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Department of Pediatrics, Medical University of Vienna, Vienna, Austria. Received January 16, 2006. Abstract: Analyzing complex protein mixtures on a ...
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Use of Solution-IEF-Fractionation Leads to Separation of 2673 Mouse Brain Proteins Including 255 Hydrophobic Structures Jae-Kyung Myung and Gert Lubec* Department of Pediatrics, Medical University of Vienna, Vienna, Austria Received January 16, 2006

Abstract: Analyzing complex protein mixtures on a single gel does not allow separation of many extracted proteins. Herein, we tried a prefractionation approach and mouse brain proteins were separated on a narrow pH range ZOOM-IEF Fractionator (MicroSol-IEF device) and run on two-dimensional gel electrophoresis. A total number of 2673 protein spots including 255 hydrophobic structures were successfully analyzed by mass spectrometry. This nonsophisticated approach to increase protein identification of a brain protein extract is a step forward in neurochemistry. Keywords: mouse brain • prefractionation • MicroSol-IEF • solution isoelectricfocusing • mass spectrometry • MALDI • twodimensional gel electrophoresis

Introduction In neuroscience, a number of recent investigations have focused on the identification of major proteins in normal brain tissue and on the construction of reference databases that catalog proteins expressed in the whole brain or brain sections,1 as well as on the change of their levels and the modifications that result from various neurological disorders in humans and in animal models until now. Further improvement of proteomics technologies to increase sensitivity and efficiency of detection of certain protein classes is necessary for a more detailed analysis of the brain proteome. In addition, the concomitant detection of several hundred proteins on a gel allows the demonstration of an expressional pattern, generated by a reliable, protein-chemical method rather than by immunoreactivity, proposed by protein-arrays.2 Proteomics employs a combination of sophisticated analytical tools. Despite promising alternative/complementary technologies (e.g., multidimensional protein identification technology, stable isotope labeling, protein arrays) that have emerged recently, two-dimensional gel electrophoresis (2-DE) is currently the mainstay for protein separation as the highest resolution technique that can be routinely applied for parallel quantitative expression profiling of large sets of complex protein mixtures such as whole cell lysates or tissue.3,4 Stained protein spots of interest are cleaved by in-gel protein digestion * To whom correspondence should be addressed. Prof. Dr. Gert Lubec, CChem, FRSC (UK), Medical University of Vienna, Department of Pediatrics, Wa¨hringer Gu ¨ rtel 18, A-1090 Vienna, Austria. Tel: +43-1-40400-3215 Fax: +43-1-40400-194. E-mail: [email protected]. 10.1021/pr060015h CCC: $33.50

 2006 American Chemical Society

and identified by mass-spectometrical approaches combined with database searching.1,5 In current proteome projects, the total number of proteins identified from 2-DE gels is mainly only a small percentage of the predicted proteome.6 This results from well-known limitations in 2-DE such as detection range, difficulties in analyzing hydrophobic, very small and large proteins, low throughput and low reproducibility but a number of improvements have been made in 2-DE technology for enhancing their profiling capacity.7 “Zoom” and “ultrazoom” gels (narrow-range immobilized pH gradient gels) expand separation distances, which minimize the problem of spot overlapping in 2-DE, an ever present hazard in 2-DE maps.8-10 Also the use of large-size gel slabs (18 cm or longer in the first dimension, 18 × 20 cm, or larger, in the second dimension), would even increase the resolution.11,12 New chaotropes, surfactants, reductants, or supporting matrixes for analyzing hydrophobic proteins,13,14 and sensitive gel-staining method that are MS friendly and offer greater linear detection ranges are introduced.15,16 Many attempts have been made developing different electrophoretic, chromatographic and a combination of hyphenated techniques to separate such complex protein mixtures. A variety of different analytical methods (Supporting Information Table 1) allowed proteins to be fractionated into different groups having similar physical and chemical properties.17 Previously reported prefractionation methods include sequential extractions with increasingly stronger solubilization solutions,18 subcellular fractionation,19 and selective removal of the most abundant protein components.20 However, these methods suffer from incomplete separation of proteins between fractions. Cross-contamination of specific proteins between fractionated pools can seriously complicate quantitative analyses and comparisons, since many proteins appear in more than one fraction and the degree of cross-contamination is often highly variable. Furthermore, when a spot appears in the same position on 2-D gels from different fractions, there is some uncertainty as to whether these spots represent two different proteins or simply cross-contamination between fractions.21 One of the most successful prefractionation techniques is IEF which shows high resolution and is compatible with subsequent separation in 2-DE analysis, a focusing step based on Immobiline technology. Upon the basis of this principle Microscale solution isoelectrofocusing (MicroSol-IEF) was developed in David W. Speicher’s laboratory recently,22 and is available from Invitrogen as the commercial version of this device, the ZOOM-IEF Fractionator. It permits harvesting a population of proteins having pI values precisely matching the Journal of Proteome Research 2006, 5, 1267-1275

1267

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technical notes

Separation of Mouse Brain Proteins Table 1. Hydrophobic Proteins: Proteins with Positive GRAVY Values acc. no.

entry name

O35215 P02088 P06151 P08249 P13707

DOPD_MOUSE HBB1_MOUSE LDHA_MOUSE MDHM_MOUSE GPDA_MOUSE

P16125 P20108

LDHB_MOUSE PRDX3_MOUSE

P52760 P56480 P62962 P67778 P99029 Q3UNI8

UK14_MOUSE ATPB_MOUSE PROF1_MOUSE PHB_MOUSE PRDX5_MOUSE Q3UNI8_MOUSE

Q542 × 7

Q542 × 7_MOUSE

Q545Y4

Q545Y4_MOUSE

Q549D9 Q54AI0 Q569N4 Q5D0E8 Q5SX50 Q76MZ3

Q549D9_MOUSE Q54AI0_MOUSE Q569N4_MOUSE Q5D0E8_MOUSE Q5SX50_MOUSE 2AAA_MOUSE

Q7TMX2

Q7TMX2_MOUSE

Q8 µBD3

Q8 µBD3_MOUSE

Q8 µBU7

Q8 µBU7_MOUSE

Q8BWM5

Q8BWM5_MOUSE

Q8BWX0

Q8BWX0_MOUSE

Q8C5R8

Q8C5R8_MOUSE

Q8CHP8 Q8CI65 Q8CIE8 Q8QZT1 Q8R1P0 Q91Z53 Q91ZB1 Q920Z2 Q924B0 Q99L13

Q8CHP8_MOUSE Q8CI65_MOUSE Q8CIE8_MOUSE THIL_MOUSE Q8R1P0_MOUSE GRHPR_MOUSE Q91ZB1_MOUSE Q920Z2_MOUSE Q924B0_MOUSE 3HIDH_MOUSE

Q99LW9 Q99LX0 Q99N15

Q99LW9_MOUSE PARK7_MOUSE Q99N15_MOUSE

Q9CQ60

6PGL_MOUSE

1268

protein name

MW

pI

GRAVY

D-dopachrome tautomerase Hemoglobin beta-1 subunit L-lactate dehydrogenase A chain Malate dehydrogenase, mitochondrial [Precursor] Glycerol-3-phosphate dehydrogenase [NAD+], cytoplasmic L-lactate dehydrogenase B chain Thioredoxin-dependent peroxide reductase, mitochondrial [Precursor] Ribonuclease UK114 ATP synthase beta chain, mitochondrial [Precursor] Profilin 1 Prohibitin Peroxiredoxin 5, mitochondrial [Precursor] CDNA, RIKEN full-length enriched library, clone:G430090B05 product:D-dopachrome tautomerase, full insert sequence Adult male kidney cDNA, RIKEN full-length enriched library, clone:0610010P14 product chaperonin subunit 2 (beta), full insert sequence Adult male kidney cDNA, RIKEN full-length enriched library, clone:0610006N18 product:lactate dehydrogenase 2, B chain, full insert sequence Hemoglobin beta minor Beta-1-globin [Fragment] Heat-responsive protein 12 Hbb-b1 protein Profilin 1 Serine/threonine protein phosphatase 2A, 65 kDa regulatory subunit A, alpha isoform Alpha isoform of regulatory subunit A, protein phosphatase 2 Mus musculus 12 days embryo male wolffian duct includes surrounding region cDNA, RIKEN full-length enriched library, clone:6720460A04 product:ELECTRON TRANSFER FLAVOPROTEIN ALPHA-SUBUNIT, MITOCHONDRIAL (ALPHA-ETF) homolog Mus musculus adult male small intestine cDNA, RIKEN full-length enriched library, clone:2010200I21 product:ELECTRON TRANSFER FLAVOPROTEIN ALPHA-SUBUNIT, MITOCHONDRIAL (ALPHA-ETF) homolog Mus musculus 2 days neonate thymus thymic cells cDNA, RIKEN full-length enriched library, clone:C920001L03 product:similar to GLYCEROL-3-PHOSPHATE DEHYDROGENASE Mus musculus 12 days embryo spinal cord cDNA, RIKEN full-length enriched library, clone:C530043L06 product:similar to HCDI PROTEIN Mus musculus adult male testis cDNA, RIKEN full-length enriched library, clone:4933430D15 product:ribose-phosphate pyrophosphokinase (EC 2.7.6.1) catalytic chain I homolog RIKEN cDNA 1700012G19 Atp5b protein [Fragment] Dlst protein Acetyl-CoA acetyltransferase, mitochondrial [Precursor] Malate dehydrogenase 2, NAD Glyoxylate reductase/hydroxypyruvate reductase Dihydrolipoamide S-acetyltransferase [Fragment] B7 olfactory receptor Myo-inositol monophosphatase 1 3-hydroxyisobutyrate dehydrogenase, mitochondrial [Precursor] Pdhb protein [Fragment] DJ-1 protein 17beta-hydroxysteroid dehydrogenase type 10/short chain L-3-hydroxyacyl-CoA dehydrogenase 6-phosphogluconolactonase

12946.0 15708.9 36367.3 35596.4 37441.5

6.15 7.26 7.76 8.82 6.83

0.075 0.064 0.059 0.141 0.097

36441.1 28127.0

5.70 7.15

0.043 0.016

14124.2 56300.4 14826.0 29820.1 21897.4 13077.2

8.73 5.19 8.50 5.57 9.10 6.09

0.122 0.033 0.018 0.009 0.176 0.091

57477.2

5.97

0.003

36572.3

5.70

0.049

15878.2 15708.9 14255.4 15834.2 14957.2 65191.4

7.85 7.26 8.73 8.56 8.46 5.00

0.022 0.064 0.136 0.060 0.031 0.072

65266.4

4.96

0.068

34995.4

8.62

0.129

34950.3

8.42

0.139

34634.3

6.46

0.108

31458.1

9.33

0.075

34820.2

6.08

0.052

34540.6 56666.8 22229.8 44816.1 35611.4 35328.8 59084.7 35149.9 30429.2 35439.9

5.20 5.24 6.43 8.71 8.93 7.57 5.71 8.95 5.08 8.37

0.004 0.032 0.050 0.080 0.118 0.029 0.001 0.733 0.018 0.030

34836.2 20021.3 27273.5

5.63 6.32 8.88

0.028 0.022 0.203

27254.4

5.55

0.149

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technical notes

Myung and Lubec

Table 1 (Continued) acc. no.

entry name

protein name

MW

pI

GRAVY

Q9CQR4 Q9CRZ2

THEM2_MOUSE Q9CRZ2_MOUSE

15182.8 14394.5

8.95 8.09

0.009 0.072

Q9CXF8

Q9CXF8_MOUSE

12432.4

9.24

0.141

Q9CY12

Q9CY12_MOUSE

15763.0

7.13

0.042

Q9CZC8 Q9D051

SCRN1_MOUSE ODPB_MOUSE

56300.4 38937.0

5.19 6.41

0.033 0.057

Q9D3D9 Q9D7G0 Q9JLZ3

ATPD_MOUSE PRPS1_MOUSE AUHM_MOUSE

Thioesterase superfamily member 2 Mus musculus 13 days embryo liver cDNA, RIKEN full-length enriched library, clone:2510040N07 product:hemoglobin, beta adult major chain, full insert sequence. [Fragment] Mus musculus 14 days embryo liver cDNA, RIKEN full-length enriched library, clone:4430402N11 product:synuclein, alpha, full insert sequence Mus musculus 13 days embryo liver cDNA, RIKEN full-length enriched library, clone:2510039D09 product:hemoglobin, beta adult major chain, full insert sequence Secernin 1 Pyruvate dehydrogenase E1 component beta subunit, mitochondrial [Precursor] ATP synthase delta chain, mitochondrial [Precursor] Ribose-phosphate pyrophosphokinase I Methylglutaconyl-CoA hydratase, mitochondrial [Precursor]

17600.0 34717.0 33394.9

5.03 6.56 9.56

0.169 0.027 0.020

pH gradient of any narrow (or wider) IPG strip and it can be loaded onto IPG strips without any need for further treatment. As a corollary of the above point, much reduced chances of protein precipitation will occur. In fact, when an entire cell lysate is analyzed in a wide gradient, there are fewer risks of protein precipitation; on the contrary, when the same mixture is analyzed in a narrow gradient, massive precipitation of all nonisoelectric proteins could occur. Much higher sample loads can be operative, permitting detection of low-abundance proteins because only proteins co-focusing in the same IPG interval will be present. In addition, it helps when analyzing the proteome by MS that may suffer from signal suppression due to the presence of given species that are well ionized and present in high amounts. By properly exploiting this prefractionation device, Pedersen et al. have been able to capture and detect a large number of the previously “undetected” yeast membrane proteins23 and Westman and co-workers24 succeeded in the identificstion of a relatively large glioma cell proteome. In this study, we undertook such an approach in brain tissue in order to improve final resolution and to increase the probability of detection of as many proteins as possible including low-abundance proteins and hydrophobic proteins such as membrane proteins performing prefractionation followed by 2-DE and MALDI-MS analysis. The methodological design of protein expression studies in mouse brain identified 2673 proteins covering several protein classes and cascades representing improvements in the area of brain protein profiling leading to a revamp of traditional approaches for 2-DE.

Experimental Section Whole Tissue Lysate from Mouse Brain. The animal studies were conducted according to the guidelines of the American Physiological Society. Whole brains from 12-week-old male C57BL/6JHim (C57) mice were obtained and stored in liquid nitrogen. This strain was selected because it is widely used in neuroscience and well-documented in biochemical, behavioral, and morphological terms. Brains were kept at -80 °C until biochemical assays were performed. The freezing chain was never interrupted until analysis Prefractionation Using Zoom-IEF Fractionator. Mouse brain samples were prefractionated using the Zoom-IEF Frac-

tionator according to the suppliers’ protocol. Briefly, mouse brains were homogenized, powderized in liquid nitrogen and dissolved in 7 M urea 2 M thiourea 4% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate) (Invitrogen, Vienna, Austria), 65 mM DTT (dithiothreitol) and 1 mM EDTA (ethylenediaminetetraacetic acid) (Merck KGaA, Darmstadt, Germany), protease inhibitors cocktail (Roche, Basel, Switzerland) and 1 mM PMSF (phenylmethylsulfonyl chloride) (Sigma, Carl Roth, Germany) and sonicated on ice for 10 rounds of 10 s each, at ∼50% power (Sonoplus GM70 from Bandelin Electronics, Berlin, Germany). The lysate was incubated for 60 min at room temperature and centrifuged at 20 000 × g for 60 min at 12 °C. The supernatant was transferred to Ultrafree-4 centrifugal filter units (Millipore, Bedford, USA) for the purpose of concentration and desalting. To determine protein concentration of the lysate, the Bradford protein assay method with BSA as a standard was used.25 Samples were diluted to 0.6 mg/ mL for IEF. 670 µL of sample was loaded onto five different pH chambers in the Zoom-IEF Fractionator (pH 3.0-4.6, pH 4.6-5.4, pH 5.4-6.2, pH 6.2-7.0, and pH 7.0-10.0) and subsequent fractionations processed at 100 V for 20 min, 200 V for 80 min followed by 600 V for 80 min. The fractionated samples were collected, desalted, concentrated, and stored at -80 °C until used for 2-DE. Two-Dimensional Gel Electrophoresis (2-DE). Samples prepared from each fraction were subjected to 2-DE as described elsewhere.26 0.6 mg protein was applied on immobilized nonlinear gradient strips (18 cm). Solubilized samples were combined with rehydration buffer (7 M urea, 2 M thiourea, 65 mM DTT, 0.2% w/v Pharmalyte 3-10 and trace amounts of bromophenol blue) to a final volume of 350 µL and rehydrated for 12 h. A 600-µg portion of total protein from each lysate were applied to the IPG strips (pH 3-10, pH 4.5-5.5, pH 4-7, pH 6-9, and pH 6-11). The resulting sample preparations were loaded into the IPG strip holder and subsequent rehydration of the IPG strips proceeded. Proteins were isoelectrically focused on an IPGphore electrophoresis unit (Amersham Bioscience, Uppsala, Sweden) and focused strips were equilibrated for 15 min at room temperature with continuous shaking in equilibration buffer containing 6 M urea, 20% glycerol, 2% SDS, 2% DTT, and subsequently for 15 min in the same buffer but containing 2.5% (w/v) iodoacetamide instead Journal of Proteome Research • Vol. 5, No. 5, 2006 1269

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Separation of Mouse Brain Proteins

of DTT. Upon completion of equilibration the IPG strips were embedded in 9-16% gradient sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) for second-dimensional separation. The gels (180 × 200 × 1.5 mm) were run at 40 mA per gel by the method of Laemmli27 until the dye front reached the bottom of the gel. Molecular masses were determined by markers according to the supplier’s instruction (Bio-Rad, Hercules, USA). Experiments were run in triplicate. Protein Visualization. Immediately after second dimensional running, gels were fixed for 18 h in 50% methanol, containing 10% acetic acid. Protein spots separated on 2-D gels were visualized by Colloidal Coomassie Blue staining kit (Novex, San Diego, CA) for 12 h on a rocking shaker and excess dye was destained with distilled water. 2-D gels were scanned on an ImageScanner (Amersham Bioscience). MALDI-TOF and MALDI-TOF/TOF. Protein spots stained by Colloidal Coomassie Blue were excised with a spot picker (PROTEINEER sp from Bruker Daltonics, Bremen, Germany), placed into 96-well microtiter plates and destaining, in-gel digestion and sample preparation for MALDI analysis were performed by an automated procedure (PROTEINEER dp, Bruker Daltonics).28 Briefly, spots were excised and washed with 10 mM ammonium bicarbonate and 50% acetonitrile in 10 mM ammonium bicarbonate. After washing, gel plugs were shrunk by addition of acetonitrile and dried by blowing out the liquid through the pierced well bottom. The dried gel pieces were reswollen with 40 ng/µL trypsin (Roche) in enzyme buffer (consisting of 5 mM Octyl β-D-glucopyranoside (OGP) and 10 mM ammonium bicarbonate) and incubated for 4 h at 30 °C. Peptide extraction was performed with 10 µL of 1% TFA (trifluoroacetic acid) (TFA is highly corrosive and acute toxic through inhalationa and work was carried out in compliance with. Material Safety Data Sheets, www.msdsonline.com) in 5 mM OGP. Extracted peptides were directly applied onto a target (AnchorChip, Bruker Daltonics) that was load with CHCA (Rcyano-4-hydroxy-cinnamic acid) (Bruker Daltonics) matrix thinlayer. The mass spectrometer used in this work was an Ultraflex TOF/TOF (Bruker Daltonics) operated in the reflector for MALDI-TOF peptide mass fingerprint (PMF) or LIFT mode for MALDI-TOF/TOF with a fully automated mode using the FlexControl software. An accelerating voltage of 25 kV was used for PMF. Calibration of the instrument was performed externally with [M+H]+ ions of angiotensin I, angiotensin II, substance P, bombesin, and adrenocorticotropic hormones (clip 1-17 and clip 18-39). All MALDI spectra were internally calibrated with tryptic auto digest ions. Each spectrum was produced by accumulating data from 200 consecutive laser shots. Those samples which were analyzed by PMF from MALDI-TOF were additionally analyzed using LIFT-TOF/TOF MS/MS from the same target. A maximum of three precursor ions per sample were chosen for MS/MS analysis. In the TOF1 stage, all ions were accelerated to 8 kV under conditions promoting metastable fragmentation. After selection of jointly migrating parent and fragment ions in a timed ion gate, ions were lifted by 19 kV to high potential energy in the LIFT cell. After further acceleration of the fragment ions in the second ion source, their masses could be simultaneously analyzed in the reflector with high sensitivity. PMF and LIFT spectra were interpreted with the Mascot software (Matrix Science Ltd, London, UK). Database searches, through Mascot, using combined PMF and MS/MS datasets were performed via BioTools 2.2 software (Bruker Daltonics). 1270

Journal of Proteome Research • Vol. 5, No. 5, 2006

System for Identification of Proteins. Identification of proteins from MALDI-MS spectra was achieved using Peptide Mass Fingerprint (PMF) of Mascot (http://www.matrixscience. com) and Peptide Mapping of Profound (http://www.unb.br/ cbsp/paginiciais/profound.htm). Monoisotopic peptide masses were matched against the MSDB database using 25 ppm mass tolerance, limited to the Mus musculus L. species. Oxidation of methionine residues was considered but no missed cleavage was allowed. The probability score calculated by the software was used as a criterion for correct identification in Mascot.29 MS/MS tolerance of 0.5 Da and 1 missing cleavage site for MS/ MS search was allowed. Database searches were performed based on Swiss-Prot database (http://www.expasy.org/sprot) and PubMed search (http://www.ncbi.nlm.nih.gov/entrez/ query.fcgi?db)PubMed). The algorithm used for determining the probability of a false positive match with a given mass spectrum is described elsewhere.30 Computational Analysis of Hydrophobicity and Predicting of Transmembrane Domains. The Grand Average of Hydropathy (GRAVY) value for a peptide or protein is calculated as the sum of hydropathy values31 of all the amino acids, divided by the number of residues in the sequence and shows the hydrophobicity of proteins. GRAVY value as well as theoretical pI and molecular weight were calculated in http://www. expasy.org/tools/ protparam.html and the alignment of two protein sequences was done with BLAST engine at NCBI website (http:// www. ncbi.nlm.nih.gov/blast/ bl2seq/ bl2.html).32 Prediction of the number of transmembrane domains in all proteins was investigated using the TMHMM (http:// www.cbs.dtu.dk/services/TMHMM/)33 method which is considered the most selective method for avoiding false positive predictions.34 All proteins within positive GRAVY values were applied to TopPred (http://bioweb.pasteur.fr/seqanal/ interfaces/toppred.html)35 and the ConPredII (http://bioinfo. si.hirosaki-u.ac.jp/∼ConPred2/)36 method to compare the prediction of the number of transmembrane domains.

Results and Discussion Proteomic Profiling of Mouse Brain Lysates. Taking into account the total number of proteins found in all five chambers, a total of 4968 spots were excised from 5 gels and 2673 spots corresponding to 581 proteins could be identified (Figure 1). On average, each protein was represented by 4.6 spots. A 600µg portion of mouse brain lysate protein in each chamber were separated by the isoelectric point. Proteins collected from each fraction were separated according to their pI and apparent molecular weight by 2-DE and digested in solution by trypsin to generate complex peptide mixtures. Peptides produced by trypsin digestion were then introduced into a mass spectrometer where mass-to-charge (m/z) ratios are measured and fragmentation spectra created through mass spectrometry. Peptide sequences and subsequently proteins are identified by unaided searching of uninterpreted mass spectra through NCBI protein database to identify proteins37,38 and the identifications were finally mapped onto analytical gel images. Identified proteins in mouse brain were categorized into several functional groups based on their representative biological roles. Figure 2 shows that the majority of proteins (31.2%) were classified as metabolism proteins, followed by 17.4% involved in miscellaneous proteins, 14.8% signaling proteins and neuronal proteins except hypothetical proteins revealed the smallest percentage (approximately 2%) of proteins identified. All identified proteins with SwissProt accession number,

technical notes

Myung and Lubec

Figure 1. Schematic illustration of a strategy for comprehensive analysis of mouse brain proteomes using MicroSol-IEF prefractionation andIPG-based 2-DE gel electrophoresis. Typically, five pH ranges were selected for mouse brain fractionation using MicroSol-IEF as indicated by the values above immobiline gel partitions in the diagram. An advantage of this scheme is that the number of fractions and their pH ranges can easily be adjusted to fit varying experiments.

Figure 2. Distribution of the identified individual proteins (A) and all expressions form (B) from mouse brain assigned to functional groups.

entry name, theoretical pI and molecular weight (MW) values, GRAVY values, the number of identified spots were summarized in Supporting Information Table 2 and a representative 2-D gel of mouse brain proteins annotated with their Swiss-Prot accession numbers is shown in Supporting Information Figure

1. Data obtained from MS and MS/MS analysis by MASCOT search are presented in Supporting Information Table 3. A including score, matched peptides numbers (matches) and sequence coverage (%) and data searched by PROFOUND are shown in Supporting Information Table 3B. Supporting Information Figure 2 shows the distribution of mouse brain proteins identified and sorted according to their theoretical molecular mass (Supporting Information Figure 2A) and pI (Supporting Information Figure 2B) values. Most proteins have masses between 10 and 100 kDa. Only one protein with a mass below 10 kDa and a small number of proteins with masses above 150 kDa were observed. Similarly, no protein with pI below 4 and only a few with pI values higher than 10 were detected. A large part of proteins was represented by several spots with different pI’s. Multiple Protein Expression Forms. On 2-D gels, the proteins are often represented by more than one spot, so that the number of expressed products is much higher than the number of the corresponding encoding genes. In prokaryotes, usually 1-2 proteins correspond to one gene and in eukaryotes 5-20 proteins may be derived from one gene.39 In this study, 294 proteins (about 51% of all identified proteins) showed more than one spot. In this study, the nature of heterogeneity was not studied. Identification of Proteins with Positive GRAVY Value and Predicted to have Transmembrane Domains. Frequently, unknown proteins are identified by high-throughput profiling of hydrophobic or membrane proteins. Analysis of protein sequences using computational tools is performed to calculate GRAVY values for prediction of hydrophobic proteins and to predict the presence of transmembrane domains. Along with technological innovations, advancements in the areas of sample preparation and computational prediction will lead to exciting discoveries.40 Journal of Proteome Research • Vol. 5, No. 5, 2006 1271

technical notes

Separation of Mouse Brain Proteins

Table 2. Proteins with Predicted Transmembrane Domains Using All TMHMM, ConPredII, and TopPred Databasesa,b acc. no

entry name

O35435

PYRD_MOUSE

P24369 P56395 P57759 Q8CBX6

PPIB_MOUSE CYB5_MOUSE ERP29_MOUSE Q8CBX6_MOUSE

Q8CIB4 Q920Z2 Q9CQ92 Q9D0M3

Q8CIB4_MOUSE Q920Z2_MOUSE TTC11_MOUSE CY1_MOUSE

Q9EQ20

Q9EQ20_MOUSE

protein name

TM

CP

TP

Dihydroorotate dehydrogenase, mitochondrial [Precursor] Peptidyl-prolyl cis-trans isomerase B [Precursor] Cytochrome b5 Endoplasmic reticulum protein ERp29 [Precursor] Mus musculus adult male diencephalon cDNA, RIKEN full-length enriched library, clone:9330181J03 product:glycerol phosphate dehydrogenase 1, mitochondrial, full insert sequence Aldehyde dehydrogenase family 6, subfamily A1 B7 olfactory receptor Tetratricopeptide repeat protein 11 Cytochrome c1, heme protein, mitochondrial [Precursor] Methylmalonate-semialdehyde dehydrogenase

1

1

3

1 1 1 1

1 1 1 1

1 1 1 4

1 7 1 1

1 6 1 2

5 7 1 4

1

1

5

a Abbreviations used: Acc. No, Swissprot accession number; TM, TMHMM; CP, ConPredII; TP, TopPred. b Prediction of the number of transmembrane domains (TMD) in all proteins was investigated using TMHMM (http://www.cbs.dtu.dk/services/TMHMM)33 method which is the most selective method for avoiding false positive predictions,34 ConPredII and TopPred.

GRAVY values of all identified proteins were calculated in http://www.expasy.org /tools/prot param.html and 255 protein spots corresponding to 50 proteins showed positive GRAVY values (Table 1). The highest GRAVY value was 0.733 of B7 olfactory receptor (Acc. No. Q920Z2) and second, 17 betahydroxysteroid dehydrogenase type 10/short chain L-3-hydroxyacyl-CoA dehydrogenase (Acc. No. Q99N15) showed a GRAVY value of 0.203. Prediction of the presence of transmembrane domains in all proteins was investigated using the TMHMM (http:// www.cbs.dtu.dk/services/TMHMM/) method, the most selective method for avoiding false positive predictions34 and predictions showed that only 10 proteins had 1 transmembrane domain. 27 proteins including those proteins found by using TMHMM were predicted to have transmembrane domains when all proteins with positive GRAVY values were applied to ConPreII (http://bioinfo.si.hirosaki-u.ac.jp/∼ConPred2/). When searched by TopPred (http://bioweb.pasteur.fr/seqanal/ interfaces/toppred.html), 48 proteins including those proteins that were predicted by using TMHMM and ConPredII were shown to have transmembrane domains. The proteins that have transmembrane domains are shown in Table 2, Supporting Information Tables 4 and 5 and are presented in Figure 3. The presence of transmembrane domains in B7 olfactory receptor (Acc. No. Q920Z2) protein was also predicted, which was showed to have 7, 6, and 7 transmembrane domains by using TMHMM, ConPredII and TopPred respectively (Supporting Information Figure 3). In addition, 17 beta-hydroxysteroid dehydrogenase type 10/short chain L-3-hydroxyacyl-CoA dehydrogenase (Acc. No. Q99N15) was predicted to have 2 and 3 transmembrane domains by ConPredII and TopPred methods and the properties are shown in Supporting Information Figure 4. Identification of Low Molecular Weight Proteins and Very Acidic and Basic Proteins. The utility of many approaches is usually limited by the inability to reliably monitor very low molecular weight proteins as well as proteins that are very acidic or basic. Herein, cytochrome c oxidase polypeptide Vib protein (Acc. No. P56391) whose molecular weight is 9940.2 Da was identified. It was not significantly identified with MS analysis because of the small size of sequence although 4 peptides are matched covering 38% of the sequence. But after database search with PMF, a MS/MS spectrum from m/z 1272

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Figure 3. Distribution of mouse brain proteins predicted to have transmembrane domains by TMHMM, ConPredII, and TopPred (A) and diagram (B).

1552.70 was acquired that assigned the identified sequence NCWQNYLDFHR to the fragmented form of cytochrome c oxidase polypeptide Vib protein (Supporting Information Figure 5); combined MS and MS/MS database search provided a unique and safe assignment with significant Mascot score. In addition, other low molecular weight proteins, such as BolAlike protein My016 homologue (Q8BGS2; theoretical MW 10214.5 Da), 10 kDa heat shock protein, mitochondrial (Q64433; 10831.5 Da), Alpha-tubulin [Fragment] (O89052; 10951.1 Da) and 60S acidic ribosomal protein P2 (P99027; 11650.9 Da) were unambiguously identified in mouse brain. In case of very basic proteins, we identify 1.2% of proteins with pI values higher than 10. Splice isoforms of myelin basic protein (Acc. No. P043707) and Histone H2A [Fragment] were unambiguously assigned with combined MS and MS/MS database searches and spectra were shown in Supporting Information Figures 6 and 7.

technical notes Conclusions A major finding of this study is proteomic profiling of mouse brain remarkably extended the amount of brain proteins usually presented using two-dimensional gel electrophoresis (2-DE). Moreover, a series of proteins containing at least one transmembrane domain (TMD) and of proteins with positive GRAVY indices, hydrophobic proteins, was identified thus representing a considerable step forward. Researches using three different databases for prediction of TMDs resulted into detection of 10 individual proteins with positive predictions of at least one TMDs in all three databases (Table 2). Among these proteins, the B7 olfactory receptor containing 6-7 TMDs was observed.41 When proteins were computed into three databases and the result predicted the presence of a TMD in at least one database, 48 structures were identified as membrane proteins (defined by the presence of a transmembrane domain; Supporting Information Tables 4 and 5). This shows the enormous difference in the outcome of database searches and this fact must be taken into consideration when a protein is classified as a membrane protein. In this context, it is stated that in many publications also membrane-associated proteins are classified as membrane proteins. The 50 proteins with positive GRAVY values are thus unambiguously predicted as hydrophobic proteins and indeed, the B7 olfactory receptor with strong hydropathy was included in this panel. It should be made clear that membrane proteins are not necessarily hydrophobic proteins and vice versa; moreover, not all highly insoluble proteins are hydrophobic proteins. Prefractionation using this principle has already been reported: Zuo and Speicher have developed a solution isoelectrofocusing device that can reproducibly prefractionate protein extracts into well-defined pools of individual pI’s.42 When this prefractionation by pI is applied to complex proteomes, resolution and spot recovery at relatively high protein loads21 and allows determination of some low abundance proteins.43 Using this technology, also larger proteins can be separated.44 A solution isoelectrofocusing device was created by the authors and is now a commercially available product.22 The use of this principle with adaptation to the brain matrix,45 herein allowed identification of approximately 2673 brain protein spots, that is much more than obtained with separation of brain proteins with the same solvents and detergents on an one-dimensional gel. This and the fact that hydrophobic and transmembrane proteins can be determined, represents an advancement in terms of nonsophisticated and nontime-consuming protein separation by 2-DE technology. The method also allowed identification of proteins that either have not been described at the protein chemical level or have never been described in the brain: Herein rare proteins as Sar1, promoting vesicle budding46 and charged multivesicular body protein 4b47 were observed. Signaling cascade proteins MDP-1, a novel eukaryotic magnesium-dependent phophatase,48 dopamine-and cAMP-regulated phosphoprotein of 32 kDa,49 neurocalcin delta, a calcium sensor of signaling,50 epsin-1, a novel adaptor protein for signaling,51 ras-related protein Rab14,52 oncostatin M receptor of interleukin signaling,53 anamorsin, a cytokine-induced apoptosis inhibitor 154 could be detected in the brain at the and revealed by a fair proteomic method rather than immunochemical techniques.

Myung and Lubec

The method as it stands can be easily extended by the use of different tissue extraction techniques, detergents and solvents. It is particularly useful for determination of brain proteins including myelins (Supporting Information Table 2) as the brain represents a lipidic and hydrophobic matrix and therefore isoelectrofocusing prior to 2DE with narrow pI strips may be considered a valuable tool for neuroproteomics.

Acknowledgment. The authors are grateful to MarieChristine Horer and Daniela Pollak for their technical assistance. We acknowledge the partial contribution from the Verein zur Durchfu ¨hrung der Wissenschaftlichen Forschung auf dem Gebiet der Neonatologie und Kinderintensiv-medizin (“Unser Kind”). Supporting Information Available: (A) tabular presentation of data and information. List of various fractionation methods used to separate proteins based on a particular physical or chemical property17 List of identified mouse brain proteins by ZOOM-IEF Fractionator except hydrophobic and membrane proteins. List of identified mouse brain proteins with the data from mass spectrometry analysis including score, pI, peptides matched, sequence coverage by MASCOT(A) and PROFOUND(B). Proteins with transmembrane domains predicted using ConPredII and TopPred. Proteins with transmembrane domains predicted using only TopPred. (B) Figures providing supplemental information and data. The 2-D maps of fractionated mouse brain proteins. 2-DE was performed in an immobilized pH 3-10 (A), pH 4.5-5.5 (B), pH 4-7 (C), pH 6-9 (D) and pH 6-11 (E) linear gradient strips respectively, followed by 9-16% SDS-PAGE, and separated proteins were detected by colloidal coomassie blue staining. The spots were analyzed by MALDI-MS or MS/MS. The identified proteins are designated by their Swiss-Prot accession number. The names of the proteins are listed in Supporting Information Table 2. White and black arrows are only provided to cope with dark or bright background. MS-TOF Distribution of mouse brain proteins in relation to their molecular mass and pI. The about 581 mouse brain proteins were sorted according to their molecular mass (A) and pI values (B). The number of proteins found in the indicated molecular mass and pI intervals is shown. The Kyte & Doolittle Protscale for GRAVY value (A) and the profiles of prediction of transmembrane domains by TMHMM (B), ConPredII (C) and TopPred (D) of B7 olfactory receptor (Acc. No. Q920Z2). The Kyte & Doolittle Protscale for GRAVY value (A) and the profiles of prediction of transmembrane domains by TMHMM (B), ConPredII (C) and TopPred (D) of 17beta-hydroxy steroid dehydrogenase type 10/short chain L-3-hydroxyacyl-CoA dehydrogenase (Acc. No. Q99N15). MS-TOF spectrum of cytochrome c oxidase polypeptide Vib (A) and LIFT-TOF/TOF spectrum of m/z 1552.698 (B) that unambiguously assigned the identified sequence NCWQNYLDFHR to cytochrome c oxidase polypeptide Vib (Acc. No. P056391). MS-TOF spectrum of splice isoform 7 of myelin basic protein (A) and LIFT-TOF/TOF spectrum of m/z 1460.745 (B) that unambiguously assigned the identified sequence TQDENPVVHFFK to splice isoform 7 of myelin basic protein (Acc. No. P04370-7). MS-TOF spectrum of histone H2a (A)-613 (A) and LIFT-TOF/TOF spectrum of m/z 1931.190 (B) that unambiguously assigned the identified sequence VTIAQGGVLPNIQAVJournal of Proteome Research • Vol. 5, No. 5, 2006 1273

Separation of Mouse Brain Proteins

LLPK to Histone H2a (A)-613 (Acc No Q64523). This material is available free of charge via the Internet at http://pubs.acs.org.

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