databases were only observed using the 1-DLC. Overall the results show the power of integrating different separation technologies and also suggest caution against making the assumption that one can validate findings using different separation methods.

	ACKNOWLEDGMENTS

 
We acknowledge the use of the mass spectrometers at The Johns Hopkins Technical Implementation and Coordination Core proteomics core. Finally we acknowledge David Graham for critical review and editing of the manuscript.

	FOOTNOTES

 
Received, November 3, 2005, and in revised form, August 15, 2006.

Published, MCP Papers in Press, September 25, 2006, DOI 10.1074/mcp.T500036-MCP200

¹ The abbreviations used are: 2-DLC, two-dimensional LC; IMM, inner mitochondrial membrane; PF2D, ProteomeLab^TM PF 2D Protein Fractionation System (Beckman Coulter); 1-DLC, one-dimensional reversed phase HPLC; 2-DE, two-dimensional gel electrophoresis; RP, reversed phase; CF, chromatofocusing; PTM, post-translational modification; 1-D, one-dimensional; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.

^* This work was supported by the Canadian Foundation for Medical Research (to J. E. V. E.), NHLBI, National Institutes of Health, Proteomics Initiative Contract N0-HV-28180) (to J. E. V. E. and R. N. C.), National Institutes of Health Grant CA10951 (to P. P.), and the Donald P. Amos Family Foundation (to J. E. V. E.). The Johns Hopkins Technical Implementation and Coordination Core proteomics core is supported by the NHLBI, National Institutes of Health, Proteomics Initiative and The Johns Hopkins Institute for Cell Engineering.

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

^¶ These authors made equal contributions to this work.

To whom correspondence should be addressed: Dept. of Medicine, The Johns Hopkins University, 602 Mason F. Lord Bldg., center tower, 5200 Eastern Ave., Baltimore, MD 21224. Tel.: 410-550-8510; Fax: 410-550-8512; E-mail: jvaneyk1{at}jhmi.edu

	REFERENCES

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 

1. Roberts, G.C., and Smith, C. W. (2002) Alternative splicing: combinatorial output from the genome. Curr. Opin. Chem. Biol. 6, 375 –383[CrossRef][Medline]

2. Farriol-Mathis, N., Garavelli, J. S., Boeckmann, B., Duvaud, S., Gasteiger, E., Gateau, A., Veuthey, A.-L., and Bairoch, A. (2004) Annotation of post-translational modifications in the Swiss-Prot knowledge base. Proteomics 4, 1537 –1550[CrossRef][Medline]

3. Van den Bergh, G., and Arckens, L. (2005) Recent advances in 2D electrophoresis: an array of possibilities Expert Rev. Proteomics 2, 243 –252[CrossRef][Medline]

4. Rabilloud, T. (2002) Two-dimensional gel electrophoresis in proteomics: old, old fashioned, but it still climbs up the mountains. Proteomics 1, 3 –10

5. Neverova, I., and Van Eyk, J. E. (2005) Role of chromatographic techniques in proteomic analysis. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 815, 51 –63[Medline]

6. Morris, D. L., Jr., Sutton, J. N., Harper, R. G., and Timperman, A. T. (2204) Reversed-phase HPLC separation of human serum employing a novel saw-tooth gradient: toward multidimensional proteome analysis. J. Proteome Res. 3, 1149 –1154[CrossRef]

7. Fujii, K., Nakano, T., Hike, H., Usui, F., Bando, Y., Tojo, H., and Nishimura, T. (2004) Fully automated online multi-dimensional protein profiling system for complex mixtures. J. Chromatogr. A 1057, 107 –113

8. Garbis, S., Lubec, G., and Fountoulakis, M. (2005) Limitations of current proteomics technologies. J. Chromatogr. A 1077, 1 –18

9. Baumann, M., and Meri, S. (2004) Techniques for studying protein heterogeneity and post-translational modifications. Expert Rev. Proteomics 1, 207 –217[CrossRef][Medline]

10. Swanson, S. K., and Washburn, M. P. (2005) The continuing evolution of shotgun proteomics. Drug Discov. Today 10, 719 –725[CrossRef][Medline]

11. Wang, Y., Wu, R., Cho, K. R., Shedden, K. A., Barder, T. J., and Lubman, D. M. (2006) Classification of cancer cell lines using an automated two-dimensional liquid mapping method with hierarchical clustering techniques. Mol. Cell. Proteomics 5, 43 –52[Abstract/Free Full Text]

12. Sheng, S., Chen, D., and Van Eyk, J. E. (2006) Multi-dimensional liquid chromatography separation of intact proteins by chromatographic focusing and reversed phased of the human serum proteome: optimization and protein database. Mol. Cell. Proteomics 5, 26 –34[Abstract/Free Full Text]

13. Zheng, S., Schneider, K. A., Barder, T. J., and Lubman, D. M. (2003) Two-dimensional liquid chromatography protein expression mapping for differential proteomic analysis of normal and O157:H7 Escherichia coli. BioTechniques 35, 1202 –1212[Medline]

14. Lubman, D. M., Kachman, M. T., Wang, H., Gong, S., Yan, F., Hamler, R. L., O’Neil, K. A., Zhu, K., Buchanan, N. S., and Barder, T. J. (2002) Two-dimensional liquid separations-mass mapping of proteins from human cancer cell lysates. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 782, 183 –196[Medline]

15. Chen, E. I., Hewel, J., Felding-Habermann, B., and Yates, J. R., III (2006) Large scale protein profiling by combination of protein fractionation and multidimensional protein identification technology (MudPIT). Mol. Cell. Proteomics 5, 53 –6[Abstract/Free Full Text]

16. Komatsu, S., Zang, X., and Tanaka, N. (2006) Comparison of two proteomics techniques used to identify proteins regulated by gibberellin in rice. J. Proteome Res. 5, 270 –276[CrossRef][Medline]

17. Gibson, B. W. (2005) The human mitochondrial proteome: oxidative stress, protein modifications and oxidative phosphorylation. Int. J. Biochem. Cell Biol. 37, 927 –934[CrossRef][Medline]

18. Pagliarini, D. J., and Dixon, J. E. (2006) Mitochondrial modulation: reversible phosphorylation takes center stage? Trends Biochem. Sci. 31, 26 –34[CrossRef][Medline]

19. Hurd, T. R., Filipovska, A., Costa, N. J., Dahm, C. C., and Murphy, M. P. (2005) Disulphide formation on mitochondrial protein thiols. Biochem. Soc. Trans. 33, 1390 –1393[CrossRef][Medline]

20. Fukada, K., Zhang, F., Vien, A., Cashman, N. R., and Zhu, H. (2004) Mitochondrial proteomic analysis of a cell line model of familial amyotrophic lateral sclerosis. Mol. Cell. Proteomics 12, 1211 –1223

21. Taylor, S. W., Fahy, E., Zhang, B., Glenn, G. M., Warnock, D. E., Wiley, S., Murphy, A. N., Gaucher, S. P., Capaldi, R. A., Gibson, B. W., and Ghosh, S. S. (2003) Characterization of the human heart mitochondrial proteome. Nat. Biotechnol. 3, 281 –286

22. Gaucher, S. P., Taylor, S. W., Fahy, E., Zhang, B., Warnock, D. E., Ghosh, S. S., and Gibson, B. W. (2004) Expanded coverage of the human heart mitochondrial proteome using multidimensional liquid chromatography coupled with tandem mass spectrometry. J. Proteome Res. 3, 495 –505[CrossRef][Medline]

23. Rezaul, K., Wu, L., Mayya, V., Hwang, S. I., and Han, D. (2005) A systematic characterization of mitochondrial proteome from human T leukemia cells. Mol. Cell. Proteomics 4, 169 –181[Abstract/Free Full Text]

24. McDonald, T. G., and Van Eyk, J. E. (2003) Mitochondrial proteomics. Undercover in the lipid bilayer. Basic Res. Cardiol. 98, 219 –227[Medline]

25. Taylor, S. W., Fahy, E., and Ghosh, S. S. (2003) Global organellar proteomic. Trends Biotechnol. 21, 82 –88[CrossRef][Medline]

26. Gabaldon, T., and Huynen, M. A. (2004) Lineage-specific gene loss following mitochondrial endosymbiosis and its potential for function prediction in eukaryotes. Biochim. Biophys. Acta 1659, 212 –220[Medline]

27. Santoni, V., Molloy, M., and Rabilloud, T. (2000) Membrane proteins and proteomics: un amour impossible? Electrophoresis 21, 1054 –1070[CrossRef][Medline]

28. Mootha, V. K., Bunkenborg, J., Olsen, J. V., Hjerrid, M., Wisniewski, J. R., Stahl, E., Bolouri, M. S., Ray, H. N., Sihang, S., Kamal, M., Patterson, N., Landers, E. S., and Mann, M. (2003) Integrated analysis of protein composition, tissue diversity, and gene regulation in mouse mitochondria. Cell 115, 629 –640[CrossRef][Medline]

29. Da Cruz, S., Xenarios, I., Langridge, J., Vilbois, F., Parone, P. A., and Martinou, J. C. (2003) Proteomic analysis of the mouse liver mitochondrial inner membrane. J. Biol. Chem. 278, 41566 –41571[Abstract/Free Full Text]

30. Da Cruz, S., Parone, P. A., and Martinou, J. C. (2005) Building the mitochondrial proteome. Expert Rev. Proteomics 4, 541 –551

31. Calvo, S., Mohit, J., Xie, X., Sheth, S. A., Chang, B., Goldberger, O. A., Spinazzola, A., Zeviani, M., Carr, S. A., and Mootha, V. K. (2006) Systematic identification of human mitochondrial disease genes through integrated genomics. Nat. Genet. 38, 576 –582[CrossRef][Medline]

32. Pedersen, P. L, Greenawalt, J. W., Reynafarje, B., Hullihen, J., Decker, G. L., Soper, J. W., and Bustamente, E. (1978) Preparation and characterization of mitochondria and submitochondrial particles of rat liver and liver-derived tissues. Methods Cell Biol. 20, 411 –481[Medline]

33. Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68, 850 –858[Medline]

34. Gharahdeghi, F., Weinberg, C. R., Meagher, D. A., Imai, B. S., and Mische, S. M. (1999) Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: a method for the removal of silver ions to enhance sensitivity. Electrophoresis 20, 601 –605[CrossRef][Medline]

35. Cserzo, M., Wallin, E., Simon, I., von Heijne G., and Elofsson, A. (1997) Prediction of transmembrane alpha-helices in prokaryotic membrane proteins: the dense alignment surface method. Prot. Eng. 10, 673 –676[Abstract/Free Full Text]

36. Pirondini, A., Visioli, G., Malcevschi, A., and Marmiroli, N. (2006) A 2-D liquid-phase chromatography for proteomic analysis in plant tissues. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 833, 91 –100[Medline]

37. Linke, T., Ross, A. C., and Harrison, E. H. (2006) Proteomic analysis of rat plasma by two-dimensional liquid chromatography and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. J. Chromatogr. A 1123, 160 –169

38. Zhu, K., Miller, F. R., Barder, T. J., and Lubman, D. M. (2004) Identification of low molecular weight proteins isolated by 2-D liquid separations. J. Mass Spectrom. 39, 770 –778[CrossRef][Medline]

39. Betgovargez, E., Knudson, V., and Simonian, M. H. (2005) Characterization of proteins in the human serum proteome. J. Biomol. Tech. 16, 306 –310[Medline]

40. Zhu, K., Zhao, J., Lubman, D. M., Miller, F. R., and Barder, T. J. (2005) Protein pI shifts due to posttranslational modifications in the separation and characterization of proteins. Anal. Chem. 77, 2745 –2755[Medline]

41. Clauser, K. R., Baker, P. R., and Burlingame, A. L. (1999) Role of accurate mass measurement (±10 ppm) in protein identification strategies employing MS or MS/MS and database searching. Anal. Chem. 71, 2871 –2882[Medline]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				J. Feng, M. Zhu, M. C. Schaub, P. Gehrig, B. Roschitzki, E. Lucchinetti, and M. Zaugg Phosphoproteome analysis of isoflurane-protected heart mitochondria: phosphorylation of adenine nucleotide translocator-1 on Tyr194 regulates mitochondrial function Cardiovasc Res, July 2, 2008; (2008) cvn161v2. [Abstract] [Full Text] [PDF]

				P. Matt, Z. Fu, Q. Fu, and J. E. Van Eyk Biomarker discovery: proteome fractionation and separation in biological samples Physiol Genomics, March 10, 2008; 33(1): 12 - 17. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Supplemental Data All Versions of this Article: T500036-MCP200v1 5/12/2392    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Glossary Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by McDonald, T. Articles by Van Eyk, J. E. Search for Related Content PubMed PubMed Citation Articles by McDonald, T. Articles by Van Eyk, J. E. Social Bookmarking