Analysis of High-Density Lipoprotein Apolipoproteins Recovered from

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Analysis of High-Density Lipoprotein Apolipoproteins Recovered from Specific Immobilized pH Gradient Gel pI Domains by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Zachlyn N. Farwig, Anna V. Campbell, and Ronald D. Macfarlane*

Department of Chemistry, Texas A&M University, College Station, Texas 77843

The proteins associated with the circulating lipoproteins in the blood function not only for mediating lipid metabolism but also for maintaining structural stability of the micellelike structure. Any modifications of these proteins, by mutation or posttranslational modification, could compromise the function of these proteins and contribute to the development of cardiovascular disease. Because of the presence of extensive lipophilic domains, these proteins, when recovered from the lipoprotein particle (apolipoproteins) present an analytical challenge because of low solubility and proclivity toward aggregate formation. Our goal is to characterize these proteins by a combination of high-accuracy pI measurement coupled with MALDI analysis. In this report, we demonstrate the high resolution of immobilized pH gradient isoelectric focusing (IPGIEF) for the analysis of these apolipoproteins isolated from serum HDL collected from a density gradient ultracentrifugation-based separation. The IPG separation of the HDL apolipoproteins was imaged and combined with digital analysis to produce a detailed pI profile of the apolipoproteins in the high-density lipoprotein (HDL) fraction. This is the first time that a high-resolution pI profile has been obtained for the HDL apolipoproteins. The feasibility of linking the pI profile to a MALDI-based molecular weight determination was achieved by incorporating passive elution of the intact proteins from the IPG gel with a four-component solvent mixture that solved the problem of recovery of the apolipoproteins from the IPG matrix. Twenty-five bands were observed in the pI profile. A survey analysis of 12 of these bands by MALDI indicated that they were associated with the known HDL apolipoproteins. While there is considerable overlap in the pI profiles of the apolipoproteins, linking the analysis with a MALDI-based second dimension in m/z is shown * Corresponding author. Phone: (979)-845-2021. Fax: (979)-845-8987. Email: [email protected]. (1) Gorg, A.; Obermaier, C.; Boguth, G.; Harder, A.; Scheibe, B.; Wildgruber, R.; Weiss, W. Electrophoresis 2000, 21, 1037-1053. (2) Righetti, P. G. Immobilized pH Gradients: Theory and Methodology; Elsevier Science: Amsterdam, 1990. 10.1021/ac026273p CCC: $25.00 Published on Web 06/25/2003

© 2003 American Chemical Society

to be an efficient method to characterize this complex mixture of apolipoproteins. Development of immobilized pH gradients (IPG) has been a major development in the characterization of the proteome.1 The high resolving power of the IPG isoelectric focusing (IEF) method allows the separation of isoforms with isoelectric points differing by as little as 0.001 pH unit.2 In the rapidly growing field of proteomics, the classical approach is the use of IPG-IEF combined with SDS-PAGE followed by protein isolation, digestion, and analysis via mass spectrometry.3 One drawback of SDS-PAGE is the poor mass accuracy of the intact protein.3,4 Analysis of the pI profile of the intact proteins in the IPG-IEF separation provides a detailed measurement of the pI distribution for a protein mixture. A second dimension (MW) is necessary to unambiguously identify a protein with a particular pI value. Mass spectrometry addresses the mass accuracy problem with SDSPAGE methods. The specificity of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for the analysis of proteins has been widely described.5-7 Combining the high resolution of the IPG-IEF technology with the specificity and accuracy of MALDI-TOF-MS would make it possible to identify particular proteins within a complex mixture. Recovery of the protein directly from the IPG strip is challenging due to the strong interaction between the proteins and the gel support.1 This is particularly the case for lipophilic proteins of the type found in lipoprotein particles, the focus of this paper. Passive elution has been used successfully for the recovery of intact proteins from SDS-PAGE gels8 and from IPG gels containing immobilized ampholytes9 for mass spectral analysis. Passive elution of proteins from the commercially available Immobiline DryStrips (Amersham Pharmacia Biotech) for electrospray mass (3) Rabilloud, T. Proteomics 2002, 2, 3-10. (4) Sarioglu, H.; Lottspeich, F.; Walk, T.; Jung, G.; Eckerskorn, C. Electrophoresis 2000, 21, 2209-2218. (5) Hillenkamp, F.; Karas, M.; Beavis, R. C.; Chait, B. T. Anal. Chem. 1991, 63, 1193A-1202A. (6) Bonk, T.; Humeny, A. Neuroscientist 2001, 7, 6-12. (7) Beavis, R. C.; Chait, B. T. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 68736877. (8) Galvani, M.; Hamdan, M.; Righetti, P. G. Rapid Commun. Mass Spectrom. 2000, 14, 1889-1897. (9) Breme, U.; Breton, J.; Visco, C.; Orsini, G.; Righetti, P. G. Electrophoresis 1995, 16, 1381-1384.

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spectrometry (ESI-MS) analysis was reported by Loo et al. following separation on the Multiphor II instrument.10 Direct desorption from the strips for MALDI-TOF-MS was also described.10 Separation and passive elution of proteins using the novel instrument IPGphor has not been reported. Protein must be recovered from the IPG strip in sufficient yield to perform the MALDI-TOF-MS analysis. The problem is that the ampholytes are covalently bound to the gel matrix. This means that a protein focused in the IPG gel has several sites of attachment making it more difficult to recover the protein from the gel. In this paper, a method for recovery of apolipoproteins from the IPG gel is described that has a sufficient recovery yield that interpretable MALDI-TOF mass spectra have been obtained. This class of hydrophobic apolipoproteins (apos) isolated from human high-density lipoprotein (HDL) is a particular challenge because of low solubility and tendency to form aggregates. The apolipoproteins associated with HDL represent a challenge for identification by the pI and MW vectors. Potentially atherogenic mutations are of particular interest. Although pI profiles have been obtained for these proteins by gel electrophoresis using carrier ampholytes, linking this mode of pI profiling with mass spectrometry is problematic. In preliminary studies, we attempted to carry out pI profiling using capillary isoelectric focusing and carrier ampholytes. While model proteins were successfully focused, the apolipoproteins precipitated out at a pH near their pI values. The study reported here, using the IPG-IEF method and linking to MALDI analysis, is a significant development in the characterization of the isoforms of the proteins associated with serum HDL. EXPERIMENTAL SECTION Chemicals and Materials. Reagent grade acetonitrile, formic acid, methanol, methyl sulfoxide (DMSO), and trichloroacetic acid were obtained from EM Science (Gibbstown, NJ). Sinapinic acid, 2-propanol, trifluoroacetic acid (TFA), phosphoric acid, Amberlite ion-exchange resin, myoglobin, and Sudan Black B were from Sigma (St. Louis, MO). Urea, (3-[3-cholamidopropyl)dimethylammonio]propanesulfonate (CHAPS), and mineral oil were from Amersham Pharmacia Biotech (Uppsala, Sweden). Acid Violet 17 was purchased from Serva (Heidelberg, Germany). Commercial apolipoprotein A-I standard was purchased from CalBiochem (San Diego, CA). The cesium bismuth EDTA complex (CsBiEDTA) was synthesized in our laboratory.11,12 Deionized water used in all experiments was from a Milli-Q water purification system (Millipore, Bedford, MA). Immobiline DryStrips pH 4-7, 13 cm, were purchased from Amersham Pharmacia Biotech. For the determination of percent recovery of apolipoprotein A-I from the gels, a commercial kit based on immunoturbidimetry was purchased from Sigma. Ultracentrifugation. The serum for this study was obtained from a normolipidemic subject following a 12-h fast. Serum samples were collected by a blood draw into a 9.5-mL Vacutainer treated with polymer gel and silica activator (366510, Becton Dickinson Systems, Franklin Lakes, NJ). Serum was separated (10) Loo, J. A.; Brown, J.; Critchley, G.; Mitchell, C.; Andrews, P. C.; Ogorzalek Loo, R. R. Electrophoresis 1999, 20, 743-748. (11) Hosken, B. D.; Macfarlane, R. D., inventors. Density gradient solutions of metal ion chelate complexes. U.S. Patent 10/092,032, 2002. (12) Hosken, B. D. Density gradient ultracentrifugation of lipoproteins using metal ion complex solutes. Ph.D. Dissertation, Texas A&M University, 2002.

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from the red blood cells by centrifugation at 3200 rpm for 20 min at 4 °C. The supernatant (serum) was aspirated from the red blood cells. A 100-µL aliquot of the serum was stained (for visualization) with 8 µL of 0.1% (w/v) Sudan Black B in DMSO and incubated at room temperature for 30 min. A 992-µL aliquot of deionized water was added, and then the samples were spun for 3 min at 7g. A 550-µL aliquot of 20% (w/w) CsBiEDTA11 was transferred to a 34 × 11 mm polycarbonate open-top centrifuge tube (Beckman Instruments, Fullerton, CA). A 550-µL aliquot of the stained serum sample was added to the tube and mixed with the CsBiEDTA to form a homogeneous solution. The tube was centrifuged in a Beckman Optima TLX-120 ultracentrifuge equipped with a 30° fixed angle TLA 120.2 rotor at 627379g at 20 °C for 4 h, 40 min.12 Following the UC spin, the tube was frozen slowly in liquid nitrogen. The lipoprotein fractions were collected quantitatively by cutting the frozen tube at positions 20.60 and 27.93 mm from the top of the tube with a high-speed thin blade (0.254 mm) scroll saw (Dremel, model 1672) using a custom-made brass adjustable block. The high-density lipoprotein fraction was collected between 1.045 and 1.144 g/mL. The fraction was thawed and centrifuged at 7g for 3 min to remove any particulates in the sample from the fraction collection. C18 Solid-Phase Extraction, Desalting, and Delipidation. The samples were desalted or delipidated as previously described.13 Briefly, a sample was prepared for delipidation with the use of a tC18 Light cartridge (Sep-Pack, No. 36805, Waters, Milford, MA) by mixing 150 µL of the HDL fraction with 40 µL opf 1% (v/v) TFA and 300 µL of water. The cartridge was conditioned with 5 mL of 0.1% (v/v) TFA in acetonitrile followed by 5 mL of 0.1% (v/v) TFA in water. After the sample was loaded onto the cartridge, the cartridge was rinsed with 5 mL of 0.1% (v/v) TFA in water to remove any salts and contaminants from the UC spin. Proteins were then eluted in 50-µL aliquots of 0.1% TFA in acetonitrile. The majority of the apolipoproteins were eluted in the second, third, and fourth aliquots, which were collected and combined for subsequent analyses. The commercial apo A-I standard was desalted using this method prior to IPG analysis. Immobilized pH Gradient Analysis. IPG analysis was performed using a standard method on the IPGphor (Amersham Pharmacia Biotech). Aliquots from the tC18 Light cartridge were evaporated to dryness and reconstituted in 255 µL of an 8 M urea, 2% (w/v) CHAPS solution. The sample was sonicated for 30 min at 20 °C, incubated at room temperature for 30 min, and centrifuged for 5 min at 7g. A 250-µL aliquot was loaded onto the precast gel. IEF parameters for the separation were 50 µA/strip at 20 °C with a rehydration step for 12 h followed by steps of 500, 1000, and 32000 V‚h. The gel was stained with Acid Violet 17 as described by Patestos et al.14 Briefly, the gel was fixed for 30 min in 20% (w/v) trichloroacetic acid. The gel was then rinsed in 3% (v/v) phosphoric acid for 1 min. The gel was stained for 10 min using a freshly prepared colloidal Acid Violet solution composed of 100 mg of Acid Violet 17 dissolved in 50 mL of deionized water mixed with 50 mL of 20% (v/v) phosphoric acid. The gel was rinsed three times for 10 min in 3% (v/v) phosphoric acid followed by three 5-min rinses in deionized water. All steps were performed (13) Watkins, L. K.; Bondarenko, P. V.; Barbacci, D. C.; Song, S.; Cockrill, S. L.; Russell, D. H.; Macfarlane, R. D. J. Chromatogr., A 1999, 840, 183-193. (14) Patestos, N. P.; Fauth, M.; Radola, B. J. Electrophoresis 1988, 9, 488-496.

with gentle shaking of the container. An image of the gel was recorded with a digital camera (Kodak DC120). Imaging analysis was performed with Kodak Digital Science 1D software (Kodak Scientific Imaging Systems, New Haven, CT) and Origin (Microcal Software, Inc., Northamption, MA) to produce a protein pI profile. Passive Elution. Following the IPG run, an unfixed and unstained gel was removed from the holder and placed on a flat surface for excising portions of the gel. The gel was excised and collected in individual sections based on the corresponding band in the stained gel and placed in tubes. Deionized water was used to wash the collected segments of the gel. Collected gel pieces were dehydrated with 200 µL of acetonitrile for 20 min, vortexing occasionally, and then washed with deionized water. The dehydration step was repeated with 200 µL of methanol for 20 min, vortexing occasionally, followed by a wash with deionized water. Proteins were extracted from the gel pieces with 100 µL of FAPH (mixture of 50% (v/v) formic acid, 25% (v/v) acetonitrile, 15% (v/ v) 2-propanol, 10% (v/v) deionized water)15 for 15 min under sonication at 35 °C. Following a 2-min spin at 10 000 rpm, the supernatant was removed and evaporated to dryness. Samples were reconstituted in 6 µL of FAPH. For the standard apolipoprotein A-I, recovery was determined with a commercial immunoassay. Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry. The samples were analyzed with a commercial MALDITOF mass spectrometer (Voyager, PerSeptive Biosystems, Framingham, MA)16 in the linear mode with delayed extraction. Samples were prepared as previously described.17 Briefly, the samples were prepared by mixing 2 µL of the sample with 3 µL of a 20 mg/mL sinapinic acid in methanol matrix solution and 6 µL of deionized water. A 0.5-µL aliquot of 20 mg/mL sinapinic acid was deposited on the target and allowed to dry. A 0.5-µL aliquot of the sample mixture was applied to the plate and allowed to dry. A 2-µL deionized water rinse was applied to the spot and shaken off after 5 s. Myoglobin was used as an external mass calibration standard. Because this was a feasibility study, no attempt was made to optimize mass accuracy because of the wide mass range encompassing the apolipoproteins and the influence of the delayed extraction method. RESULTS AND DISCUSSION Overview. The apolipoproteins of HDL are comprised mainly of apo A-I and -II and apo C-I, -II, and -III. Recent MALDI analysis of this mixture has shown that each of these apolipoproteins exists in different isoforms due to posttranslational modification.18 These isoforms can be resolved by mass spectrometry so long as the mass differences are large enough that they can be resolved. By linking the mass spectrum of the HDL apolipoproteins with its pI profile, a more complete analysis of the isoform distribution could be obtained. However, in this study, no attempt was made to optimize mass accuracy because the objective was to determine feasibility of analyzing apolipoprotein mixtures by two-dimensional pI-MALDI profiling.

Analysis of Apo A-I Standard. The first phase of the study was to analyze a commercial high-purity apo A-I standard by both MALDI analysis and IPG-IEF. A solution of the standard was run through the same preparation protocol as for the HDL fraction: solid-phase extraction, analysis of an aliquot by MALDI, and treatment of a second aliquot with urea/CHAPS prior to IPG-IEF analysis. MALDI-TOF-MS. Figure 1a shows the MALDI mass spectrum obtained for the standard. The spectrum is composed of two ions, both associated with apo A-I, the singly and doubly charged molecular ion of apo A-I. IPG-IEF. In the development of the IPG-IEF separation, the goal was a procedure that is simple and efficient with as few steps as possible. We chose not to employ carrier ampholytes because they produce a higher background during staining and have the potential for interfering with the elution of the proteins from the gel. According to Breme et al.,9 carrier ampholytes increase the background in ESI-MS analysis, an observation confirmed by Loo.10 We determined that the IPG-IEF separation was possible for the apolipoproteins without using carrier ampholytes. Further, reducing agents were not used because we wanted to study the native apolipoproteins. For example, we discovered by MALDI analysis in a separate study that apo A-II consists of two monomers linked by a disulfide bond.17 We chose colloidal Acid Violet for visualization rather than the traditional Coomassie Blue because of its superior sensitivity (5-10 ng/band14) and low background. A photograph of the stained IPG-IEF strip is shown between panels a and b of Figure 1. Several dark bands in the vicinity of pI 5 are present along with a collection of faint bands between pI 4.5 and 6.4. Figure 1b is the profile obtained from a digital analysis of the IPG strip. The assigned pI values are based on a pH gradient analysis provided by APBiotech for the run conditions described in the Experimental Section (8 M urea, 20 °C). The high resolution of the IPG-IEF method made it possible to measure the pI values to two significant figures. The pI profile indicated that as many as 12 isoforms of apo A-I may exist in the pI region between 4.52 and 5.33. It is possible that other proteins are contributing, but the MALDI analysis indicates that apo A-I is the dominant component. In previous IEF studies, apo A-I was found to have up to five isoforms19-21 with three isoforms due to single and double deamidation, a modification that only changes the molecular mass of the protein by 1-2 Da.22 The results obtained here suggest that many more isoforms may exist that can be resolved by the IPG-IEF method. It is also possible that many of these isoforms are due to other in vitro modifications that do not significantly change the MW of the apo A-I or to modification by the FAPH solvent system. This question cannot be adequately addressed in this study because the method used for MW determination was not optimized for the mass accuracy required. The unexpected appearance of these multiple bands in a mass spectrometrically homogeneous, highly purified sample illustrates the potential utility of a method that can give additional information

(15) Ehring, H.; Stromberg, S.; Tjernberg, A.; Noren, B. Rapid Commun. Mass Spectrom. 1997, 11, 1867-1873. (16) Vestal, M. L.; Juhasz, P.; Martin, S. A. Rapid Commun. Mass Spectrom. 1995, 9, 1044-1050. (17) Bondarenko, P. V.; Cruzado, I. D.; Cockrill, S. L.; Watkins, L. K.; Macfarlane, R. D. J. Lipid Res. 1999, 40, 543-555. (18) Bondarenko, P. V.; Farwig, Z. N.; McNeal, C. J.; Macfarlane, R. D. Int. J. Mass Spectrom. Ion Processes 2002, 219, 671-680.

(19) Brewer, H. B., Jr.; Fairwell, T.; Kay, L.; Meng, M.; Ronan, R.; Law, S.; Light, J. A. Biochem. Biophys. Res. Commun. 1983, 113, 626-632. (20) Ghiselli, G.; Rohde, M. F.; Tanenbaum, S.; Krishnan, S.; Gotto, A. M., Jr. J. Biol. Chem. 1985, 260, 15662-15668. (21) Nestruck, A. C.; Suzue, G.; Marcel, Y. L. Biochim. Biophys. Acta 1980, 617, 110-121. (22) Robinson, A. B.; Scotchler, J. W.; McKerrow, J. H. J. Am. Chem. Soc. 1973, 95, 8156-8159.

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Figure 1. (a) MALDI-TOF mass spectrum of commercial apolipoprotein A-I standard eluted from tC18 Light cartridge. (b) IPG-IEF pI profile of apo A-I standard, digital image of gel stained with Acid Violet.

on specific bands in the pI profile. We selected four bands in the profile, presumably apo A-I isoforms, to illustrate the method we have developed to identify a pI-specific band by MALDI analysis. Passive Elution. Prior to the development of the passive elution approach, an attempt was made to obtain MALDI spectra directly from the gel as previously described.10 This approach was not successful. The approach here was to select bands from the IPG-IEF strip, recover the protein intact, and determine whether the protein had a MW close to that of apo A-I. Four bands were selected for the study, those at pI 4.87, 4.94, 5.02, and 5.10. The method for excising each band is described in the Experimental Section. Several solvent systems used with success in 3826 Analytical Chemistry, Vol. 75, No. 15, August 1, 2003

previous studies proved to be unsuccessful for the recovery of the apolipoproteins. This includes the use of 0.1% TFA in acetonitrile, which was successfully used in the recovery of the apolipoproteins from the C18 solid-phase extraction cartridge. A similar solvent system was used previously for elution of hydrophilic proteins from IPG gels.9,10 A detergent system (urea/ CHAPS) was able to recover a small fraction of the apo A-I from the bands (∼5%). What proved to give the highest recovery yield (25%) was a mixture of formic acid, acetonitrile, 2-propanol, and water (FAPH).15 Details of the recovery conditions are given in the Experimental Section. This solvent system was used for the remainder of the study.

Figure 2. MALDI-TOF mass spectra of recovered proteins from labeled excised bands (a-d) from unstained IPG gel of apo A-I standard.

The MALDI spectra obtained from elution of the four bands using FAPH are shown in Figure 2. The recovered protein in FAPH was used directly for analysis without further purification. The MALDI analysis showed that each of the four bands has a MW close to that of of apo A-I and is presumably an isoform. There is some indication of a small amount of degradation of apo A-I in the recovery, possibly due to hydrolysis by FAPH. Table 1 shows the experimental and theoretical masses of apo A-I. In

comparison with the calculated molecular weight for the mature protein, mass accuracy was on the order of (0.3%. The mass differences may in part be due to the isoform differences of apo A-I separated in the gel although this difference should be 1-2 mass units due to deamidation. Loo was able to obtain mass accuracy of ( 0.1% for proteins of 20-30 kDa with direct (23) Swiss Institute of Bioinformatics and European Bioinformatics Institute. SWISS-PROT Database, 2002.

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Figure 3. IPG-IEF pI profile produced from an Acid Violet-stained pH 4-7 gel of a delipidated high-density lipoprotein fraction with digital image of the stained gel. Table 1. Experimental Molecular Weights for Standard Apo A-I and the Recovered Intact Proteins Compared to the Calculated Molecular Weight. Designations Correspond to Figure 2 molecular weight

discrepancy (Da)

apo A-I

exptl

calcd23

standard (Figure 1) a b c d

28 101.7

28 078.6

23.1

28 236.0 28 179.2 28 133.4 28 114.6

28 078.6 28 078.6 28 078.6 28 078.6

157.4 100.6 54.8 36.0

from calcd

from std

134.4 77.5 31.7 13.0

desorption from the gel.10 Breme et al. reported high mass accuracies using liquid chromatography followed by ESI-MS.9 In our studies, the focus was on the feasibility of carrying out an IPG pI/MALDI profile analysis on some of the most difficult and medically relevant proteins. It remains an open question whether, in the process, there may be some chemical modification of the apolipoproteins by the FAPH solvent system. This question will be addressed in a future study that will include a more detailed analysis of the isoforms with higher mass accuracy. The next step was to carry out the IPG-IEF/MALDI analysis for the delipidated fraction from a serum sample. Analysis of HDL Fraction. To demonstrate the feasibility of linking the IPG-IEF pI profile with MALDI for mixture analysis, we analyzed the delipidated HDL fraction following the procedure described in the Experimental Section. The results are shown in 3828 Analytical Chemistry, Vol. 75, No. 15, August 1, 2003

Figure 3. A photograph of the stained IPG strip has been inserted at the top of the figure. The digitized profile, aligned with the strip, comprises the remainder of the figure. Approximately 25 components were observed. The major components, clustered in the pI range 5.00-5.33, had the same pI values as those observed for the apo A-I standard. But in addition, components were also observed between pI 4.37 and 6.47. The IPG strip was then divided into eight segments, and each segment was subjected to passive elution followed by MALDI analysis of the FAPH extract. The first section (labeled “a” on the IPG strip in Figure 3) covers a pI range from 4 to 4.35. Although the bands in this section had low intensity, the MALDI spectrum for the apolipoproteins recovered from this section (Figure 4a) contained identifiable components with m/z values corresponding to three known apo C-III isoforms (apo C-III0.Glyc, apo C-III1, apo C-III2). The section excised from the gel strip labeled “b” covers a pI range from 4.35 to 4.50. MALDI analysis showed that this pI range includes additional isoforms of apo C-III0,Glyc and apo C-III1 as well as proapo C-II. The intense band in the pI range 4.50-4.60 (labeled “c”) was confirmed by MALDI analysis to be apo A-II. A typical peak distribution for both the dimer and monomer of the disulfide-linked homodimer is present in the mass spectrum as illustrated by the inset (Figure 4c).18 The doublet peaks at ∼17 300 are due to the intact apo A-II dimer and a truncated isoform of the dimer.18 The next section selected for analysis (labeled “d”) covers a pI range from 4.90 to 5.05 and encompasses an intense band at pI 5.01 that was also observed

Figure 4. MALDI-TOF mass spectra of recovered proteins from labeled excised bands (a-h) from unstained IPG gel of a delipidated highdensity lipoprotein fraction.

in the pI profile of the apo A-I standard (Figure 1b). MALDI analysis of the sample recovered from this section confirms that the band is due to an isoform of apo A-I (Figure 4d). The second intense band in the apo A-I region covering a pI range from 5.05 to 5.15 (labeled “e” on the gel) was also confirmed by MALDI analysis to indeed be an isoform of apo A-I (Figure 4e). The two weak bands in the pI range from 5.15 to 5.28 (section “f”) with pI values of 5.20 and 5.24 were confirmed by MALDI to also be isoforms of apo A-I (Figure 4f). The next section selected labeled “g” includes two weak peaks in the pI profile (Figure 3) at 5.54 and 5.58. The MALDI analysis of this section showed these species to be due to human serum albumin (HSA) (Figure 4g). (The low level of contamination of the HDL fraction by HSA is due to the excellent separation achieved using the new CsBiEDTA density gradient-forming solute.) The last section studied was in the pI range of 6.25-6.35, where one band was detected. The MALDI spectrum (Figure 4h) shows that the dominant species is apo C-I. In addition, a small peak was observed at m/z 12,887 due to the constitutive isoform of serum amyloid A, an acute phase protein associated with systemic inflammation that was found to be present in the serum sample used in this study. Shown in Table 2 are the experimental and theoretical masses for the apolipoproteins detected in the HDL fraction. Mass accuracy for 5-10-kDa proteins was (0.14% and for proteins in the 10-20-kDa range, (0.09%. For proteins in the 20-30-kDa range, mass accuracy was (0.2%. Overall the mass accuracy was (0.15%. While the mass accuracy is sufficient to associate the

Table 2. Experimental Molecular Weight from Recovered Intact Proteins Compared to the Calculated Molecular Weighta molecular weight protein

exptl

calcd23

discrepancy (Da)

A-I (d) A-I (e) A-I (f) A-II (c) C-I (h) pro C-II (b) C-III0 (a,b) C-III1 (a,b) C-III2 (a) SAA4 serum albumin

28 174.1 28 065.4 28 153.3 17 373.5 6 642.1 8 214.4 9 139.1 9 430.3 9 742.4 12 882.8 66 557.4

28 078.6 28 078.6 28 078.6 17 379.8 6 630.6 8 204.1 9 130.0 9 421.3 9 712.6 12 863.2 66 472.2

95.5 13.2 74.7 6.3 11.5 0.5 9.1 9.0 29.8 19.6 85.2

a

Designations correspond to Figure 4.

isoforms with the major HDL apos, it is not sufficient to unambiguously rule out the possibility of modification of these apos by the FAPH recovery solvent. Improved mass accuracy using internal standards covering the same m/z range as the apos will address this question. CONCLUSIONS The primary objectives of this study were to develop an IPGIEF separation for apolipoproteins and a method for recovering these apolipoproteins from immobilized pH gradient gels. ApoliAnalytical Chemistry, Vol. 75, No. 15, August 1, 2003

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poproteins provide a unique mixture of hydrophobic proteins that are important markers for cardiovascular disease as well as a challenge for analysis. A straightforward and relatively simple method for the separation of apolipoproteins from the HDL fraction using IPG-IEF has been developed for this study. We found a four-component solvent system consisting of formic acid, acetonitrile, 2-propanol, and water (FAPH) to give recovery yields on the order of 25% for apo A-I, the largest and most complex protein in HDL. FAPH does not appear to alter the structure of apo A-I in the recovery based on MALDI data for the dominant isoform at pI 5.10 (Figure 4b). Although the study was directed toward solving a specific technical problem, these data already demonstrate the analytical power of linking the IPG pI profile with MALDI. The high resolution of the IPG pI profile is capable of resolving isoforms differing by 0.05 pI unit while the MALDI analysis for a particular pI isoform determines how much of a mass difference is associated with these isoforms. Improvements in the mass accuracy and resolution are goals for future investigation. These first results have already revealed new isoforms of apo A-I not previously detected. The sensitivity of this method is

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at the picomole level. The IPG-pI/MALDI protocol will have particular application to detecting mutations and posttranslational modifications in the key apolipoproteins that comprise lipoprotein particles. In the literature, there has been variation in the reported pI values for many of the apolipoproteins. We have concluded that the use of commercial strips with a fixed pI scale for set conditions on the IPGphor provides standardization for the determination of the pI values of these proteins. ACKNOWLEDGMENT This work was supported by the NIH, Heart & Lung Institute (HL 54566, HL68794) and the Welch Foundation (A-0258). We thank Dr. David H. Russell and Dr. Shane Tichy for the use of the MALDI-TOF MS instrument in The Laboratory for Biological Mass Spectrometry.

Received for review October 30, 2002. Accepted May 15, 2003. AC026273P