High Recovery HPLC Separation of Lipid Rafts for Membrane

High Recovery HPLC Separation of Lipid Rafts for Membrane Proteome Analysis James Martosella,*,† Nina Zolotarjova,† Hongbin Liu,† Susanne C. Moyer,† Patrick D. Perkins,‡ and Barry E. Boyes†,§ Agilent Technologies, Proteomics Reagents and Separations, 2850 Centerville Road, Wilmington, Delaware 19808, Proteomics Applications Development, 5301 Stevens Creek Blvd., Santa Clara, California 95051, and Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens, Georgia 30602 Received February 16, 2006

Proteomic analysis of complex samples can be facilitated by protein fractionation prior to enzymatic or chemical fragmentation combined with MS-based identification of peptides. Although aqueous soluble protein fractionation by liquid chromatography is relatively straightforward, membrane protein separations have a variety of technical challenges. Reversed-phase high performance liquid chromatography (RP-HPLC) separations of membrane proteins often exhibit poor recovery and bandwidths, and generally require extensive pretreatment to remove lipids and other membrane components. Human brain tissue lipid raft protein preparations have been used as a model system to develop RP-HPLC conditions that are effective for protein fractionation, and are compatible with downstream proteomic analytical workflows. By the use of an appropriate RP column material and operational conditions, human brain membrane raft proteins were successfully resolved by RP-HPLC and some of the protein components, including specific integral membrane proteins, identified by downstream SDS-PAGE combined with in-gel digestion, or in-solution digestion and LC-MS/MS analysis of tryptic fragments. Using the described method, total protein recovery was high, and the repeatability of the separation maintained after repeated injections of membrane raft preparations. Keywords: proteomics • pre-fractionation • reversed-phase chromatography • integral membrane proteins • lipid rafts • mass spectroscopy • gel electrophoresis • HPLC

Introduction Cellular membrane proteins are an important class of proteins that play a critical role in cellular processes, and thus account for a large percentage of drug targets.1,2 However, despite their biological and biomedical significance, this class of proteins remains an under-represented subset of studied proteins by available proteomic technologies. Membrane protein structure analysis remains a considerable challenge, due in part to problems associated with their inherent amphipathic character, and correspondingly poor solubility in aqueous solutions.3 Often, detergents are required for solubility in aqueous, or aqueous/organic mixtures, and are needed to dissociate heterogeneous complexes of lipids, associated proteins, and other biological macromolecules from protein targets.4 A consequence of this complex solution behavior, and complexity of structure, is considerable difficulty in developing efficient chromatographic methods for separation and recovery of membrane proteins. Although progress has been made with membrane protein separations in certain liquid chromatogra* To whom correspondence should be addressed. Tel: (302) 633-8666. Fax: (302) 633-8908. E-mail: [email protected]. † Proteomics Reagents and Separations, Agilent Technologies. ‡ Proteomics Applications Development, Agilent Technologies. § Complex Carbohydrate Research Center, University of Georgia. 10.1021/pr060051g CCC: $33.50

 2006 American Chemical Society

phy (LC) modes of operation, such as size exclusion and ion exchange,5-9 high efficiency separations generally require the addition of detergents to the mobile phase.5,10 Reversed-phase (RP) separations can be highly efficient for protein separations, but poorly tolerate the presence of detergent in samples, and are all but inoperable with the addition of detergent to the mobile phase. Whitelegge and colleagues3,9 have recently reported on the successful use of RP for the analysis of membrane proteins, but have yet to show protein recovery information and compatibility with a bottom-up proteomic strategy and use in a global proteomic analysis. The goal of this work was to apply RP methods to high efficiency separations of membrane proteins. For our purposes, lipid rafts are a readily accessible source of membranous material enriched in integral membrane proteins, and as such, a good starting material to define separations conditions that may have broad utility. Lipid rafts, also referred to as detergent resistant membrane fragments, are localized cell membrane regions, or subdomains, and are enriched in cholesterol, glycosphingolipids, and integral membrane proteins.11-15 These heterogeneous membrane fragments were originally associated with lipid trafficking, but now appear to be implicated in a variety of biological processes, such as signal transduction, endocytosis, protein processing, and pathogen entry.16-19 Lipid Journal of Proteome Research 2006, 5, 1301-1312

1301

Published on Web 05/13/2006

research articles rafts are commonly prepared from cultured cells and disrupted tissues by selective solubilization with detergents, followed by differential centrifugation. Significant dependencies on protein and lipid compositions of such preparations are seen based on the conditions used for detergent extraction and sample workup.6,13 A variety of biophysical, biochemical, and proteomic approaches have been applied to the definition of the composition of lipid rafts. Proteomic analyses of lipid rafts have mainly used electrophoretic separations at the protein level, using either 1D or 2D PAGE, and/or cleavage of heterogeneous mixtures of lipid raft proteins, followed by separation of tryptic or chemical cleavage fragments by 1D and/or 2D LC, usually with online electrospray MS detection.11,20-24 These approaches have largely supplied protein identification biased toward peripheral membrane proteins, particularly outer membrane proteins, with a distinct tendency to under-represent integral membrane proteins.25,26 Alternatively, RP separations can be highly efficient for protein separations, but poorly tolerate the presence of detergent in samples, and are all but inoperable with the addition of detergent to the mobile phase. To be applicable for membrane protein separations, the RP separation should resolve proteins effectively, tolerate the presence of residual detergents, complex lipids and other naturally occurring membrane materials. To be useful for proteomic analyses, the separation needs to be both highly reproducible and to demonstrate high protein recoveries, thereby avoiding the problem of introducing systematic bias for proteomic comparative studies. This work initially focused on the conditions needed for the complete solubilization of lipid raft proteins, starting with a lipid raft-enriched membrane fraction obtained from frozen post-mortem mouse brain. Proper membrane protein solubilization is a necessary prerequisite to reversed-phase analysis of this material. Useful RP operational conditions have been developed using a recently described column packing material that yields high recovery of the soluble proteins of human serum and plasma.27 As observed in this previous study, elevated column temperature operation is required to yield high protein recoveries, and to prevent rapid column fouling. Although these methods provided useful starting conditions, they were not sufficient for the reversed-phase separation of membrane proteins. We observed that specific sample treatment and mobile phases are needed to enable partial or full disassociation of protein from the membrane lipids, as well as for the regeneration of the RP surface by eluting strongly absorbed lipids and related materials. On-column delipidation resulted in resolved protein fractions with reduced lipid content and delayed elution of very hydrophobic components occurring late in the separation. The method described yields highly reproducible separations with protein fractions that are readily solubilized for downstream SDS-PAGE or gel-free proteomic analysis. In-gel tryptic digests of RP human brain raft protein fractions were analyzed by LC-MS/MS, leading to the identification of integral membrane proteins.

Experimental Section Materials. HPLC-grade acetonitrile and 2-propanol were purchased from VWR International (West Chester, PA). Formic acid (98-100%) was obtained from Sigma-Aldrich. The water used was Milli-Q grade (Millipore, Bedford, MA). Trifluoroacetic acid and trifluoroethanol were purchased from Sigma (St. Louis, MO). Sequencing grade-modified trypsin was from Promega (Madison, WI). Trypsin In-Gel Digestion Kit was from Agilent 1302

Journal of Proteome Research • Vol. 5, No. 6, 2006

Martosella et al.

(Palo Alto, CA). The 50 mL conical BD polystyrene tubes were obtained from VWR International. Pre-cast gels were obtained from Invitrogen (Carlsbad, CA). The reversed-phase column used in this study was a 4.6 × 50 mm I.D. macroporous reversed-phase C18 column from Agilent Technologies (Wilmington, DE). The 5 peptide mixture used is the Alberta Peptide Institute Standard RPS-P0010 (API, Edmonton, AB).

Methods Preparation of Lipid Raft-Enriched Fraction. The lipid raftenriched fraction was prepared by detergent extraction on ice and flotation on a sucrose gradient. Frozen mouse brains (sectioned at the pons, with cerebellum removed, and hereafter referred to as cerebra; obtained from PelFreez, Rogers, AR), or neurologically normal human brain neocortex (Superior Temporal Gyrus, kindly supplied by Drs. T. Beach and D. G. Walker, Sun Health Research Institute, Sun City AZ) were employed for the preparation of membrane rafts. All steps were carried out on ice or at 4 °C. Pieces of frozen brain tissue (1.2 g) were homogenized on ice in 20 mL of cold TBS (10 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 7.4) with Complete Protease Inhibitor Cocktail (Roche Diagnostics, Indianapolis, IN) using a Polytron homogenizer (Brinkmann, Westbury, NY) (2 times for 10 s). A chilled solution of 20 mL of 2% Triton X-100 in TBS buffer (final concentration was ∼3 mg/mL in 1% Triton X-100) was added to the sample. The sample was mixed by inversion 5 times and incubated on ice for 30 min. After incubation the sample was adjusted to 40% sucrose by the addition of equal volume (40 mL) of 80% sucrose in TBS buffer. A discontinuous sucrose gradient was formed in the centrifuge tubes (Rotor JS - 24.38, Beckman Coulter, Fulerton, CA). A 12-mL portion of sample solution was placed on the top of 1.6 mL of 80% sucrose followed by 12 mL of 35% sucrose and 12 mL of 5% sucrose, all in the presence of TBS. The gradients were centrifuged for 42 h at 103 900 × g. During high-speed centrifugation, lipid rafts migrated to the low-density region of the gradient due to their high lipid content. The low-density material floating at the 5-30% interface was collected, diluted 4 times in TBS and centrifuged for 2 h at 200 000 × g to pellet the detergentresistant fraction. The resulting pellet was resuspended in 2 mL of TBS and pelleted by centrifugation as above. One additional washing of the pellet was performed. Final pellets were dispersed in 2 mL of H2O, then stored at -80 °C until use. Electrophoretic Analysis. SDS-PAGE analysis was performed on Invitrogen Tris-glycine precast gels (4-20% acrylamide, 10 wells, 1 mm). Samples from reversed-phase separations were dried in SpeedVac on low heat. Following resuspension in 2× sample preparation buffer, samples were heated for 1 min at 50 °C, and then loaded onto the gel. Gel-separated proteins were visualized by Coomassie Blue staining using Pierce GelCode Blue. Sample Preparation for HPLC. A 100-µL portion (500 µg protein) of membrane rafts were dried in a centrifugal vacuum concentrator (Thermo-Savant, Millford, MA), resolubilized in 200 µL of 80% formic acid (FA) and sonicated in a water bath for 30 s. The samples were dried in the centrifugal vacuum concentrator, resolubilized in 500 µL of 80% FA and sonicated in a water bath for approximately 30 s or until a clear mixture resulted. The final sample protein concentration was approximately 1 mg/mL in 80% FA. HPLC Separation and Fraction Collection. HPLC separations were performed on an automated Agilent 1100 LC system

research articles

Reversed-Phase Chromatography of Lipid Rafts

with an autosampler equipped with a 900 µL capillary loop. All the RP separations for membrane rafts were performed at 80 °C under a set of optimized conditions using a combined multi-segmented and linear elution gradient, with eluent A (0.1% TFA), eluent B (0.08% TFA in acetonitrile (ACN), v/v), eluent C (20% formic acid in ACN v/v) and eluent D (2propanol). The gradient flow rate was 0.75 mL/min and elution conditions consisted of two segments with increasing concentrations of eluent B: 20-50% B in 40 min, 50-100% B in 10 min and a hold of 100% B and two subsequent linear elutions with increasing and decreasing concentrations of eluent C: 0-100% in 5 min, 100-0% in 5 min and eluent D: 0-100% in 5 min, 100-0% in 5 min for a total runtime of 80 min. For consecutive runs, a 30 min. postrun comprised of 3 min. 100% eluent D, followed by 20% eluent B (80% eluent A) for column reequilibration. Lipid raft separations were monitored at 280 nm and fractions were automatically collected at 1-2 min intervals. Each fraction was dried in a centrifugal vacuum concentrator (Thermo-Savant, Milford, MA) and stored at -80 °C for subsequent SDS-PAGE and LC-MS/MS analysis. RP separations of the five peptide standard was performed using a linear elution gradient of eluent A (0.1% TFA) and eluent B (0.085% TFA in 80/20 ACN/water, v/v) with increasing eluent B from 3 to 20% in 30 min at 0.75 mL/min Chromatograms were monitored at 210 nm. Safety Concern: Working with high concentrations of formic acid at high temperature and pressure is fully cautioned. The HPLC effluent bottle and fraction collector were contained in a fume cupboard in order to trap any volatile acid or vapors. The abatement of such vapors gives assurance to the operators of the LC sytem. Protein Recovery. Lipid rafts were fractionated on a 4.6 mm I.D. × 50 mm mRP-C18 column under the conditions described above. The eluent was collected and samples analyzed according to a procedure described by Martosella et al.27 Briefly, total column effluent was collected into 50 mL polystyrene conical tubes (VWR International). Control runs were performed in the same manner, however, with the column removed from the flow path. Control and column eluates (approximately 47 mL) were dried in a speed vacuum concentrator at medium drying temperatures overnight. Dried samples of column and control runs were solubilized with 0.5 mL of 6 M urea, 1% Triton X-100, and 0.25% acetic acid. Samples were vortexed extensively to solubilize all protein and remove any material adhering to the tube walls. Samples were transferred to Eppendorf tubes and sonicated in a water bath for approximately 2 min. Protein quantitation was performed by BCA protein assay (Pierce). A 50-µL portion of sample was mixed with deionized H2O to a final volume of 100 µL and the protein assay performed according to manufacturer’s suggested protocol. Recovery results were also evaluated using the EZQ Protein Quantitation kit from Invitrogen/Molecular Probes (Carlsbad, CA). A 1-µL portion of each sample was spotted onto assay paper and the samples were fixed, washed and stained with EZQ stain according to the manufacturer’s protocol. Each sample was then processed in quadruplicate and fluorescence measured by scanning on a Typhoon 8600 laser scanner (Amersham Biosciences, Piscataway, NJ) using a 532 nm laser for the excitation and the 580 nm emission filter. Protein In-Gel Digestion and LC-MS/MS. A total of 48 gel bands were cut and digested with trypsin according to the instructions provided in the Trypsin In-Gel Digestion Kit. The

digested peptides were extracted and proteins were identified by LC-MS/MS analysis (Agilent 1100 nano-LC and 1100 MSD trap XCT equipped with a nanoelectrospray ionization source). 70% of the total volume of extracted peptides from each gel band were loaded onto a Zorbax 300SB-C18 (3.5 µm, 0.075 mm I.D. × 150 mm) capillary column. Proteolytic peptide fragments were gradient eluted where Buffer A was 3.0% ACN and 0.1% FA, and Buffer B was 90% ACN and 0.1% FA. The gradient was 5-12% B in 5 min, 12-35% B in 35 min, 35-65% B in 10 min, 65-100% B in 5 min, followed by 5 min 100% B, and reequilibration at 5% B, for a total run time of 62 min. The XCT ion trap mass spectrometer was operated in standard scan mode for MS analysis and in ultra scan mode for MS/MS. Protein In-Solution Digestion and Chip-Based nano-LCMS/MS. Dried human lipid raft protein fractions were resolubilized, reduced, alkylated and then digested with trypsin according to previously published procedures.28 The peptide digests of each mRP-C18 fraction were analyzed with a microfluidic chip-based nano-LC-MS/MS system (Agilent Zorbax 300SB-C18, 5 µm, 0.075 X 0.043 mm HPLC chip, HPLC-Chip/ MS system with 1100 ion trap XCT Ultra). The RP buffers were the same as those described above and the gradient was 5-10% B in 2 min, 10-35% B in 18 min, 35-50% B in 2 min, 50-95% B in 0.5 min, followed by one minute at 95% B and reequilibration at 5% B, for a total run time of 26 min. The ion trap mass spectrometer was calibrated using a builtin automated calibration algorithm, with the tuning mix specifically designed for this type of instrument. MS/MS data were searched against the SwissProt Human database (total of 12015 entries), using Spectrum Mill computer database search algorithm, with the “Calculate Reversed Database Scores” option “on”. The peptide/protein hits were filtered with the “autovalidation” option using the following parameters: minimum score for peptides: +1, 7.0; +2, 8.0 (if SPI larger than 90%, the score was lowered to 7.0); +3, 9.0; +4, 9.0. All peptide matches were required that “Forward-Reverse Score” is larger than 1.0, and “Rank 1-2 score” is larger than 1.0. The protein score was set at minimum of 15.0. Only fully tryptic peptides were considered, with two missed cleavages allowed. Lipid Partitioning and MS Analysis The final set of combined fractions from the mRP-C18 column, which eluted in ACN/FA and 2-propanol, was injected onto a model 1100 capillary LC/MSD quadrapole SL system (Agilent Technologies) using a 0.5 mm I.D. × 150 mm Zorbax SB-C18 column, (3.5 µm, 300 Å), at room temperature. An injection volume of 5 µL was used. The mobile phase was 80% methanol/15% tetrahydrofuran/5% water (v/v/v) containing 0.1% FA at a flow rate of 12 µL/min. The MSD was operated using an ESI source with positive ion polarity. The nebulizer pressure was 30 psi, drying gas flow rate was 7 µL/min, drying gas temperature was 300 °C, and the capillary voltage was 4000 V. The MSD was scanned over the range of 150-1200 m/z, while setting the fragmentor voltage to +100 or +400 V to obtain either molecular ion information or fragmentation information in alternate scans, respectively.

Results and Discussion During the past few years, a variety of RP column materials and operational conditions have produced encouraging results for protein fractionation, prior to protein identification by MS or LC/MS approaches.29-38 The current work is based on our experience in previous RP-HPLC methods development for fractionation of soluble proteins in human serum, plasma and Journal of Proteome Research • Vol. 5, No. 6, 2006 1303

research articles

Martosella et al.

Figure 1. Chromatogram showing a reversed-phase (RP) separation of 500 µg human brain lipid raft proteins. The sample was solubilized in 80% formic acid and separated on a 4.6 mm I.D. × 50 mm mRP-C18 column at 80 °C under optimized gradient elution conditions. The majority of protein elution is complete by 47.0 min.

CSF samples, prior to protein identification by LC/MS methods. These previous studies have employed a reversed-phase microparticulate macroporous silica column (mRP-C18) operated at 80 °C.27 The combination of column packing material and operational conditions has been observed to yield reproducible and high recovery separations of complex protein samples. Sample Preparation Considerations for Lipid Raft Protein Separations. A major difficulty arises in preparing detergent resistant membrane raft preparations as dispersed solutions for injection on an LC system, as many sample components exhibit visible solubility problems. Initial work using membrane rafts derived from frozen mouse cerebra employed various combinations of chaotropes (Urea up to 6 M, urea/thiourea mixtures), organic solvents (hexafluoro2-propanol), trifluoroacetic acid, as well as 2% SDS. The efficiency of solubilization was determined by extraction, centrifugation, and BCA assay of soluble supernatants and where possible, SDS-PAGE analysis. For a typical experiment, crude membrane raft preparations contained approximately 0.94 µg/µL of protein. After pelleting and solubilization in test solutions of the same volume, concentrations ranged from, a minimum of 0.38 µg/µL using 2% SDS to a maximum of 1.67 µg/µL, using 80% FA. Other samples were found to have concentrations somewhere between these two extremes. During protein recovery analysis lipid raft samples solubilized in FA repeatedly resulted in higher protein concentrations when measured after the reversedphase separation as compared with the sample run without the column. This may be explained by RP column dissociation of tightly bound lipid from proteins, causing a concomitant reduction in interference during the protein assay. This explanation is consistent with gel band intensities, when samples were examined by SDS-PAGE and stained with colloidal Coomassie Blue. SDS-PAGE analysis uniformly indicated much higher protein band intensities for the FA extracts, although proteins solubilized with 2% SDS gave the best protein separation on these gels (data not shown). Both extracts were observed to yield clear solutions, with no apparent flocculates 1304


or precipitate when centrifuged at 10 000 × g. Due to the greatly improved recovery with 80% FA, as well as it’s compatibility with RP-HPLC separation, this sample matrix was used for all subsequent work. Separations Method Development. Previous work with serum protein separations employed elevated column temperature with a multi-segmented linear gradient of water (0.1%TFA) and ACN (0.1% TFA). Although initially promising for FA solubilized lipid raft proteins, repeated injections exhibited inconsistent peak shapes and excessive band broadening, both of which grew worse with increasing sample numbers. A likely explanation is that the high lipid and cholesterol content (50% or greater by mass), combined with highly hydrophobic membrane proteins, as well as residual Triton X-100 in the raft preparations, leads to irreversible coating of the surface of the column packing material. By analogy with approaches successfully used for 2D-PAGE sample preparations, acetone and ethanol delipidation procedures were attempted for both the crude lipid raft preparation, as well as the FA solubilized lipid rafts.39 HPLC of the resolubilized pellets after delipidation however, showed a loss of protein mass by SDS-PAGE, diminished UV absorbance by HPLC, as well as significant band broadening and elevated column back pressures. This suggests that delipidation was incomplete, protein components are lost during the procedures, and/or irreversible aggregation of proteins in the sample may occur during these sample cleanup procedures. An RP approach has the benefit of reducing the loss of proteins in the sample and also minimizes sample handling processes. Since sample injections at elevated temperature using binary water/ACN gradient elution generate acceptable initial separations, with good recovery, subsequent method development was concerned with defining mobile phases more capable of an effective elution of strongly retained components, in a fully soluble form. For comparison of the utility of conditions, we employed both lipid raft protein samples, as


research articles gradient segment of 0.08% TFA in ACN to 20% FA in ACN. Full regeneration of the separation system further required an additional gradient segment from the FA/ACN mixture to neat 2-propanol. Attempts to eliminate these latter two segments resulted in unstable separations of the lipid raft protein samples, with drift in retention and increasing bandwidth as the sample numbers increased. Operation of the system at 80 °C is an absolute requirement, as injection of the lipid raft protein preparation at room-temperature resulted in immediate column failure resulting from sample precipitation on the column.

Figure 2. SDS-PAGE analysis of 47 RP fractions from the separation shown in Figure 1. Fractions were collected at 1.0 min time intervals and combined based on prior electrophorectic analyses. A total of 70 RP fractions were collected, however fractions corresponding from 48 to 70 min (gels not shown) did not show any presence of protein components.

well as synthetic peptide standards, to evaluate the regeneration of column packing surfaces by examining elution repeatability. Previous reports have described the use of FA and 2-propanol as mobile phase components for membrane protein separations.40,41 We found that with the use of a quaternary system we were able to completely regenerate the column packing material, with an elution program that includes a

The chromatogram in Figure 1 shows the RP-HPLC separation of human brain membrane raft proteins using the conditions described above. In Figure 2, SDS-PAGE analysis of column fractions was informative to define the properties of this separation. Examination of the gel band pattern on SDSPAGE for the 1 min fractions collected across this chromatogram shows that protein elution is completed by 45-47 min, corresponding to 50-55% ACN. The separation can be described as exhibiting an early region in which essentially all of the proteins are eluted by increasing ACN, and a later region of eluting lipid material, driven by FA and 2-propanol, which is well away from the protein elution region. Systematic changes in the gradient composition to improve this separation, and further remove the lipid region, resulted in the preferred separation conditions detailed in the “Experimental” section. The area extending from 48 to 70 min contained a significant amount of lipids or lipid raft components which did not exhibit any significant quantity by protein assay or visible staining on SDS-PAGE analysis. This late eluting area of the separation was collected and further analyzed by ESI-LC-MS as discussed below. Separation of the raft lipid components resulted from the combined ACN-FA/2-propanol solvent system and elevated temperature. On subsequent blank runs the chromatograms showed no evidence of peak ghosting or carryover.

Figure 3. Comparison of chromatograms from three consecutive reversed-phase separations of 100 µg lipid raft proteins, separated on a 4.6 mm I.D. × 50 mm mRP-C18 column under optimized chromatographic conditions. Shaded regions represent fractions analyzed for repeatability by LC-MS/MS. Journal of Proteome Research • Vol. 5, No. 6, 2006 1305

research articles

Martosella et al.

Figure 4. Extracted ion chromatograms (EIC) of a tryptic peptide generated from 2′, 3′-cyclic-nucleotide 3′-phosphodiesterase, (R)VELSEQQLGLWPSDVDK(L). The three EIC represent LC-MS analysis of mRP-C18 column fractions collected at 30 min from three repeat RP separations (shown in Figure 3). Table 1. Thirty-Six Human Brain Lipid Raft-Associated Integral Membrane Protein Identifications from 48 1D SDS-PAGE Bands protein

Microsomal glutathione S-transferase 3 Flotillin-1 Protein C8orf2 Thy-1 membrane glycoprotein precursor Sodium/potassium-transporting ATPase beta-1 chain Dihydrolipoyl dehydrogenase, mitochondrial precursor Cytochrome c oxidase polypeptide VIc precursor ADP,ATP carrier protein, heart/skeletal muscle isoform T1 Cytochrome c oxidase subunit IV isoform 1, mitochondrial Neural cell adhesion molecule 1, 120 kDa isoform precursor Neural-cadherin precursor Vacuolar ATP synthase subunit B, brain isoform Vacuolar ATP synthase subunit C Voltage-dependent anion-selective channel protein 1 Oligodendrocyte-myelin glycoprotein precursor ATP synthase B chain, mitochondrial precursor ATP synthase alpha chain, mitochondrial precursor Prohibitin ATP synthase gamma chain, mitochondrial precursor Vacuolar ATP synthase subunit E Vacuolar ATP synthase catalytic subunit A, ubiquitous isoform Voltage-dependent anion-selective channel protein 2 Dihydropyridine-sensitive L-type, calcium channel subunits precursor Vacuolar ATP synthase subunit d Contactin associated protein 1 precursor Contactin 2 precursor Mitochondrial Opioid binding protein/cell adhesion molecule precursor (OBCAM) Myelin-oligodendrocyte glycoprotein precursor 2-oxoglutarate/malate carrier protein (OGCP) Homer protein homolog 1 Vacuolar proton translocating ATPase 116 kDa subunit a isoform 1 Toll-interacting protein Pleckstrin homology domain-containing protein family B member 1 Bassoon protein (Zinc-finger protein 231) Vacuolar ATP synthase subunit D

Chromatographic repeatability was examined using the preferred quaternary elution conditions and elevated temperature. Repeated separations of human membrane raft preparations for three injections were compared and are shown in Figure 3. The RP separations are reproducible and show no changes from run to run in either retention times, apparent selectivity, or bandwidths. Blank runs performed after each separation are reproducible and show no indications of protein carryover or ghosting. We selected 4 fractions from the separation, shown in Figure 3, and evaluated protein identification repeatability by LC-MS/MS. Proteins from the lipid rafts samples were RP fractionated three times under the same chromatographic conditions at 20, 26, 30, and 40 min (corresponding to fractions 11, 14, 16, and 1306


accession no.

score

protein MW

no. of spectra

mean intensity

O14880 O75955 O94905 P04216 P05026 P09622 P09669 P12235 P13073 P13592 P19022 P21281 P21283 P21796 P23515 P24539 P25705 P35232 P36542 P36543 P38606 P45880 P54289 P61421 P78357 Q02246 Q02978 Q14982 Q16653

24.07 46.33 20.4 54.56 71.81 31.15 26.54 39.76 114.67 24.62 21.63 355.35 161.39 207.7 49.85 81.95 182.46 41.63 17.39 203.63 17.58 42.79 47.68 46.33 318.22 46.22 25.01 30.8 54.63

16516.4 47355.5 37839.8 17934.8 35061.5 54150.5 8781.5 32933.5 19576.8 83770.5 99851.9 56501 43941.8 30641.5 49608.2 28908.8 59750.9 29804.2 32996.2 26145.5 68304.5 38092.9 123184 40329.3 156267.5 113393.9 33930.7 38007.8 28179.2

2 4 2 61 10 2 13 3 66 2 2 118 23 103 4 12 14 4 2 85 2 8 4 3 42 4 2 4 8

58100000 19600000 26800000 1150000000 426000000 90100000 303000000 16800000 77100000 53000000 8740000 166000000 263000000 51100000 146000000 139000000 131000000 14200000 37900000 171000000 46900000 37300000 34000000 99100000 152000000 118000000 28900000 74600000 28500000

Q86YM7 Q93050 Q9H0E2 Q9UF11 Q9UPA5 Q9Y5K8

84.34 46.42 25.85 27.3 26.43 148.31

40277 96413.3 30282 27186.1 416370.1 28263

6 5 2 2 2 24

72500000 28500000 18800000 16600000 24100000 105000000

21). Each fraction was digested in-solution with trypsin, and analyzed by a microfluidic chip-based nano-LC/MS system. For each time point, on average, more than 80% of the identified proteins in each individual run were shared by another run and more than 70% of the identified proteins were found in all three runs. The relatively high abundant proteins in each fraction (judging by the number of matched MS/MS spectra) were found in all three repeats. Such results are close to those obtained by repeat LC-MS analysis of the sample protein digests obtained by others.42 Figure 4 shows the extracted ion chromatograms of a peptide from 2′,3′-cyclic-nucleotide 3′phosphodiesterase from fraction 16. The MS signal intensities are very similar for this peptide as well 2 others selected from this protein (data not shown). Comparisons of integrated ion


research articles

Figure 5. Two RP-HPLC elution profiles (absorbance at 210 nm) of five synthetic hydrophobic peptide standards performed before and after membrane raft protein separations on an mRP-C18 (4.6 mm I.D. × 50 mm). Panel (A) establishes an elution profile of the peptides for a new column prior to use with the membrane raft proteins. Panel (B) compares the peptide separation with (A) after 4 repeat injections of 220 µg of membrane raft proteins.

intensities for each sample indicate similar recoveries of protein in the replicates, consistent with a repeatable RP separation. Both the protein ID results and individual peptide ion chromatograms displayed repeatable separations at the protein level. Protein Recovery. High protein recovery is a critical parameter for useful protein pre-fractionation strategies, and elevated temperature plays an important role for achieving high recoveries. To test recovery of protein, a series of 4 injections of lipid raft preparations was conducted, with collection of the eluate. Starting protein concentrations were normalized against the control run (no column in the flow path). Optimized chromatographic separation provided high recovery of protein. Two methods of analysis (BCA and EZQ) yielded approximately 114% protein recovery from the column relative to the normalized injection. We believe the modest increase in recovered

protein results from the dissociation of lipids during the separation, with the effect that delipidated raft proteins are more accessible to the reactants of the protein assay. Full regeneration of the separation system can only occur if all of the components of the injected sample are eluted during the separation. A goal of the current work was to define conditions that would permit complete elution of membrane raft components, yielding a clean packing material surface, and thus a reproducible separation system. To evaluate the regeneration of the surface, separations of a mixture of synthetic peptide standards were conducted over the course of the use of a column for membrane raft protein separations. If hydrophobic proteins and/or lipids are irreversibly bound to the surface, then unstable separations will likely result. We used a mixture of 5 peptides to monitor retention and selectivity after consecutive lipid raft injections. Peptide separations were Journal of Proteome Research • Vol. 5, No. 6, 2006 1307

research articles

1308


Martosella et al.


research articles

Figure 6. Selected MS/MS peptide sequences to manually confirm the identification of proteins. Panel (A) Oligodendrocyte-Myelin Glycoprotein Precursor protein, a cell adhesion molecule attached to the cell membrane via a GPI-anchor.45 Panel (B) 2 MS/MS spectra that matched to two peptides from Dihydropyridine-Sensitive L-type, Calcium Channel R-2/δ Subunits.49,50 The peptides identified in the MS/MS spectra shown are from residues 679-692 (TPNNPSCNADLINR) and 408-420 (GYYEIPSIGAIR). Insert shown in bottom panel details the lower mass region of the bottom MS/MS spectra. Panel (C) Myelin-Oligodendrocyte Glycoprotein Precursor51 may be involved in completion and/or maintenance of the myelin sheath and in cell-cell communication. This 247 amino acid residue protein is reported to have two transmembrane regions spanning from residues 155-175 and 211-231, a cytoplasmic domain from residues 176-210, and two extracellular domains covering residues 30-154 and 232-247. The tryptic peptides from this protein were from each of the two extracellular domains (FSDEGGFTCFFR, residues 119-130, and LAGQFLEELR, residues 235-244).

compared for a new mRP-C18 column, and after 4 consecutive injections of 220 µg of membrane raft proteins. As shown in Figures 5A and 5B, no significant shift in retention, selectivity or bandwidth results from this challenge with the membrane protein sample. In both pre and postcolumn injections, peptide peaks have retained nearly identical retention times. The same column was used extensively over several weeks, with no evidence of degradation of separation performance. Column cleaning or regeneration methods with TFE have been proposed as a wash regime to remove irreversibly bound proteins.43 To investigate the possible adsorption of proteins during the separation, the column eluent was collected during a blank run (immediately after a lipid raft sample), as well as following an injection of 300 µL neat trifluorethanol. These samples were dried, then analyzed by SDS-PAGE to detect proteins that may have been carried over between raft sample separations. The gel lanes from each method showed no evidence of protein (data not shown). We did observe blue streaking in the blank injection gel lane which may have resulted from some excess lipid material remaining after previous injections. However, this was not present in the TFE

elution and may have been carryover caused by shortening the postcolumn run time. Mass Spectrometry. A total of 48 randomly cut gel bands were analyzed by nano-RP-LC-MS/MS using data dependent acquisition mode, and the resulting MS/MS spectra were analyzed with the Spectrum Mill database search algorithm. A total of 158 proteins were identified, from 940 unique peptides and 5945 matched MS/MS spectra. More than 85% of the proteins were identified with two or more unique peptides. The protein list was uploaded into GOMiner to analyze their biological functions and cellular localizations.44 The identified proteins were also manually examined by reference to the primary literature, as available, to assess the accuracy of localization in the appropriate subcellular compartment. The majority of these proteins returned with their predicted/known molecular functions and cellular localizations, 151 and 149, respectively. A total of 73 proteins were identified as membrane proteins and 35 as integral membrane proteins (Table 1). Moreover, a large number of proteins were associated with the cytoskeleton (36) and mitochondrion (37). Overall, there were 103 proteins that were associated with intracellular organelles. Journal of Proteome Research • Vol. 5, No. 6, 2006 1309

research articles

Martosella et al.

Figure 7. LC-MS EIC spectra (fragmentor voltage of +100 V) identifying phosphatidylcholine (PC) and sphingomyelin (SM) from combined late-eluting RP fractions collected from an mRP-C18 column. The fractions were collected from the region of the chromatogram in Figure 1 from 48-70 min.

The following are the major protein categories, according to their biological functions: protein binding, 63, transporter, 43 and hydrolase, 31. Since the gel bands were randomly cut from more than two-thirds of the gel lanes, the results shown here are presumably a fair representation of the proteins present in this sample. Additional protein identifications could likely result from systematic gel slicing, but this was not the purpose of this work. Many of the membrane proteins identified in this study had low protein identification scores, due to the relatively low numbers of spectra obtained and peptides identified for these proteins. To confirm these membrane protein identifications, the MS/MS data for these proteins were manually verified. Figure 6 shows examples of three of these identified membrane proteins, where sequences of two of the detected peptide MS/ MS spectra were used to manually confirm the identification of the proteins. For example, the spectra in Figure 6A were used to confirm the identification of the 440 amino acid residue Oligodendrocyte-Myelin Glycoprotein Precursor protein, which is a cell adhesion molecule attached to the cell membrane via a GPI-anchor, and contributes to myelination in the central nervous system.45 The two peptides shown in these MS/MS spectra correspond to residues 247-262 (AHVIGTPLSTQISSLK), which is located within a Ser/Thr-rich domain, and residues 90-99 (LESLPAHLPR), which is found within a leucine-rich repeat region. MS Identification of Phospholipids in Late-Eluting Column Fractions. Late eluting column fractions collected after 47 min were analyzed by LC/MS in order to generally confirm the presence of lipids. Phosphatidylcholines (PC) and sphingomyelins (SM) are significant constituents of the human brain. When analyzed by LC/MS in acidic media in the absence of alkali ions, they undergo loss of the charged phosphocholine headgroup under CID conditions to yield an ion at 184 m/z.46,47 1310


This information, combined with the knowledge of the molecular ion, was used for identification. Figure 7 shows the EICs obtained at a fragmentor voltage of +100V; molecular ions corresponding to several species of PC and SM were detected. These traces resemble those reported previously.48 Under insource CID conditions using a fragmentor voltage of +400V, these species yielded an intense ion at 184 m/z, confirming the presence of the phosphocholine headgroup. Two examples are shown in Figure 7 (inset).

Conclusions We have developed RP-HPLC conditions that are effective for protein fractionation of highly complex and hydrophobic protein mixtures, such as those demonstrated by lipid raft proteins. The purpose of the present study was to define useful operational conditions for RP separations of membrane protein samples, including consideration of protein recovery and operational compatibility with downstream proteomic workflows. For our studies, lipid rafts were chosen as a readily accessible membrane-rich source and an appropriate starting material to define our separation conditions and proteomic strategy. This work was not intended to be a comprehensive survey of membrane protein identifications from lipid rafts, but rather, the development of a viable protocol for the effective separation and recovery of hydrophobic proteins. We achieved efficient high-recovery separation of membrane proteins using the combination of a macroporous column material (mRP-C18) and FA and organic solvent elution conditions at elevated temperature. Sample solubilization with 80% FA combined with the mobile phase elution scheme, enabled a highly resolved and reproducible membrane protein separation, delivering column fractions with reduced lipid content. The method presented here yielded high protein recovery and fractions that


were readily analyzed by electrophoresis and MS methods for protein identification. Although we have shown evidence of high bulk protein recovery it is likely that unknown specific lower abundance proteins could be recovered with lower efficiency.

References (1) Calmarini, C. M.; Galmarini, F. C. Curr. Opin. Investig. Drugs 2003, 4, 1416-1421. (2) Rahbar, A. M.; Fenselau, C. Unbiased examination of changes in plasma membrane proteins in drug resistant cancer cells. J. Proteome Res. 2005, 4, (6), 2148-2153. (3) Whitelegge, J. P.; Gomez, S. M.; Faull, K. F. Proteomics of membrane proteins. Adv. Protein Chem. 2003, 65, 271-307. (4) Whitelegge, J. P. Tandem mass spectrometry of integral membrane proteins fot top-down proteomics. Trends Anal. Chem. 2005, 24 (7). (5) Schluesener, D.; Fischer, F.; Kruip, J.; Rogner, M.; Poetsch, A. Mapping the membrane proteome of Corynebacterium glutamicum. Proteomics 2005, 5 (5), 1317-1330. (6) Josic, D.; Brown, M. K.; Huang, F.; Callanan, H.; Rucevic, M.; Nicoletti, A.; Clifton, J.; Hixson, D. C. Use of selective extraction and fast chromatographic separation combined with electrophoretic methods for mapping of membrane proteins. Electrophoresis 2005, 26 (14), 2809-2822. (7) Oh-Ishi, M.; Maeda, T. Separation techniques for high-molecularmass proteins. J Chromatogr B Anal. Technol. Biomed. Life Sci. 2002, 771, (1-2), 49-66. (8) Welling, G. W.; van der Zee, R.; Welling-Wester, S. Column liquid chromatography of integral membrane proteins. J. Chromatogr. 1987, 418, 223-243. (9) Whitelegge, J. P.; Zhang, H.; Aguilera, R.; Taylor, R. M.; Cramer, W. A. Full subunit coverage liquid chromatography electrospray ionization mass spectrometry (LCMS+) of an oligomeric membrane protein: cytochrome b(6)f complex from spinach and the cyanobacterium Mastigocladus laminosus. Mol. Cell. Proteomics 2002, 1 (10), 816-827. (10) Bledi, Y.; Inberg, A.; Linial, M. PROCEED: A proteomic method for analysing plasma membrane proteins in living mammalian cells. Briefings Funct. Genomics Proteomics 2003, 2 (3), 254-265. (11) Foster, L. J.; De Hoog, C. L.; Mann, M. Unbiased quantitative proteomics of lipid rafts reveals high specificity for signaling factors. Proc. Natl. Acad. Sci. U.S.A. 2003, 100 (10), 5813-5818. (12) Pike, L. J. Lipid rafts, bringing order to chaos. J. Lipid Res. 2003, 44, 655-667. (13) Pike, L. J. Lipid rafts: heterogeneity on the high seas. Biochem. J. 2004, 378, (Pt 2), 281-292. (14) Simons, K.; Ikonen, E. Functional rafts in cell membranes. Nature 1997, 387, (6633), 569-572. (15) Simons, K.; Toomre, D. Lipid rafts and signal transduction. Nat. Rev. Mol. Cell Biol. 2000, 1(1), 31-39. (16) Becher, A.; Green, A.; Ige, A. O.; Wise, A.; White, J. H.; McIlhinney, R. A. Ectopically expressed gamma-aminobutyric acid receptor B is functionally down-regulated in isolated lipid raft-enriched membranes. Biochem. Biophys. Res. Commun. 2004, 321 (4), 981987. (17) Manie, S. N.; Debreyne, S.; Vincent, S.; Gerlier, D. Measles virus structural components are enriched into lipid raft microdomains: a potential cellular location for virus assembly. J. Virol. 2000, 74 (1), 305-311. (18) Nebl, T.; Pestonjamasp, K. N.; Leszyk, J. D.; Crowley, J. L.; Oh, S. W.; Luna, E. J. Proteomic analysis of a detergent-resistant membrane skeleton from neutrophil plasma membranes. J. Biol. Chem. 2002, 277 (45), 43399-43409. (19) Oh, p., Schnitzer, J. E. Segregation of Heterotrimetric G Proteins in cell Surface Microdomains. Mol. Biol. Cell 2001, 12, 685-698. (20) Tu, X.; Huang, A.; Bae, D.; Slaughter, N.; Whitelegge, J.; Crother, T.; Bickel, P. E.; Nel, A. Proteome analysis of lipid rafts in Jurkat cells characterizes a raft subset that is involved in NF-kappaB activation. J. Proteome Res. 2004, 3 (3), 445-454. (21) Slaughter, N.; Laux, I.; Tu, X.; Whitelegge, J.; Zhu, X.; Effros, R.; Bickel, P.; Nel, A. The flotillins are integral membrane proteins in lipid rafts that contain TCR-associated signaling components: implications for T-cell activation. Clin. Immunol. 2003, 108 (2), 138-151. (22) Becher, A.; White, J. H.; McIlhinney, J. The gamma-aminobutyric acid receptor B, but not the metabotropic glumaate receptor type1, associates with lipid rafts in the rat cerebellum. J. Neurochem. 2001, 79, 787-795.

research articles (23) Karsan, A.; Blonder, J.; Law, J.; Yaquian, E.; Lucas, D. A.; Conrads, T. P.; Veenstra, T. Proteomic analysis of lipid microdomains from lipopolysaccharide-activated human endothelial cells. J. Proteome Res. 2005, 4 (2), 349-357. (24) Riddell, D. R.; Christie, G.; Hussain, I.; Dingwall, C. Compartmentalization of beta-secretase (Asp2) into low-buoyant density, noncaveolar lipid rafts. Curr. Biol. 2001, 11 (16), 1288-1293. (25) Blonder, J.; Goshe, M. B.; Moore, R. J.; Pasa-Tolic, L.; Masselon, C. D.; Lipton, M. S.; Smith, R. D. Enrichment of integral membrane proteins for proteomic analysis using liquid chromatography-tandem mass spectrometry. J. Proteome Res. 2002, 1 (4), 351-360. (26) Goshe, M. B.; Blonder, J.; Smith, R. D. Affinity labeling of highly hydrophobic integral membrane proteins for proteome-wide analysis. J. Proteome Res. 2003, 2 (2), 153-161. (27) Martosella, J.; Zolotarjova, N.; Liu, H.; Nicol, G.; Boyes, B. E. Reversed-phase high-performance liquid chromatographic prefractionation of immunodepleted human serum proteins to enhance mass spectrometry identification of lower-abundant proteins. J. Proteome Res. 2005, 4 (5), 1522-1537. (28) Meza, J. E.; Miller, C. A.; Fischer, S. M. Improved Tryptic Digestion of Proteins Using 2,2,2-Trifluoroethanol (TFE). 2004. (29) Whitelegge, J. P.; Gundersen, C. B.; Faull, K. F. Electrosprayionization mass spectrometry of intact intrinsic membrane proteins. Protein Sci. 1998, 7 (6), 1423-1430. (30) Whitelegge, J. P.; Jewess, P.; Pickering, M. G.; Gerrish, C.; Camilleri, P.; Bowyer, J. R. Sequence analysis of photoaffinity-labeled peptides derived by proteolysis of photosystem-2 reaction centres from thylakoid membranes treated with [14C]azidoatrazine. Eur. J. Biochem. 1992, 207 (3), 1077-1084. (31) McDonald, W.; Ohi, R.; Miyamoto, D.; Mitchison, T.; Yates, J. Comparison of three directly coupled HPLC MS/MS strategies for identification of proteins from complex mixtures: singledimension LC-MS/MS, 2-phase MudPIT, and 3-phase MudPIT. Int. J. Mass Spectrom. 2002, 219 (1), 245-251. (32) Badock, V.; Steinhusen, U.; Bommert, K.; Otto, A. Prefractionation of protein samples for proteome analysis using reversed-phase high-performance liquid chromatography. Electrophoresis 2001, 22 (14), 2856-2864. (33) Janini, G. M.; Chan, K. C.; Conrads, T. P.; Issaq, H. J.; Veenstra, T. D. Two-dimensional liquid chromatography-capillary zone electrophoresis-sheathless electrospray ionization-mass spectrometry: evaluation for peptide analysis and protein identification. Electrophoresis 2004, 25 (13), 1973-1980. (34) Lee, H.; Yi, E. C.; Wen, B.; Reily, T. P.; Pohl, L.; Nelson, S.; Aebersold, R.; Goodlett, D. R. Optimization of reversed-phase microcapillary liquid chromatography for quantitative proteomics. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2004, 803 (1), 101-110. (35) Lescuyer, P.; Hochstrasser, D. F.; Sanchez, J. C. Comprehensive proteome analysis by chromatographic protein prefractionation. Electrophoresis 2004, 25 (7-8), 1125-1135. (36) Neverova, I.; Van Eyk, J. E. Application of reversed phase high performance liquid chromatography for subproteomic analysis of cardiac muscle. Proteomics 2002, 2 (1), 22-31. (37) Righetti, P. G.; Castagna, A.; Herbert, B.; Reymond, F.; Rossier, J. S. Prefractionation techniques in proteome analysis. Proteomics 2003, 3 (8), 1397-1407. (38) Van den Bergh, G.; Clerens, S.; Vandesande, F.; Arckens, L. Reversed-phase high-performance liquid chromatography prefractionation prior to two-dimensional difference gel electrophoresis and mass spectrometry identifies new differentially expressed proteins between striate cortex of kitten and adult cat. Electrophoresis 2003, 24 (9), 1471-1481. (39) Mastro, R.; Hall, M. Protein delipidation and precipitation by trin-butyl phosphate, acetone, and methanol treatment for isoelectric focusing and two-dimensional gel electrophoresis. Anal. Biochem. 1999, 273 (2), 313-315. (40) Heukeshoven, J.; Dernick, R. Reversed-phase high-performance liquid chromatography of virus proteins and other large hydrophobic proteins in formic acid containing solvents. J. Chromatogr. 1982, 252, 241-54. (41) Lee, R. P.; Doughty, S. W.; Ashman, K.; Walker, J. Purification of hydrophobic integral membrane proteins from mycoplasma hyopneumoniae by reversed-phase high-performance liquid chromatography. J. Chromatogr. A 1996, 737, 273-279. (42) Liu, H.; Sadygov, R. G.; Yates, J. R. 3rd, A model for random sampling and estimation of relative protein abundance in shotgun proteomics. Anal. Chem 2004, 76 (14), 4193-4201. (43) Bhardwaj, S.; Day, R. A. Trifluoroethanol Removes Bound Proteins from Reversed-Phase Columns. LC-GC 1999, 17 (4), 354-355.

Journal of Proteome Research • Vol. 5, No. 6, 2006 1311

research articles (44) Zeeberg, B. R.; Feng, W.; Wang, G.; Wang, M. D.; Fojo, A. T.; Sunshine, M.; Narasimhan, S.; Kane, D. W.; Reinhold: W. C.; Lababidi, S.; Bussey, K. J.; Riss, J.; Barrett, J. C.; Weinstein, J. N. GoMiner: a resource for biological interpretation of genomic and proteomic data. Genome Biol. 2003, 4 (4), R28. (45) Mikol, D. D.; Stefansson, K. A phosphatidylinositol-linked peanut agglutinin-binding glycoprotein in central nervous system myelin and on oligodendrocytes. J. Cell. Biol. 1988, 106 (4), 12731279. (46) Griffiths, W. J. Tandem mass spectrometry in the study of fatty acids, bile acids, and steroids. Mass. Spectrom. Rev. 2003, 22, 81152. (47) Hsu, F. F.; Turk, J., Electrospray ionization/tandem quadrupole mass spectrometric studies on phosphatidylcholines: the fragmentation processes. J. Am. Soc. Mass Spectrom. 2003, 14 (4), 352-363. (48) Issac, G.; Bylund, D.; Mansson, J.-E.; Markides, K. E.; Bergquist, J. Analysis of Phosphatidylcholine and Sphingomyelin

1312


Martosella et al. Molecular Species from Brain Extracts Using Capillary LC/ESI/ MS 2003. (49) Biel, M.; Hullin, R.; Freundner, S.; Singer, D.; Dascal, N.; Flockerzi, V.; Hofmann, F. Tissue-specific expression of high-voltageactivated dihydropyridine-sensitive L-type calcium channels. Eur. J. Biochem. 1991, 200 (1), 81-88. (50) Williams, M. E.; Feldman, D. H.; McCue, A. F.; Brenner, R.; Velicelebi, G.; Ellis, S. B.; Harpold, M. M. Structure and functional expression of alpha 1, alpha 2, and beta subunits of a novel human neuronal calcium channel subtype. Neuron 1992, 8 (1), 71-84. (51) Albouz-Abo, S.; Wilson, J. C.; Bernard, C. C.; von Itzstein, M. A conformational study of the human and rat encephalitogenic myelin oligodendrocyte glycoprotein peptides 35-55. Eur. J. Biochem. 1997, 246 (1), 59-70.

PR060051G

High Recovery HPLC Separation of Lipid Rafts for Membrane

Recommend Documents