Application of Proteomic Tools To Detect the Nonspecificity of a Polyclonal Antibody against Lipoprotein Lipase Albert Casanovas,† Montserrat Carrascal,‡ Joaquı´n Abia´n,‡ M. Dolores Lo ´ pez-Tejero,† and ,† Miquel Llobera* Departament de Bioquı´mica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, E-08028 Barcelona, Spain, and CSIC/UAB Proteomics Laboratory, IIBB-CSIC-IDIBAPS, Universitat Auto`noma de Barcelona, E-08193 Bellaterra, Spain Received February 18, 2008
Abstract: Specific antibodies are essential tools for studying proteins. P66 is a chicken polyclonal antibody raised against bovine lipoprotein lipase (LPL) that has been used in earlier studies. Here, we developed a two-dimensional (2D) Western blot with reducing gels, using commercial bovine LPL (53 kDa) as a standard and P66 for detection. Our results revealed incomplete purification of commercial LPL and nonspecificity of P66, both undetectable in one-dimensional analysis. Antithrombin III (ATIII) was identified as both a major contaminant in commercial LPL and a cross-reacting protein with P66. Although LPL purification methods were presumably designed to eliminate ATIII, here we demonstrate that some procedures fell short of this objective and thus led to the production of a nonspecific antibody. Our results define 2D electrophoresis/Western blot and mass spectrometric protein identification as the most reliable procedure for validating LPL purity and the specificity of antibodies against this enzyme. Keywords: two-dimensional Western blot • antithrombin III • bovine • P66 • 5D2
Introduction Lipoprotein lipase (LPL) plays a central role in lipid metabolism by hydrolyzing circulating triacylglycerides (TAG) from chylomicra and VLDLs. The enzyme is synthesized by parenchymal cells but functionally it is located in homodimeric form bound to heparan sulfate proteoglycans on the endothelial cell surface. A reduction in LPL activity produces marked lipemia and increased levels of TAG in plasma, whereas its absence causes neonatal death in knockout mice.1 In addition, the relationship between LPL and several pathologies such as atherosclerosis, diabetes, obesity and Alzheimer’s disease shows the importance of this enzyme.2 For these and many other reasons, LPL has been widely studied at physiological, cellular and molecular level, for which specific antibodies against LPL are an essential tool. * To whom correspondence should be addressed. Miquel Llobera, Departament de Bioquı´mica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal 645, E-08028 Barcelona, Spain. Tel.: 3493-402 15 22. Fax: 34-93-402 15 59. E-mail:
[email protected]. † Universitat de Barcelona. ‡ Universitat Auto`noma de Barcelona. 10.1021/pr800131n CCC: $40.75
2008 American Chemical Society
Since there is no commercially available antibody against LPL, for years researchers have developed their own antibodies. The antigens used were synthetic peptides corresponding to specific regions of LPL,3 a portion of human LPL as a bacterial fusion protein,4 recombinant human LPL3 or, more commonly, native LPL purified from a variety of sources including milk, postheparin plasma and adipose tissue from different species.5–8 During the 1980’s and 1990’s, a commonly used method for antibody production consisted of first-affinity purification of LPL from bovine milk, which was subsequently used for immunization of hens.8,9 Some authors used a commercially available affinity-purified LPL from bovine milk (Sigma, St. Louis, MO) as an alternative to their own purification of LPL.10,11 Bovine milk was a preferred source of LPL because, on a weight basis, it contains the same amount of LPL as other tissues.12 Chickens were a preferred species for developing polyclonal antibodies, due to the phylogenetic distance between mammals and birds, and because higher amounts of antibody can be obtained from them without invasive procedures, since antibodies produced are transferred from serum to egg yolk.13 To complete this process, LPL-specific antibodies were affinity-purified from egg yolks9–11 or sera8 using bovine LPL coupled with CNBr-activated Sepharose. In our laboratory, for years, we used P66, a polyclonal antibody obtained following this protocol, for LPL immunodetection by Western blot.14,15 The obtaining of P66 was described in a previous study,9 in which this antibody was used for coating in a sandwich ELISA combined with the widely used monoclonal antibody (mAb) 5D2 for detection. In our research, we were interested in development of twodimensional Western blot against LPL using P66. For this, we used commercially available affinity-purified bovine LPL as a standard. Unexpectedly, silver-stained gels showed two major spots at similar molecular weight, but different isoelectric point. Moreover, the polyclonal antibody P66 recognized both spots, showing cross-reactivity that was undetectable in one-dimensional Western blot. The aims of the present study were (i) to clarify the specificity of antibodies and the purity of commercial LPL in order to reevaluate systems for LPL mass detection, and (ii) to propose a useful methodology for validating the LPL purification process and testing the specificity of antibodies against LPL. Journal of Proteome Research 2008, 7, 4173–4177 4173 Published on Web 07/17/2008
technical notes Experimental Procedures Sample. LPL was from bovine milk (Sigma, St Louis, MO). The enzyme is provided as a suspension in ammonium sulfate 3.8 M, Tris-HCl 0.02 M, pH 8 buffer. Rat plasma: male Wistar rats were anesthetized by intraperitoneal injection of ketamine (90 mg/kg body weight) and xylacine (10 mg/kg body weight). Blood was extracted from the tail vein using a syringe pretreated with EDTA solution. Blood samples were centrifuged (2000g, 15 min, 4 °C) and the separated plasma was stored at -80 °C. This procedure was approved by the Committee on Animal Bioethics and Care of the University of Barcelona and the Generalitat (Autonomous Regional Government) of Catalonia, Spain. One-Dimensional Electrophoresis. Bovine LPL (0.75 µg) or rat plasma (110 µg) was mixed with sample buffer 3:1 (v/v) (250 mM Tris-HCl, pH 6.8, 40% (v/v) glycerol, 8% (w/v) SDS, 600 mM DTT and bromophenol blue), boiled for 10 min and applied to a 9% (w/v) polyacrylamide gel. Two-Dimensional Electrophoresis (2DE). To eliminate salts that might interfere in IEF, bovine LPL suspension was first centrifuged (13 000g, 10 min, 4 °C), supernatant was discarded and pellet was resolubilized in equal volume of Tris-HCl 15 mM, pH 7.4. This LPL solution was then precipitated using TCA followed by acetone washing, as described elsewhere,16 to concentrate protein and remove remaining traces of ammonium sulfate. The pellet was then solubilized in Rehydration Buffer (7 M urea, 2 M thiourea, 2% (w/v) CHAPS, 0.5% (v/v) IPG Buffer, pH 3-10, and a trace amount of bromophenol blue) containing 18 mM DTT, sonicated as recommended,17 aliquoted and frozen at -80 °C. The 7 cm immobilized pH gradient (IPG) strips, pH 3-10 (GE Healthcare, Uppsala, Sweden), were passively rehydrated overnight in 125 µL of Rehydration Buffer containing 1.2% (v/ v) DeStreak Reagent (GE Healthcare, Uppsala, Sweden). Six micrograms of the precipitated protein (bovine LPL) was applied to the rehydrated IPG strip by cup-loading. The strips were focused according to the following protocol: linear ramp to 1000 V over 2 h, linear ramp to 5000 V over 1 h, and 5000 V for 25 kVh. For rat plasma samples, 0.3 mg of protein was mixed with Rehydration Buffer containing DTT (18 mM final concentration), cup-loaded onto rehydrated IPG strip and focused by linear ramp to 300 V over 30 min, constant voltage at 300 V for 4 h, raise to 1000 V, 2000 V and 4000 V in 3 h, and constant voltage at 4000 V for 40 kVh. Prior to loading the focused IPG strips on the seconddimensional gels, IPG strips were incubated for 15 min in 2.5 mL of Equilibration Buffer (50 mM Tris, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS and bromophenol blue) containing 10 mg/mL DTT. In strips used for later mass spectrometric protein identification, this was followed by a further 15-min incubation in the same equilibration buffer containing 25 mg/mL iodoacetamide. This second equilibration step was not performed in strips used for later Western blot analysis, to avoid possible alterations in epitope and antibody recognition due to alkylation. The equilibrated IPG strips were loaded in a 9% (w/v) polyacrylamide gel and sealed using a solution containing 0.5% (w/v) agarose, 50 mM Tris-base, 0.1% (w/v) SDS, 192 mM glycine and bromophenol blue. Gels were silver-stained for protein visualization using a modified staining procedure compatible with mass spectrometry18 or transferred (1 h 100 V) to a nitrocellulose membrane (GE Healthcare, Little Chalfont, U.K.) for Western blot assay.14 4174
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Casanovas et al. Western Blot Analysis. Membranes were blocked in Trisbuffered saline (TBS) containing 5% (w/v) nonfat dry milk for 1 h at 37 °C and then incubated overnight at 4 °C with either P66, chicken polyclonal antibody raised against bovine LPL (1: 4000 (v/v), kind gift of Dr. T. Olivecrona, University of Umeå, Umeå, Sweden) or monoclonal antibody (mAb) 5D2 (1:2000 (v/v), kind gift of Dr. J. D. Brunzell, University of Washington, Seattle, WA), both in blocking buffer containing 0.05% (v/v) Tween. After membranes were washed thrice with TBS containing 0.1% (v/v) Tween and thrice with TBS, they were incubated for 2 h at room temperature with different secondary antibodies: rabbit anti-chicken IgG peroxidase conjugated (1: 10 000 (v/v), Chemicon, Temecula, CA) for membranes incubated with P66 or goat anti-mouse IgG peroxidase conjugated (1:10 000 (v/v), Chemicon, Temecula, CA) for membranes incubated with mAb 5D2. The membranes were then washed as described above. Bound antibodies were detected using SuperSignal West Pico Chemiluminiscent Substrate (Pierce, Rockford, IL) as directed by the manufacturer. In-Gel Digestion. The spots/band of interest were excised and subjected to in-gel digestion with trypsin. Prior to in-gel digestion, protein spots were reduced with 10 mM DTT and alkylated with 55 mM iodoacetamide. Enzymatic digestion was performed with trypsin (Promega, Madison, WI) in an automatic digestor (INTAVIS Bioanalytical Instruments AG, Koeln, Germany) following conventional procedures as described.19,20 Samples were dried in a Speed-Vac and redissolved in 5 µL of metanol/water (1/2 (v/v)) containing 0.1% (v/v) TFA. Mass Spectrometry Protein Identification. Proteins were identified by peptide mass fingerprinting (PMF) using matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The MALDI-TOF MS analysis of the tryptic digest was performed using a Voyager DE-PRO (Applied Biosystems, Barcelona, Spain) instrument in the reflectron mode. For the analysis, 0.5 µL of sample was loaded in a 96 × 2 well plate, mixed with 0.5 µL of matrix (3 mg/mL R-cyano-4-hydroxycinnamic acid in acetonitrile /H2O 2/1 (v/ v), 0.1% (v/v) TFA) and let to dry. Spectra were externally calibrated using a standard mixture [des-Arg1-bradiquinine (Mr 904.46), Glu1-fibrinopeptide B (Mr 1570.68), angiotensin-1, (Mr 1296.69), ACTH 1-17 (Mr 2093.09), ACTH 18-39 (Mr 2465.20), ACTH 7-38 (Mr 3657.93)] and, when the ions corresponding to known trypsin autolytic peptides (m/z 842.5100 and 2111.1046) were detected at adequate intensities, an automatic internal calibration of the spectra was performed. The MS-Tag program of Protein Prospector software (University of California, San Francisco, CA) was used for data mining in the UniProtKB/Swiss-Prot (European Bioinformatics Institute, Hinxton, U.K.) database. The parameters used for the searches were trypsin digestion, 2 missed cleavages, carbamidomethylation of cysteine and maximun error tolerance 50 ppm.
Results Figure 1 shows the results of commercial bovine LPL analysis by one-dimensional electrophoresis. Silver staining (Figure 1A) showed a single major band at an apparent molecular weight of 53 kDa (computed with Quantity One software, Bio-Rad Laboratories, Hercules, CA). In accordance with this, LPL immunodetection using either P66 (Figure 1B), a polyclonal antibody, or mAb 5D2 (Figure 1C), a monoclonal antibody, showed a single band at the same molecular weight.
LPL Analysis by 2D Western Blot and MS
technical notes
Figure 1. One-dimensional electrophoresis analysis of commercial bovine LPL. Silver-stained gel (A) is compared with the corresponding Western blot against LPL using P66 (B) or 5D2 (C) antibody. In panel A, band 1 was excised and digested with trypsin for MS analysis.
The analysis of this sample by 2DE (Figure 2) revealed that the band observed in one-dimensional electrophoresis (Figure 1A) contained two abundant proteins with similar molecular weight but different isoelectric point (Figure 2A). Moreover, P66 recognized both of these two major spots (Figure 2B), whereas mAb 5D2 only detected one (Figure 2C). PMF protein identification (Table 1 and Figure 3) confirmed that the band observed in one-dimensional electrophoresis (Figure 1A) contained two different proteins identified as Antithrombin III precursor (ATIII; Swiss-Prot P41361) and Lipoprotein lipase precursor (LPL; Swiss-Prot P11151) (Figure 3A). These two proteins could be resolved by 2DE according to their different isoelectric point and identified separately (Figure 3B,C). Then, protein identifications and 2D Western blot demonstrated that P66 recognizes both LPL and ATIII, whereas mAb 5D2 specifically detects LPL. This analysis was performed with identical results using two different batches of commercial bovine LPL. To contrast whether the nonspecificity of P66 was limited to bovine LPL, we tested this antibody by one- and twodimensional Western blot with rat plasma, a sample that we analyzed in previous studies.14 Again, the single band observed in one-dimensional Western blot (Figure 4A) corresponds to two different proteins with similar molecular weight (Figure 4B), indicating that P66 nonspecificity also affected rat plasma samples.
Discussion
Figure 2. Two-dimensional electrophoresis analysis of commercial bovine LPL. Silver-stained gel (A) is compared with the corresponding Western blot against LPL using P66 (B) or 5D2 (C) antibody. In panel A, spots 2 and 3 were excised and digested with trypsin for MS analysis. 2 DE was performed using pH 3-10, 7 cm IPG strips in the first dimension and 9% (w/v) polyacrylamide gels in the second dimension.
A classic procedure for obtaining antibodies against LPL was immunization of hens with LPL purified from bovine milk.8–10 Several recent studies have used polyclonal antibodies obtained following this strategy to immunodetect LPL by Western blot or ELISA.14,15,21–23 The present study demonstrates that one of these polyclonal antibodies, P66, is nonspecific because, as well as binding to LPL, it also recognizes ATIII (Figure 2B), which has a molecular weight similar to that of LPL. This result warns against the use of P66 to immunodetect LPL when proteins are resolved using SDS-PAGE (which is used in most LPL studies). However, since LPL is functional as a homodimer, native gels could potentially be used to distinguish dimeric LPL from ATIII on the basis of
their different molecular weight. Otherwise, P66 can be used as a primary or capture antibody in a sandwich ELISA combined with a specific antibody for detection, as reported by Vilella et al.9 The nonspecificity of a polyclonal antibody can be explained in at least two ways: (i) the antibody cross-reacts because different proteins have similar epitopes, or (ii) the antigen used to obtain the antibody was not pure. Since no significant similarity between LPL and ATIII exists (sequences compared using BLAST, National Center for Biotechnology Information, Bethesda, MD), P66 nonspecificity is probably due to incomplete purification of LPL, which was later used as immunogen. Journal of Proteome Research • Vol. 7, No. 9, 2008 4175
technical notes
Casanovas et al.
Table 1. Proteins Identified by PMF band/spot no.
protein name
accession no.a
peptides matched
peptides not matched
sequence coverage %
MOWSE score
1 1 2 3
Lipoprotein lipase precursor Antithrombin-III precursor Antithrombin-III precursor Lipoprotein lipase precursor
P11151 P41361 P41361 P11151
10 8 21 9
24 26 31 33
28.2 19.6 50.5 26.2
39371 11175 1.03 × 10E10 39371
a
Accession number in UniProtKB/Swiss-Prot.
Figure 4. Comparison of one-dimensional (A) and two-dimensional (B) Western blot of rat plasma using P66. 2 DE was performed using pH 3-10, 7 cm IPG strips in the first dimension and 9% (w/v) polyacrylamide gels in the second dimension. Proteins were transferred to nitrocellulose membrane and polyclonal antibody P66 was used for detection.
Figure 3. MALDI-TOF mass spectra of proteins in band 1 in Figure 1, and spots 2 and 3 in Figure 2. In panel A, tryptic peptides corresponding to LPL and ATIII are labeled with open circles and filled circles, respectively. In panels B and C, protein sequence coverage corresponding to each tryptic peptide is indicated in italic font. Trypsin autolysis products are labeled with a T symbol.
As a result, a mixture of antibodies against LPL and antibodies against ATIII was obtained. Zechner defined ATIII as a common contaminant in LPL isolation procedures from human milk.5 The presence of ATIII in bovine milk was also reported by Olivecrona et al.12 Most methods of LPL purification from bovine milk are based on chromatography on heparin-Sepharose. Literature describes variations and additions to this basic procedure. To obtain pure LPL protein, Bengtsson-Olivecrona24 recommended a first preparative heparin-Sepharose chromatography with a stepwise elution followed by rechromatography also on heparinSepharose, with gradient elution (with 0.95-2.0 M NaCl) selecting LPL-containing fractions from the later part of the peak to discard fractions containing ATIII. Alternatively, So4176
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corro25 purified LPL by heparin-Sepharose, including a washing cycle with Triton N-101 to elute nonspecifically bound proteins, whereas other authors combined heparin-Sepharose chromatography with dextran-sulfate-Sepharose chromatography8 or hydrophobic interaction chromatography on phenyl-Sepharose.26 However, the process of LPL purification prior to obtaining P66 antibody, described in Bengtsson-Olivecrona et al.,24 seemed to not be enough to completely purify LPL; the final result was a nonspecific polyclonal antibody. Despite the results reported here, heparin-Sepharose could potentially be used to separate LPL from ATIII, as some fractions eluted using salt gradients might contain pure LPL. However, careful screening of fractions is essential to ensure the absence of ATIII. P66 nonspecificity affected not only commercial bovine LPL, but also rat plasma samples (Figure 4). In contrast, mAb 5D2, a widely used specific antibody whose binding site has been characterized in detail,27 selectively recognizes bovine LPL (Figure 2C). When 5D2 is assayed for Western blot to immunodetect LPL in rat plasma, no proteins are detected (A. Casanovas, M. D. Lo´pez-Tejero, and M. Llobera, unpublished observations). This might be due to (i) low amounts of LPL in preheparin plasma and/or (ii) well-described 5D2 having lower recognition of rat LPL than of bovine LPL.27 Because of the difficulty in LPL purification, previous studies reported the use of commercial bovine LPL as immunogen to obtain antibodies.10,11 However, this LPL, which is also purified from bovine milk, contains high levels of ATIII (Figure 2A) as a result of an incomplete purification process. This feature could also be relevant for previous studies that used commercial bovine LPL to determine the presence of anti-LPL autoantibodies in sera from patients with Systemic Lupus Erythematosus28,29 or to evaluate differential binding of lipoproteins to LPL.30 The nonspecificity of P66 cannot be broadly extrapolated to other antibodies since LPL purity may vary greatly depending
technical notes
LPL Analysis by 2D Western Blot and MS on the source and the strategy used for purification. However, it cannot be ruled out that this nonspecificity might also apply to other antibodies, as some procedures used for validating LPL purity and/or antibody specificity are shared by different laboratories. Thus, several studies, after LPL purification, determined lipolytic activity as a sign of the presence of LPL and used SDS-PAGE to validate the purification process.3,7,8,25,31 However, our results clearly recommend the use of 2DE to ensure the purity of LPL, since the presence/absence of ATIII is difficult to assess in one-dimensional electrophoresis (Figure 1A). In a similar way, previous studies tested antibodies against LPL by inhibiting LPL activity,8,11,31 immunodetecting purified bovine LPL by one-dimensional Western blot31 or ELISA,10,11,25 or immunoprecipitating LPL to demonstrate that the antibody binds to LPL.8 These analyses can provide valuable information about affinity and LPL recognition but not about specificity, since none of these assays exclude potential cross-reactivity with other proteins.
Conclusions Our work demonstrates that P66, a polyclonal antibody against LPL that has been used in previous studies, is nonspecific; as well as binding to LPL, it also recognizes ATIII. This nonspecificity could be explained by an incomplete purification of the LPL used as an immunogen, similar to that observed in commercial bovine LPL. Since most methods used to validate LPL purity and/or antibody specificity were common to different laboratories, the nonspecificity displayed by P66 might also apply to other polyclonal antibodies. Our results define 2DE/Western blot and mass spectrometric protein identification as the most reliable procedure for validating the LPL purification process and testing the specificity of antibodies against this enzyme. Therefore, these proteomic tools might be of interest to authors who wish to reevaluate previously published work. Abbreviations: 2DE, two-dimensional electrophoresis; ATIII, Antithrombin III; IEF, isoelectric focusing; IPG, immobilized pH gradient; LPL, lipoprotein lipase; mAb, monoclonal antibody; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; MS, mass spectrometry; PMF, peptide mass fingerprinting; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Acknowledgment. This study was funded by grants SAF2005-02391 and BFU2007-65247/BMC from the Direccio´n General de Investigacio´n Cientı´fica y Te´cnica, Spanish Ministry of Education and Science. Albert Casanovas was the recipient of a fellowship from the Ministry of Universities, Research and Information Society of the Generalitat de Catalunya and from European Social Funds. The authors thank Dr. T. Olivecrona (University of Umeå, Umeå, Sweden) and Dr. J. D. Brunzell (University of Washington, Seattle, WA) for providing antibodies and the Language Advisory Service of the University of Barcelona for technical advice. The Proteomics Laboratory CSIC/UAB is a member of ProteoRed, is funded by Genoma Spain and follows the quality criteria set up by ProteoRed standards.
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PR800131N
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