ProteinChip Technology: A New and Facile Method for the Identification and Measurement of High-Density Lipoproteins apoA-I and apoA-II and Their Glycosylated Products in Patients with Diabetes and Cardiovascular Disease† Bishambar Dayal*,‡ and Norman H. Ertel*,‡,§ VA NJ Health Care System, East Orange, New Jersey 07018, UMDNJ-New Jersey Medical School, Newark, New Jersey 07103, and RWJ Medical School, New Brunswick, New Jersey 08903 Received December 18, 2001
Abstract: This paper describes a ProteinChip technology for the identification and quantification of apolipoprotein profiles in crude biological samples. Expression levels of apoA-I and apoA-II and their glycosylated products were accomplished using single 1 µL plasma samples. In the present studies, strong anionic and weak cationic exchanger ProteinChips (SAX2 and WCX2 chip surfaces) were tested, and the WCX2 chip was found to be selective for specific apolipoproteins. Using the WCX2 chip and analysis via surface-enhanced laser desorption ionization mass spectrometry (SELDI-MS), apoA-I and apoA-II were separated as sharp peaks at 28 and 17 kD and did not overlap with other serum protein peaks. Since these assays can be completed on a large number of clinical samples in approximately 1 h, further development of this technique will facilitate both epidemiological studies and therapeutic trials in assessing the role of the apolipoproteins and their glycosylated products in atherosclerosis. Keywords: diabetes • cardiovascular disease • surface-enhanced laser desorption/ionization (SELDI) • time-of-flight mass spectrometry (TOF-MS) • matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) • HDL-apolipoprotein A-I • ProteinChip
Introduction Plasma high-density lipoprotein cholesterol (HDL-C) levels are inversely correlated with risk for atherosclerosis.1-5 The mechanism(s) for this association may involve reverse transport of cholesterol. Many but not all epidemiological studies have indicated a closer association of HDL apolipoprotein A-I with heart disease risk than with HDL-C.4-8 ApoA-II associated with HDL2 does not seem to have a similar protective effect.4,7-9 In patients with diabetes, glycosylation of HDL may result in a functionally abnormal and atherogenic apolipoprotein A-I particle.10-13 * To whom correspondence should be addressed. E-mail: (B.D.)
[email protected], (N.H.E.)
[email protected]. † Presented in part at the 223rd National Meeting of the American Chemical Society, Orlando, FL, April 2002; American Chemical Society: Washington, DC, 2002; Abstract 125 (Medicinal Chemistry Division). ‡ VA NJ Health Care System and UMDNJ-New Jersey Medical School. § RWJ Medical School. 10.1021/pr010008n CCC: $22.00
2002 American Chemical Society
Figure 1. ProteinChip array (reprinted with permission from Ciphergen Biosystems).
Traditionally, protein expression levels in plasma samples have been compared using one-dimensional or two-dimensional (1D and 2D) gel electrophoresis.14-16 We have used 1D gel electrophoresis to identify the apolipoproteins in hypercholesterolemia patients.17 However, these methods are laborintensive and time-consuming, limiting their usefulness in epidemiologic studies. The lack of gel reproducibility and low dynamic range are drawbacks while comparing the expression levels of large numbers of known and unknown proteins. Specific immunoassays (competitive enzyme-linked immunosorbent assay, ELISA)18,19 have been used to measure apoA-I and apoA-II levels in purified HDL lipoprotein subfractions. However, due to significant cross-reactivity between specific antibodies used in these studies, the immunoassays do not accurately reflect the level of apolipoproteins in the plasma.18-29 Also, accurate measurement of both apolipoprotein isoforms could be of metabolic interest since the two major HDL apolipoproteins have been shown to differentially modulate cholesteryl ester transfer protein (CETP) activity and phospholipid transfer protein (PLTP) activity.20-31 It is now well established that plasma lipoproteins do not constitute stable entities in vivo but rather are continuously modified in the blood stream through the action of various Journal of Proteome Research 2002, 1, 375-380
375
Published on Web 05/30/2002
ProteinChip Technology
technical notes
Figure 2. Molecular mass of apolipoprotein A-I and Apo A-II obtained on the WCX2 SELDI ProteinChip array system. Data is obtained for control, type I, and type II diabetes patients.
enzymes, lecithin:cholesterol acyltransferase (LCAT), lipoprotein lipase (LP), cholesteryl ester transfer protein (CETP), and the phospholipid transfer protein (PLTP).4,20-31 Recent studies have demonstrated that the two distinct plasma lipid transfer proteins, CETP and PLTP, markedly alter the size distribution of plasma lipoprotein fractions by shuttling lipid components from one lipoprotein substrate to another.23-26,28,30,31 Preliminary results from our laboratory have indicated that both CETP and PLTP are involved in remodeling HDL, which then may account in vivo for the heterogeneity in the size and composition of plasma HDL.32,33 376
Journal of Proteome Research • Vol. 1, No. 4, 2002
With these factors in mind, we have utilized SELDI (surface enhanced laser desorption ionization) ProteinChip technology to determine the differential protein expression profile in patients with types 1 and 2 diabetes who also have cardiovascular disease. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) has been utilized effectively as a powerful technique for the identification of proteins and has become a unique and a novel procedure in recent years for protein analyses, study of structure, purity, heterogeneity, and posttranslational modification. Some minor limitations of MALDI,
technical notes
Dayal and Ertel
Figure 3. Differential apolipoprotein expression profile: ApoA-I, ApoA-II in control (black) and in patients with type I (red) and type II (blue) diabetes.
such as extensive sample preparation and presence of various matrix adducts and background problems due to inorganic and organic contaminants, have been overcome by the SELDI technique. The ProteinChip (SELDI) has been recently employed with considerable success for the detection, identification, and quantitation of proteins in complex mixtures.34-36 Although ProteinChip is essentially same as the MALDI-MS technique, there are specific advantages that make Proteinchip technology a facile, convenient, and less time-consuming methodology. This technology essentially utilizes the reversal of the MALDI sample preparation. By combining an array of different surfaces and wash conditions, high-resolution chromatographic separations are accomplished on the chips. The position of an individual protein in the spectrum corresponds to its “time of flight” because the small proteins fly faster and the large proteins fly more slowly. SELDIsa refinement of MALDIs preselects the proteins in the sample by allowing them to bind to the treated surface of a metal bar, which is coated with a specific chemical that binds a subset of the proteins within the serum sample. Finally, EAM, the energy absorbing molecule, the SELDI equivalent of MALDI matrix, is added to the chip and subjected
to on-chip laser desorption mass analysis to provide molecular weight information that then relates to the protein profile.
Materials and Methods The ProteinChip array (Ciphergen Biosystems, Palo Alto, CA) is able to determine molecular mass with deviations of less than 0.3% (300 ppm), detects femtomoles of protein, and estimates the quantity of large numbers of proteins simultaneously (refs 34-37, Figure 1). Figure 1 shows a series of protein chip arrays that are used in combination with different wash conditions to optimize a method for protein molecular mass and relative concentration determination from serum samples. SELDI ProteinChip Analysis. Proteins bind differentially to novel and unique chromatographic surfaces of the chips. These surfaces could be chemical (hydrophobic, hydrophilic, cationic, anionic, etc.) or biochemical (antibody, receptor, DNA, etc.) and interact variably with the proteins of interest. Different types of ProteinChip arrays, H4 (aliphatic hydrophobic), NP (normal phase), SAX2 (strong anionic exchange), WCX2 (weak cationic exchange), and IMAC3 (immobilized metal affinity chromatography) (Ciphergen Biosystems) were tested for usefulness in apolipoprotein analysis.34 Different chips have been used to fractionate proteins in tissue lysates,34,35 Figure 1). In Journal of Proteome Research • Vol. 1, No. 4, 2002 377
technical notes
ProteinChip Technology
Figure 4. Positive-ion MALDI-mass spectrum of standard apolipoprotein A-I.
our experiments, strong anionic and weak cationic exchanger (SAX2 and WCX2 chips) surfaces were used (Figures 2 and 3). For apolipoprotein assays using antibodies, the PS1 (preactivated chip, Ciphergen Biosystems) was utilized.37 For analysis using the WCX2 and SAX2 chips, the samples were prepared by equilibrating them with appropriate buffer systems (NaOAc, pH 4.5 for WCX2 and Tris‚HCl, pH 8.5 for SAX2). Comparative studies showed that the WCX-2 chip was superior to the SAX-2 chip in the analysis of apolipoproteins (Figures 2 and 3). The WCX2 chips used in our experiments were washed with buffer (NaOAc, pH 4.5) just before use, and excess buffer was then removed from the spots. We added 1 µL of diluted plasma (×10 mL of NaOAc buffer) onto the chip from patients with types 1 and 2 diabetes, cardiovascular disease without diabetes, and control subjects, transferred the chip into a humidity chamber, and then incubated with agitation for 1 h at room temperature. Each spot on the chip was washed with 10 mL of buffer, followed by washing with water and wiping dry around the spots. An aqueous solution of energy-absorbing molecules, CHCA for SAX2 (R-cyano-4-hydroxycinnamic acid, mw 189.17 Da) or SPA (sinapinic acid, mw 224.21, 2 mg/mL) for WCX2 was prepared containing 50% acetonitrile and 0.5% trifluoroacetic acid and added 1 µL each solution to each spot. After the spots were air-dried completely, the chip was analyzed using the SELDI ProteinChip system (PBS-1, Ciphergen). Patient Selection. Serum samples from the following patient groups were studied: (1) type 1 diabetics; (2) type 2 diabetics; (3) nondiabetic controls; and (4) cardiovascular disease patients. 378
Journal of Proteome Research • Vol. 1, No. 4, 2002
Table 1. Molecular Mass of Apolipoprotein A-I by Protein Chip and MALDI/ MS
control type I DM type II DM
molecular mass (Da) of apoA-I via ProteinChip (apolipoprotein A-I relative intensity)
molecular mass (Da) of apo A-I via MALDI/ MS
27 927 (7.5) 27 887 (3.5) 27 894 (5.0)
28 117 28 095 28 080
Results The differential protein profiles in various patient groups (Figures 2 and 3) were read via surface-enhanced laser desorption ionization-time-of-flight mass spectrometry (SELDITOF). Data were collected using adjustable laser intensity (15%, 30%, and 90%) and detector sensitivity to get easily identifiable peaks without generating either too many doubly charged ions or causing background from matrix-related peaks.34-37 The HDL apolipoproteins A-I (MW ) 28 099 Da) and A-II (MW ) 17 263 Da) were well resolved on this chip (Figure 2) and their molecular masses confirmed via matrix-assisted laser desorption ionization-time-of-flight mass spectrometry32,33,38-44 (Table 1). Plasma samples of the above-mentioned groups of patients were studied and results recorded in terms of molecular weights and peak heights using Ciphergen Biosystems computer software (refs 34-36, Figures 2 and 3). As depicted in the Table 1 and Figures 2 and 3, there are measurable and reproducible intensity variations in apolipo-
technical notes protein A-I on SELDI/MS analysis in different patient groups. The lower levels seen in the patients with diabetes could be in part due to increased catabolism of apolipoproteins with posttranslational modifications such as glycosylation.45 Table 1 provides comparison between the apoA-I levels in three subjects. Relative intensity of apoA-I as seen in Figure 2 in the nondiabetic control has maximum relative intensity (7.5). The intensity in the type 1 diabetic patient is about 4.0 and in the type 2 diabetic patient 5.5. MALDI-MS analysis of the standard HDL apoA-I using sinapinic acid as the matrix exhibited molecular mass values as 28 099 Da (Figure 4). MALDI-MS analysis on the control, type 1, and type 2 patients provided comparable molecule mass values as depicted in Table 1. SELDI/MS Immunoassay. Our recent experiment described in ref 37 utilizes the standard anti-apoA-I, human type 1 (mouse) antibody and demonstrated no cross-reactivity with apoA-II or apoB. The results obtained in terms of molecular mass of apolipoprotein A-I with WCX-2 concur with those from PS-1. Both chips used in these studies possess traditional chromatographic properties, and they did not exhibit any cross reactivity attributable to their surface.
Discussion A major advantage in using ProteinChip technology for the measurement of HDL apolipoproteins was found to be the ease and speed of doing the assays and the ability to obtain results directly from crude biological samples such as serum. There were no steps involved in pretreating the plasma samples such as SDS-PAGE purification or immunoprecipitation.14-17 In addition, SELDI ProteinChip technology offered significant advantages over existing ELISAs techniques18,19 that rely on indirect chemical or radioactive methods of detection. We compared two chips, the strong anionic SAX2 and the weak cationic WCX2, for their ability to separate the HDL apolipoproteins. We found the latter to be superior and used this chip for the clinical studies. Using the ProteinChip technology, we were able to show that apoA-I, apoA-II, and human serum albumin were covalently bound on the chip directly from serum samples. The apolipoproteins A-I and A-II were detected directly using small amounts of serum (1 µL). The apoA-I signals and the area under the peaks were higher for the patients with type 2 diabetes compared with type 1 diabetes in keeping with the relative levels of HDL-C (Figure 3). Thus, using specific WCX2 ProteinChip arrays, we have identified and quantified the apolipoprotein A-I and A-II (MW 28 kD and 17kD) expression levels (Figures 2 and 3) in patients with diabetes and cardiovascular disease. Measurement and comparison of apoA-I and apoA-II expressions in the disease states shown in Figure 3 can be correlated with both cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP) activities.23-26,30-31 The lower levels of apoA-I reflect increased CETP and decreased PLTP activities and all correlate with the higher incidence of coronary artery disease in such states.22-26 In addition, since the ratio of apoA-I to apoA-II (Figures 2 and 3) determines HDL functional and antiatherogenic properties,4,9 measuring this ratio by the ProteinChip method34-37 may be a powerful tool in studying atherogenic cardiovascular disease.46-48
Acknowledgment. We are indebted to Dr. Sameer Bajaj, MD, for his valuable suggestions and fine editing of this
Dayal and Ertel
manuscript. We also gratefully acknowledge the technical assistance provided by Dr. Trigun Bhatt, MD, and Mr. Robert Heinze (Undergraduate Seton Hall University). We thank Mr. Kenneth Mizrach, Medical Center Director, for his continuous support of this research program.
References (1) Frank, P. G.; Marcel, Y. L. Apolipoprotein A-1: structure-function relationships. J. Lipid Res. 2000, 41, 853-872 and references cited theirin. (2) Koppaka, V.; Axelsen, P. H. Lipoprotein A-1 structure. Trends Cardiovasc. Med. 1999, 9, 192-195. (3) Segrest, J. P.; Li, L.; Anantharamaiah, G. M.; Harvey, S. C.; Liadaki, K. N.; Zann. Structure and function of human apolipoprotein A-1 and high-density lipoprotein. Curr. Opin. Lipidol. 2000, 11, 105115. (4) Hedric, C. H.; Castellani, L. W.; Wong, H.; Lusis, A. J. In vivo interactions of apoA-II, apoA-I, and hepatic lipase contributing to HDL structure and antiatherogenic functions. J. Lipid Res. 2001, 42, 563-570. (5) Young, S. G.; Fielding, C. J. The ABC’s of cholesterol efflux. Nature Genet. 1999, 22, 316-31846. (6) Bielicki, J. K.; McCall, M. R.; Stoltzfus, L. J, Ravandi, A.; Kuksis, A.; Rubin, E. M.; Forte, T. M. Evidence that apolipoprotein A-Imilano has reduced capacity, compared wtih wild-type apolipoprotein A-I, to recruit membrane cholesterol. Arterioscler. Thromb. Vasc. Biol. 1997, 17, 1637, 1643. (7) Brinton E. A.; Eisenberg S.; Breslow J. L. Elevated high-density lipoprotein cholesterol levels correlate with decreased apolipoprotein A-I and A-II fractional catabolic rate in women. J. Clin. Invest. 1989, 84, 262-269. (8) Nakamura, T.; Fox-Robichaud A.; Kikkawa R.; Kashiwagi A.; Kojima H.; Fujimiya, M.; Wong N. C. W. Transcription factors and age-related decline in apolipoprotein A-I expression. J. Lipid Res. 1999, 40, 1709-1718. (9) Horowitz, B. S.; Goldberg I. J.; Meran J.; Vanni, T. M.; Ramakrishnan, R.; Ginsberg, H. N. Increased plasma and renal clearance of an exchangeable pool of apolipoprotein A-I in subjects with low levels of high-density lipoprotein cholesterol. J. Clin. Invest. 1993, 91, 1743-1752. (10) Curtis, L. K.; Witztum, J. L. Plasma apolipoproteins Al, All, B, Cl, and E are glucosylated in hyperglycemic diabetic subjects. Diabetes 1985, 34, 452-461. (11) Brownlee, M. Advanced protein glycosylation in diabetes and aging. Annu. Rev. Med. 1995, 46, 223-234. (12) Schmidt, A. M.; Yan, S. D.; Wautier, J. L.; Stern, D. Activation of receptor for advanced glycation end products. Circ. Res. 1998, 84, 489-497. (13) Rudd, M. P.; Endo, T.; Colominas, C.; Groth, D.; Wheeler, S. F.; Harvey, D. J.; Wormald, M. R.; Serban, H.; Prusiner, S. B.; Kobata, A.; Dwek, R. A. Glycosylation differences between the normal and pathogenic prion protein isoforms. Biochemistry 1999, 23, 1304413049. (14) Sprecher, D. L.; Taam, L.; Brewer, H. B., Jr. Two-dimensionalelectrophoresis of human plasma apolipoproteins. Clin. Chem. 1984, 30, 2084-2092. (15) Karpe, F.; Hamsten, A. Determination of apolipoprotein B-48 and B-100 in triglyceride-rich lipoproteins by analytical SDS-PAGE. J. Lipid Res. 1994, 35, 1311-1317. (16) Gauss, C. et al. Analysis of the mouse proteome. Brain proteins: separation by two-dimensional gel electrophoresis and identification by mass spectrometry and genetic variation. Electrophoresis 1999, 20, 575-600. (17) Dayal, B.; Ertel, N. H. Matrix-assisted laser desorption/ionization mass spectrometry of apolipoproteins extracted directly from sodium dodecyl sulfate-polyacrylamide gels: studies performed in patients with diabetes and cardiovascular disease. Unpublished results. (18) Oka, T.; Kujiraoka, T.; Ito, M.; Nagano, M.; Ishihara, M.; Iwasaki, T.; Egashira, T.; Miller, N. E.; Hattori, H. Measurement of human phospholipid transfer protein by sandwich enzyme-linked immunosorbent assay. Clin. Chem. 2000, 46, 1357-1364. (19) Desrumaux, C.; Athias, A.; Bessede, G.; Verges, B.; Farnier, M.; Persegol, L.; Gambert, P.; Lagrost, L. Mass concentration of plasma Phospholipid Transfer Protein in normolipidemic, type IIa hyperlipidemic, type IIb hyperlipidemic, and noninsulindependent diabetic subjects as measured by a specific ELISA. Arterioscler. Thromb. 1999, 19, 266-275.
Journal of Proteome Research • Vol. 1, No. 4, 2002 379
technical notes
ProteinChip Technology (20) Guyard-Dangremont, V.; Lagrost, L.; Gambert, P. Comparative effects of purified apolipoproteins A-I, A-II, and A-IV on Cholesteryl Ester Transfer Protein Activity. J. Lipid Res. 1994, 35, 982992. (21) Jauhiainen, M.; Huuskonen, J.; Baumann, M.; Metso, J.; Kka, T.; Egashira, T.; Hattori, H.; Olkkonen, V. M.; Ehnholm, C. Phospholipid transfer protein (PLTP) causes proteolytic cleavage of apolipoprotein A-I. J. Lipid Res. 1999, 40, 654-664. (22) Jauhiainen, M.; Ehnholm, C. Role of the phospholipid transfer protein (PLTP) in plasma lipid transport. In Advances in Vascular Biology: Plasma Lipids and Their Role in Disaese; Barter P. J., Rye, K. A., Eds.; Harwood Academic: Australia, 1999; pp 28597. (23) Roy P.; MacKenzie, R.; Hirama T.; Jiang, X. C.; Kussie, P.; Tall, A.; Rassart, E.; Milne, R. Structure-function relationships of human cholesteryl ester transfer protein: analysis using monoclonal antibodies. J. Lipid Res. 1996, 37, 22-34. (24) Sviridov, D.; Hoang, A.; Sawyer.; W. H.; Fidge, N. H. Identification of a sequence of apolipoprotein A-I associated with the activation of lecithin:cholesterol acyltransferase. J. Biol. Chem. 2000, 275, 19707-19712. (25) Cho, K. H.; Jonas, A. A key point mutation (V156E) affects the structure and function of human apolipoprotein A-1. J. Biol. Chem. 2000, 275, 26821-26827. (26) Asztalos, B.; Wenwu, Z.; Roheim, P. S.; Wong, L. Role of free apolipoprotein A-I in cholesterol efflux. Arterioscler. Thromb. Vascu. Biol. 1997, 17, 1630-1636. (27) Maciejko, J. J. et al. Apolipoprotein A-I as a marker of angiographically assessed CAD. N. Engl. J. Med. 1983, 309, 385-389. (28) Dalton, M. B.; Swaney, J. B. Structural and functional domains of apolipoprotein A-I within high-density lipoproteins. J. Biol. Chem. 1993, 268, 19274-19283. (29) Chiesa, G.; Stoltzfus, L. J.; Michelagnoli, S.; Bielicki, J. K.; Santi, M.; Orte, T. M.; Sirtori, C. R.; Franceschini, G.; Rubin, E. M. Elevated triglycerides and low HDL cholesterol in transgenic mice expressing human apolipoprotein A-Imilano. Atherosclerosis 1998, 136L, 139-146. (30) Francis, G. A.; Knopp, R. H.; Oram, J. Defective removal of cellular cholesterol and phospholipids by apolipoprotein A-I in Tangier disease. J. Clin. Invest. 1995, 96, 78-87. (31) Rye, K. A.; Clay, M. A.; Barter, P. J. Remodeling of high-density lipoproteins by plasma factors. Atherosclerosis 1999, 145, 227238. (32) Dayal, B.; Li, Q.; Ertel, N. H. A facile mass spectral approach for the measurement and identification of apolipoproteins A-1 (apoA-1). 221st National Meeting of the American Chemical Society, San Diego, CA, April 2001; American Chemical Society: Washington, DC, 2001; Abstract 19 (Medicinal Chemistry Division). (33) Dayal, B.; Ertel, N. H. et al. The role of phospholipid transfer proteins in remodeled HDL and their identification via matrixassisted laser desorption ionization mass spectrometry (MALDIMS). 40th Eastern Analytical Symposium & Exposition, Atlantic City, NJ, Oct 1-4, 2001, Abstract 282. (34) Davies, H.; Lomas, L.; Austen, B. Profiling of amyloid Beta peptide variants using SELDI ProteinChip arrays. Biotechniques 1999, 27, 1258-1261.
380
Journal of Proteome Research • Vol. 1, No. 4, 2002
(35) Li, X.; Mohan, S.; Gu, W.; Miyakoshi, N.; Baylink, D. J. Differential protein profile in the ear-punched tissue of regeneration and nonregeneration strains of mice: a novel approach to explore the candidate genes for soft-tissue regeneration. Biochim. Biophys. Acta 2000, 1524, 102-109. (36) Wright, G. L., Jr.; Cazares, L. H.; Leung, S. M.; Nasim, S.; Adam, B. L.; Yip, T. T.; Schellhammer, P. F.; Gong, L.; Vlahou, A. ProteinChip surface enhanced laser desorption/ionization (SELDI) mass spectroscopy: a novel protein biochip technology for detection of prostate cancer biomarkers in complex protein mixtures. 1999, 2, 264-276. (37) Dayal, B.; Ertel N. H.; Dang, A. Binding of apolipoprotein A-1 from human plasma to anti-apolipoprotein A-1 antibody and its measurement by Surface Enhanced Laser Desorption IonizationTime-of-Flight Mass Spectrometry (SELDI-TOF): (ProteinChip Technology). Manuscript in preparation. (38) Ehring, H.; Stromberg, S.; Tjernberg, A.; Noren, B. Matrix-assisted laser desorption ionization mass spectrometry of proteins extracted directly from sodium dodecyl sulphate-polyacrlyamide gels. Rapid Commun. Mass Spec. 1997, 11, 1867-1873. (39) Mirza, U. A.; Liu, Yan-Hui, Tang J. T.; Porter, F.; Bondoc, L.; Chen, G.; Pramanik, B. N.; Nagabhushan, T. L. Extraction and Characterization of Adenovirus Proteins From Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis by Matrix Assisted Laser Desorption/Ionization Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2000, 11, 356-361. (40) Bondarenko, P. V.; Cockrill, S. L.; Watkins, L. K.; Cruzado, I. D.; Macfarlane, R. D. Mass spectral study of polymorphism of the apolipoproteins of very low-density lipoprotein. J. Lipid Res. 1999, 40, 543-555 and references therein. (41) Beavis, R. C.; Chait, B. T. Rapid, sensitive analysis of protein mixtures by mass spectrometry. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 6873-6877. (42) Ito, Y.; Breslow, J. L.; Chait, B. T. Apolipoprotein C-III0 lacks carbohydrates residue: use of mass spectrometry to study apolipoprotein structure. J. Lipid Res. 1998, 30, 1781-1787. (43) Gygi, S. P. et al. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nature Biotechnol. 1999, 17, 994-999. (44) Lueking, A.; Horn, M.; Eickhoff, H.; Walter, G. Protein microarrays for gene expression and antibody screening. Anal. Biochem. 1999, 270, 103-111. (45) Witztum, J. L.; Fisher, M.; Pietro, T.; Steinbrecher, U. P.; Elam, R. L. Nonenzymatic glucosylation of high-density lipoprotein accelerates its catabolism in guinea pigs. Diabetes 1982, 31, 10291032. (46) Downs, J. R.; Clearfield, M.; Weis, S. et al. Primary prevention of acute coronary events with lovastatin in men and women with average cholesterol levels: results of AFCAPS/TexCAPS. Air Force/ Texas Coronary Atherosclerosis Prevention Study. JAMA. 1998, 279, 161. (47) Scandinavian Simvastatin Survival Study Group. Lancet 1994, 344, 1383-9. (48) Shepherd, J.; Cobbe, S. M.; Ford, I.; Isles, C. G.; Lorimer, A. R. et al. Prevention of coronary heart disease with pravastatin in men with hypercholesterolemia. N. Engl. J. Med. 1995, 333, 1301-7.
PR010008N
