Fast Proteolytic Digestion Coupled with Organelle Enrichment for Proteomic Analysis of Rat Liver Randy J. Arnold,*,† Petra Hrncirova,† Kiran Annaiah,‡ and Milos V. Novotny*,† Proteomics R&D Facility, Department of Chemistry, Indiana University, Bloomington, Indiana 47405 and School of Informatics, Indiana University, Bloomington, Indiana 47405 Received November 25, 2003
Abstract: The use of an acid-labile surfactant as an alternative to urea denaturation allows for same-day proteolytic digestion and fast cleanup of cellular lysate samples. Homogenized rat liver tissue was separated into four fractions enriched in nuclei, mitochondria, microsomes (remaining organelles), and cytosol. Each subcellular fraction was then subjected to proteolytic digestion with trypsin for 2 h after denaturing with an acid-labile surfactant (ALS), separated by nanoflow reversed phase HPLC, and mass analyzed by tandem mass spectrometry in a 3-D ion trap. The results obtained from ALS denaturation for both organelle enrichment and whole cell lysate samples were comparable to those obtained from aliquots of the same samples treated by reduction, alkylation, and urea denaturation. Each method resulted in a similar number of peptides (694 for urea, 674 for ALS) and proteins (225 for urea, 229 for ALS) identified, with generally the same proteins (47% overlap) identified. As expected, organelle enrichment enabled the identification of more proteins (66% more with urea, 60% more with ALS) compared to a whole cell lysate. With organelle enrichment, the number of proteins with equal or increased sequence coverage went up by 73% with urea and 67% with ALS compared to the whole cell lysate. Additional information regarding the subcellular location of many proteins is obtained by organelle enrichment. While organelle enrichment is demonstrated with a bottom-up proteomics approach, it should be easily amenable to top-down proteomics approaches. Keywords: Proteomics • organelle enrichment • acid-labile surfactant • subcellular fractionation • proteolysis • nano-LC • tandem mass spectrometry • rat liver
Introduction Complex biological mixtures analyzed using current proteomics approaches appear to require better separations, particularly for the bottom-up approaches used in studies of mammalian systems. In order for proteomics to reach its full analytical potential of detecting 100% sequence coverage of all proteins in a cell, including all post-translational modifications, † ‡
Proteomics R&D Facility, Department of Chemistry, Indiana University. School of Informatics, Indiana University.
10.1021/pr034110r CCC: $27.50
2004 American Chemical Society
numerous methodological advances must be made. Among them, increased mass spectrometer sensitivity, dynamic range, and scan speed, higher chromatographic resolution with lower solute losses, and more efficient proteolytic digestion will all be required to reach this challenging goal. The organelle enrichment, or subcellular fractionation, of the individual compartments of a eukaryotic cell is likely to play an important role in reducing a cell lysate complexity. Faster, cleaner, and more efficient proteolytic digestions should be used to speed sample processing and increase sample throughput. In this report, organelle enrichment and protein denaturation using an acid-labile surfactant are presented as effective tools for increasing a proteome coverage and decreasing the analysis time for a proteomic sample. It is not widely recognized that two-dimensional separations of peptides may lose the continuity of biological information. While techniques such as multidimensional protein identification technology (MudPIT)1 certainly increase the number of peptides observed, data complexity is increased by potentially spreading peptides from the same protein across a large number of salt fractions. Thus, to compare this type of mass spectral (MS) data to a protein database, MS data from all fractions must be combined and submitted in one search. An alternative bottom-up approach would first separate proteins into fractions, digest a smaller number of proteins, separate the resulting peptides in a single (or at least fewer) reversedphase LC analysis, and submit this smaller MS data set for database searching. As shown previously, organelle enrichment can serve as an additional tool in pre-fractionating the proteome prior to LC-MS/MS2 or 2-D gel3 analysis. Other prefractionation techniques have also proven useful prior to 2Dgel analysis.4 In several reports, proteomes of isolated organelles have also been studied.5-9 Enriching proteins prior to a proteolytic digestion can potentially result in greater sequence coverage of identified proteins. Organelle enrichment does share with other multidimensional separation techniques the quality that separating the sample into fractions prior to reversed-phase chromatography allows the mass spectrometer more time to acquire tandem mass spectra on low-intensity MS signals during the reversed-phase gradient, thereby increasing proteome coverage.1,10 By increasing sequence coverage, the probability that post-translational modifications can be detected also increases. Of particular interest are those modifications, such as glycosylation, found on the surface of organelles which act as signals or binding sites.11 Journal of Proteome Research 2004, 3, 653-657
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Proteomic Analysis of Rat Liver
Acid-labile surfactants have been shown previously to have beneficial effects when replacing SDS in gel-based separations12-14 prior to mass-spectrometric analysis. This study presents a comparison between the trypsin digestion performed on complex protein mixtures treated with either ALS or urea for protein denaturation. In addition, since samples treated with ALS are not reduced and alkylated, whereas the ureatreated samples are reduced and alkylated, the time and sample handling needed prior to and during digestion are reduced. Proteolytic digests performed in the presence of an ALS are also easier to recover and introduce into the reversed-phase chromatography, as they do not require another “cleanup” step prior to LC analysis.
Method Materials. TRIZMA hydrochloride (Tris HCl), ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), proteomics grade trypsin, and formic acid, 95-97% were obtained from Sigma-Aldrich Co. (St. Louis, MO). Dithiothreitol (DTT) and iodoacetamide were purchased from Bio-Rad Laboratories (Hercules, CA). Sucrose, urea, and water were obtained from EM Science (Gibbstown, NJ). Ammonium bicarbonate was purchased from Mallinckrodt Chemical (Paris, KY). Acetonitrile and hydrochloric acid solution N/10 were obtained from Fisher Scientific (Fair Lawn, NJ). All chemicals were used as obtained without further purification. Biological Samples. Liver samples were obtained from adult (90-110 days old), male alcohol nonpreferring (NP) rats of the 51st generation bred and maintained at Indiana University School of Medicine. The NP rat line has been selectively bred from Wistar stock for their alcohol nonpreference and has been extensively characterized elsewhere.15,16 The animals used in this experiment were maintained in facilities fully accredited by the American Association for the Accreditation of Laboratory Animal Care (AAALAC). Animals were killed by decapitation and the liver quickly extracted through a midline incision. Liver samples were placed in sterile tubes and immediately frozen on dry ice. Samples were stored at -70 °C until processed. Homogenization and Organelle Enrichment. Intact frozen liver tissue (100 mg) was homogenized in 200 µL of organelle isolation buffer (OIB, containing 20 mM Tris HCl, pH 7.4, 1 mM DTT, 1 mM EDTA, 1 mM EGTA, 250 mM sucrose). This sample was separated into 2 vials, each containing 50 mg of tissue homogenate (∼150 µL). Organelle enriched fractions were prepared as described previously.17 Briefly, vials were centrifuged at 1000 × g for 20 min to sediment the nuclear pellet. The supernatant and the nuclear pellet were retained. The retained supernatant was centrifuged at 15 000 × g for 20 min. The supernatant and the mitochondrial pellet were retained. The retained supernatant aliquots were combined and centrifuged at 100 000 g for 60 min. The supernatant (cytosol, ∼150 µL) and the microsomal pellet were retained. Each pellet was suspended in 50 µL of OIBNS (OIB without sucrose), centrifuged at the same speed and duration that it was first pelleted and, after discarding supernatant, re-suspended in 50 µL of OIBNS. Each pellet suspension, estimated to have a protein concentration of approximately 5 µg/µL, was frozen at -20 °C. The first two centrifugation steps were performed in a refrigerated tabletop centrifuge at 4 °C while the final highspeed centrifugation was performed in an ultracentrifuge at 4 °C. 654
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technical notes Reduced/Alkylated Urea Digestion. Whole cell lysate samples were prepared by diluting 50 µL of the concentrated (500 µg tissue/µL) homogenate with 200 µL of OIBNS. Organelle enriched samples were used as described above. A 5-µL aliquot of each fraction was mixed with 20 µL reducing buffer containing 10 M urea, 12.5 mM DTT, and 125 mM ammonium bicarbonate, and incubated at 37 °C for 2 h. After cooling, 5 µL of alkylating buffer containing 8 M urea and 60 mM iodoacetamide was added and the mixture was incubated at 4 °C for 90 min. The urea concentration was reduced by adding 70 µL of 100 mM ammonium bicarbonate. A 10-µL aliquot of 0.1 µg/µL proteomics grade trypsin was added and the mixture was incubated at 37 °C for 14 h overnight followed by the addition of 100 µL 1% formic acid to quench the reaction. Urea and excess reagents were removed from the sample by capturing the peptides on a 1 cm3 Oasis HLB Extraction Cartridge (Waters Corp., Milford, MA), washing away the contaminants, and eluting the peptides. Briefly, the samples were loaded onto the washed cartridges, washed twice with 1 mL of 0.1% formic acid, and eluted with 300 µL each of a 40% acetonitrile and a 90% acetonitrile aqueous solution, each containing 0.1% formic acid. The combined eluents were reduced to approximately 20 µL at 45 °C in an Eppendorf Vacufuge Concentrator (Brinkmann Instruments, Inc., Westbury, NY) and 10 µL of 97:3:0.1 water/ acetonitrile/formic acid was added. Subsequently, the samples were frozen at -20 °C prior to LC-MS/MS analysis. Acid-Labile Surfactant Digestion. Whole cell lysate samples were prepared by diluting 50 µL of the concentrated (500 µg tissue/µL) homogenate with 200 µL of OIBNS. Organelle enriched samples were used as described above. A 5 µL aliquot of each fraction was mixed with 5 µL of 1% RapiGest (Waters Corp., Milford, MA) acid-labile surfactant, heated at 95 °C for 5 min., followed by the addition of 10 µL of 0.1 mg/mL Proteomics grade trypsin (Sigma, St. Louis, MO). Samples were incubated at 37 °C for 2 h, followed by the addition of 20 µL 100 mM HCl, incubation at 37 °C for 45 min. and centrifugation at 13 000 rpm for 10 min. Supernatant was transferred to an autosampler vial, 10 µL 97:3:0.1 water/acetonitrile/formic acid was added, and samples were frozen at -20 °C prior to LCMS/MS analysis. LC-MS/MS Instrumentation. Either 0.5 or 1.0 µL of protein digest (∼0.25-0.50 µg of protein per injection) was loaded onto a 15 mm × 100 µm i.d. trapping column packed with 5 µm, 200 Å Magic C18AQ packing materials (Microm BioResourses Inc., Auburn, CA) and eluted through a 150 mm × 75 µm i.d. analytical column with the same packing material, except for its 100 Å pore size, using a 120-minute gradient from 100% to 65% solvent A, 97:3:0.1 water/acetonitrile/formic acid (solvent B is 0.1% formic acid in acetonitrile) at 250 nL/min using an LC Packings (Dionex, Sunnydale, CA) Famos autosampler, Switchos switching valve and pump, and UltiMate gradient pump. From the end of the column, ions were electrosprayed directly into a ThermoFinnigan (San Jose, CA) LCQ Deca XP ion-trap mass spectrometer which recorded mass spectra and data-dependent tandem mass spectra of the peptide ions. By using dynamic exclusion, the mass spectrometer was limited to acquiring only one tandem mass spectrum for a given parent m/z over a 60-second window. Database Searching. MSMS spectra were searched against protein sequences for Rattus in the Swiss-Prot database using a licensed copy of Mascot18 on a 2.0 GHz Pentium 4 Dell workstation PWS 340 with 1 GB of RAM for peptide identification. A minimum Mascot score of 28, which indicates identi-
technical notes
Arnold et al.
Table 1. Number of Proteins and Peptides Identified from Various Rat Liver Fractions by Urea or Acid-labile Surfactant Denaturation Prior to Trypsin Digestion and RP-nanoLC-MS/MSa urea fraction
whole cell nuclear mitochondrial microsomal cytosol organelle totals
ALS
both
analysis 1
proteins peptides proteins peptides proteins peptides
134 56 125 43 121 52 74 28 109 46 225 82
460 358 381 282 418 328 254 197 376 297 694 523
156 59 137 36 137 52 120 32 109 48 229 84
Table 2. Number of Proteins and Peptides Identified from Replicate Analyses of Rat Liver Whole Cell Lysates by Urea or Acid-Labile Surfactant Denaturation Prior to Trypsin Digestion and RP-nanoLC-MS/MSa
432 312 337 197 382 276 292 185 357 276 674 481
93 41 86 26 83 36 51 22 78 34 161 63
168 135 138 104 171 140 92 75 168 137
a Numbers in italics correspond to proteins with minimum of 3 peptides identified.
fication at the 95% confidence level for these searches, was used to discriminate peptide matches. Mascot result files were parsed using a Protein Results Parser program written in-house. In determining the total number of proteins and peptides observed, searches were performed with zero variable modifications selected (fixed modification of carbamidomethyl cysteine was selected for urea-denatured sample searches) and a maximum of one missed cleavage site. This mode of searching enables searches to be executed in a reasonable amount of time (∼5 min/search), but intentionally ignores peptides with posttranslational modifications. In separate database searches, acetylation of lysine side chains and protein N-termini was investigated by allowing for these variable modifications.
Results Organelle enrichment provides enhanced proteome coverage in terms of the number of proteins observed as compared to a whole cell lysate. As shown in Table 1, approximately 100 proteins can be identified by one or more peptides in each organelle-enriched fraction using either urea or ALS to denature proteins. Combining the results from organelle-enriched fractions, over 220 unique proteins are identified in the sample, compared to 134 or 156 proteins identified in the whole cell lysate using urea or ALS, respectively. Proteins identified with three or more peptides also increase from 56 to 82 using urea and from 59 to 84 using acid labile surfactant. In addition to identifying more proteins, the number of peptides observed also increases, as shown in Table 1. In the whole cell lysate samples, 460 and 432 peptides are observed by urea and ALS denaturation, respectively. Analysis of organelle enriched fractions reveals that 694 and 674 peptides are observed when the maximum numbers of peptides observed for a protein in any single fraction are summed for the urea and acid labile surfactant samples, respectively. As a result, protein sequence coverage based on the number of peptides observed remains the same or increases for 73% of proteins using urea and 67% of proteins using ALS with organelle enrichment compared to the whole cell lysate. The results shown in Table 1 for the use of urea compared to ALS for protein denaturation indicate that both methods enable the identification of similar numbers of proteins and peptides for the whole cell lysate and organelle enriched fraction analysis by trypsin digestion coupled to LC-MS/MS.
analysis 2
both
denaturant proteins peptides proteins peptides proteins peptides
urea ALS
134 56 156 59
460 358 431 309
138 48 161 54
449 334 445 309
96 39 120 44
261 207 259 187
a Numbers in italics correspond to proteins with minimum of 3 peptides identified.
With the exception of the microsomal fraction, the numbers of proteins identified from any fraction agree within a 16% difference. The numbers of peptides identified agree within 15% for all fractions, including the microsomal fraction. The fact that a substantial number of proteins appear to be unique to one of the two denaturing methods is most likely due to the inability to perform tandem mass spectrometry on all peptide ions eluting from the LC column at a given time. This conclusion is consistent with the results obtained by performing duplicate LC-MS/MS analyses on the trypsin digests of the whole cell lysates denatured by either method, as shown in Table 2. In this case, while many peptides are “missed”, there are a number of proteins that would seem to be “unique” to each of the two replicate analyses, but since we know that the same sample is used in both cases, a reasonable explanation would be that there was not sufficient time for the mass spectrometer to perform tandem mass spectrometry on all eluting peptide ions. Of the 82 proteins identified with three or more peptides using urea denaturation, 78 were identified with at least one peptide in the ALS denatured samples. Conversely, 80 of the 84 proteins identified with three or more peptides using ALS denaturation were identified with at least one peptide in the urea denatured samples. A random investigation of the peptides, namely those used to identify the proteins listed in Tables 3 and 4, reveals that reduction and alkylation of disulfide bonds, as with the urea denaturation protocol, do not significantly increase the ability of LC-MS/MS to identify proteins. Of the 84 and 83 peptides found in the fractions containing the most peptides for each protein for ALS and urea-treated samples respectively, 47 peptides are found by both methods. Of these identified peptides, nine from the urea-treated samples contained cysteine residues, while only one from the ALS-treated samples contained cysteine. Remarkably, proteins with two or more cysteine-containing peptides identified, catalase (3 peptides), arginase (2 peptides) and glutathione S-transferase Yb2 (3 peptides), were observed to have the same number or more peptides observed when treated with ALS (without reduction/ alkylation) compared to urea (with reduction/alkylation). This extensive protein and peptide coverage overlap indicates that for the most part, the same proteins are detected using either denaturing method. Despite not having sufficient time for the mass spectrometer to identify all peptides eluting from the LC, the results obtained by denaturing samples with ALS without reduction and alkylation (and using a much shorter digestion time) compare quite favorably with the standard protocol using urea denaturation, reduction, alkylation, extended digestion times and subsequent sample cleanup. In addition to the enhanced protein and sequence coverage obtained by organelle enrichment, another advantage of this Journal of Proteome Research • Vol. 3, No. 3, 2004 655
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Proteomic Analysis of Rat Liver
Table 3. Number of Peptides Observed in Each Fraction for Proteinsa Identified by Organelle Enrichment Using Acid-Labile Surfactant Denaturation Followed by Trypsin Digestion and LC-MS/MS Analysis
a
protein, Swiss-Prot acc. no.
whole
nuclear
mitoch.
micros.
cytosol
maximum
ATP synthase R-chain, P15999 glutamate dehydrogenase, P10860 catalase, P04762 protein disulfide iso. A3, P11598 arginase I, P07824 pyruvate carboxylase, P52873 isocitrate dehyd. [NADP], P41562 liver carboxylesterase B-1, Q63010 glutathione S-transf. Yb2, P08010 cytokeratin 8, Q10758
8 10 10 2 2 2 1
11 9 3 6 4 2 1 2
10 11 5 10 2 2
3 11 7 5 5 7
2 10 12 2 7 3 3
11 11 12 10 7 7 3 3 8 12
6 9
3 8
12
Searches performed with no variable post-translational modifications selected.
Table 4. Number of Peptides Observed in Each Fraction for Proteinsa Identified by Organelle Enrichment Using Urea Denaturation Followed by Trypsin Digestion and LC-MS/MS Analysis
a
protein, Swiss-Prot acc. no.
whole
nuclear
mitoch.
micros.
ATP synthase R-chain, P15999 glutamate dehydrogenase, P10860 catalase, P04762 protein disulfide iso. A3, P11598 arginase I, P07824 pyruvate carboxylase, P52873 isocitrate dehyd. [NADP], P41562 liver carboxylesterase B-1, Q63010 glutathione S-transf. Yb2, P08010 cytokeratin 8, Q10758
5 9 9 1 6
10 9 7 4 6 1
10 14 9 9 6 1
4 8 7 1 7 3
7 3
1 5
1 3 12 1
3 11
7 11 6 1 1 10
maximum
10 14 11 9 7 3 1 7 10 11
Searches performed with fixed carbamidomethyl cysteine and no variable post-translational modifications selected.
technique is the ability to obtain information pertaining to the subcellular location of proteins. For proteins identified with three or more peptides in any single organelle-enriched fraction, 44% in the urea-treated samples and 48% in the ALStreated samples had one fraction with at least twice as many peptides as any other fraction. Although a direct correlation between protein abundance and the number of peptides identified from a protein has not been established, one can assume that these two parameters are at least roughly proportional. This result seems to indicate that even though the fractionation is intended only to enrich, but not fully isolate, organelles, the overall result is that many proteins are substantially enriched in one of the four fractions. Examples of such enrichment, along with other selected proteins, are shown in Tables 3 and 4 for ALS- and urea-treated samples, respectively. Another benefit of organelle enrichment is the ability to detect post-translational modifications (PTMs). Database searches were performed on the same tandem mass spectrometry data sets allowing for variable acetylation of lysine residues and protein N-termini. Each assignment of acetylation was manually verified. These searches identified five and nine acetylated peptides in the urea- and ALS-treated whole cell lysate samples, respectively. Organelle enrichment resulted in the identification of 14 (urea) and 17 (ALS) acetylated peptides, of which, three (urea) and five (ALS) were also identified in the whole cell lysate analyses. Eleven acetylated peptides were observed in both urea- and ALS-treated samples. Six peptides were identified as acetylated in organelle-enriched samples for both denaturing treatments but not identified in the whole cell lysate for either treatment. One such example is the N-terminal peptide Ac-MEVHELFR from long-chain-fatty-acid-CoA ligase. The tandem mass spectrum of this acetylated peptide from the mitochondrial fraction of the urea-treated sample is shown in Figure 1. 656
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Figure 1. Tandem mass spectrum of modified peptide AcMEVHELFR from long-chain-fatty-acid-CoA ligase (P18163) acquired by LC-MS/MS analysis of the mitochondrial fraction of the urea-treated sample. Singly charged b and y fragment ions are labeled in the spectrum. Similar tandem mass spectra for the same acetylated peptide were observed in the nuclear fraction of the urea-treated sample and both the nuclear and mitochondrial fractions of the ALS-treated sample, but not in either whole cell lysate analysis.
Conclusions Although the use of an ALS as a protein denaturant prior to proteolytic digestions for proteomics applications is presented
technical notes here as an alternative to the standard reduction, alkylation, and urea denaturation method, the two methods potentially offer complementary information. Proteins may favor denaturation or be more susceptible to proteolysis under one of the two conditions, but not the other. Given that similar numbers of proteins and peptides can be observed, and that equally dense chromatograms (data not shown) can be obtained by either method, the application of both methods to a sample may prove beneficial in maximizing proteome coverage. As shown in this report and by others,2,3 organelle enrichment improves proteome coverage by increasing the number of peptides observed. By removing many high-abundance proteins that are found in other fractions, lower abundance proteins should have higher relative concentrations and be detected with greater sensitivity and higher sequence coverage. Increasing sequence coverage for a protein should also increase the probability of finding post-translational modifications that might otherwise be difficult to detect. Although most of the results presented here were obtained from database searches that did not include variable modifications, searches that included variable acetylation of lysines and protein N-termini demonstrate that PTMs are more readily detected with organelle enrichment. As mentioned above, it is apparent that not all peptides eluting from the LC separation are being identified by tandem mass spectrometry. Higher orders of separation should alleviate this situation, allowing the mass spectrometer more time to perform tandem mass spectrometry as peptides elute. Even with this improvement, a wide dynamic range of protein concentrations may still limit detection to only the most abundant proteins in a sample. Organelle enrichment has thus far been pursued by using a classical sedimentation technique,17 which works well for plentiful (milligrams of tissue) samples, such as rat liver. Unfortunately, not all proteomics samples are as convenient, such that alternative methods must be sought to provide organelle enrichment when starting with smaller (micrograms amounts) samples. For such samples, eliminating intermediate cleanup steps, as shown here by using an ALS, should prove beneficial.
Acknowledgment. The authors would like to acknowledge Myeong Hee Moon for many helpful discussions and for
Arnold et al.
his help in setting up the nanoflow LC. We wish to thank Dr. William McBride and the Indiana Alcohol Research Center AA07611 for the animals, and Dr. Wendy Strother-Robinson and the INIA Cellular Imaging Core AA13521 who supplied the tissue samples. We also acknowledge support from the Indiana Genomics Initiative (INGEN), a grant to Indiana University by the Lilly Endowment.
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