the Biology of Apolipoprotein M

1 INTRODUCTION Introduction to the Biology of Apolipoprotein M (ApoM) Cholesterol is an integral part of cell membranes and in the synthesis of steroids, bile, and vitamin D. Cholesterol found in the human body comes from two sources: biosynthesis and diet [1]. Cholesterol is a hydrophobic molecule and cannot travel through the bloodstream without assistance from lipoproteins. Lipoprotein particles consist of protein components called apolipoproteins that help solubilize the hydrophobic lipids [2]. Apolipoproteins transport cholesterol through the bloodstream to target organs and tissues. A schematic of cholesterol transport and cycling of lipoproteins is shown in Figure 1 [3]. Lipoprotein particles are generally classified as high, intermediate, low, and very low density lipoproteins (HDL, IDL, LDL, and VLDL, respectively), and chylomicrons [1]. Lipid-free apolipoprotein AI and lipid-poor preβ-HDL particles are precursors to the generation of mature, lipid-containing HDL particles. Apolipoprotein M is a component of preβ-HDL and is present in a sub-population of mature HDL particles. Preβ-HDL particles are thought to function primarily to induce cholesterol efflux from cells and act as the initial acceptors of cholesterol from peripheral tissues in reverse cholesterol transport (RCT) [2]. In RCT, HDL accepts cholesterol from peripheral cells, transports, and delivers it to the liver for degradation and excretion [2]. Preβ-HDL accepts unesterfied cholesterol and phospholipids, then the enzyme LCAT (lecithin cholesterol acyltransferase) esterfies the free cholesterol to modify the preβ- HDL into the spherical α-HDL (cholesterol ester core) [3]. The pre-beta form of HDL 2 exists in a lipid-poor state and may be indicative of the cholesterol-carrying capacity of the HDL in circulation. Human apoM was discovered by Xu, N. et al in 1999 [4] and is mainly associated with a small sub-population of HDL particles [5]. HDL particles lacking apoM showed a 50% decrease in cholesterol efflux from macrophages compared to apoM-containing HDL particles in vitro [2]. Human apoM is primarily associated with lipid-poor preβ- HDL, mature HDL, and to a lesser extent LDL and VLDL [4]. Wolfrum et al demonstrated that apoM is necessary for the formation of these preβ-HDL particles [2]. It has been shown that elevated levels of LDL, VLDL, and chylomicrons in circulation can lead to atherosclerotic lesion formation, whereas HDL has protective anti- atherogenic effects [6]. As a component of HDL, apoM is thought to have a significant influence on the development of coronary heart disease (CHD), caused by atherosclerosis [7]. Atherosclerotic lesions are a product of the accumulation of macrophage foam cells in blood vessels. Foam cells can be formed when excess LDL in circulation is oxidized and engulfed by macrophages [8]. Accumulation of chylomicron remnants in macrophages can also promote foam cell formation without oxidation [9]. The apoM present in LDL particles is thought to play a protective role against CHD by reducing oxidation of LDL and it has been shown that apoM-containing LDL particles are more resistant to oxidation [10]. LDL receptor knockout (ldlr-/-) mice over-expressing apoM had 70% less atherosclerotic lesion formation compared to ldlr-/- mice with endogenous normal apoM levels [2], supporting the anti-atherogenic benefits of apoM and making it a significant apolipoprotein in the study of CHD. 3 ApoM is also studied in association with other lipid metabolic diseases including diabetes, specifically mature-onset diabetes of the young (MODY3) [11, 12] and obesity [13]. Since apoM is a recently characterized protein [4], a quantitative assay for apoM has not yet been well established. Western blots have been used to demonstrate changes in apoM levels [2], but this method is not highly quantitative and suffers from low throughput. To date there is not a commercially-available quantitative assay for apoM in any species. The ability to quantitatively analyze apoM will enable a better understanding of its behavior in relationship with certain diseases. Even though development and use of an ELISA has been reported [14], the assay is not widely available. In-house efforts by a collaborator to replicate an ELISA measuring apoM in human serum has proven difficult and was ultimately unsuccessful (unpublished data). To address the need for a quantitative assay for apoM in serum, an antibody-free, high throughput, mass spectrometry-based assay was developed. Introduction to Mass Spectrometry The use of high performance liquid chromatography (HPLC) coupled to a mass spectrometer (MS) has been gaining popularity for development of quantitative assays over the past five years [15]. However, this approach often requires an antibody for selective enrichment of the target protein, especially when the protein of interest is in low abundance [16]. We developed a MS-based targeted assay to quantify apoM in human and rodent (mouse or rat) serum that does not require the use of an antibody. This label- free method to quantify apoM in serum provides a powerful tool to further understand the biology of apoM with many research applications. 4 In the initial stages of the development of a quantitative assay for a specific protein or proteins using a targeted LC-MS method, an unbiased global MS approach can be used to identify the target protein in serum. Global profiling studies are typically done to identify the entire protein content of a sample. Global profiling of a biological sample (i.e. serum) begins with enzymatic digestion of the proteins, typically with trypsin to create tryptic peptides. These peptides can be separated based on hydrophobicity using a simple, two and a half hour HPLC gradient prior to MS analysis. The peptides in the effluent of the HPLC column are ionized and sprayed into an on-line ion trap MS, such as an LTQ (Thermo). Proteins are identified from this analysis based on the identification of unique tryptic peptides from a specific protein. The ionized tryptic peptides are measured by MS as a mass-to-charge ratio (m/z) using the molecular mass and charge status of each peptide. In a global profiling study, a triple-play method can be used to collect ion spectra using three MS scans per peptide: centroid peptide full MS scan, profile zoom scan, and centroid fragment MS/ MS scan. The full MS scan captures the mass-to-charge (m/z) ratios of all ionized peptides eluted at a specific time point. The most abundant peptide in the scan is selected as a precursor ion and a zoom scan of this peptide is used to estimate the charge status and monoisotopic and average masses of the peptide. The zoom scan also evaluates the quality of the selected peptide to avoid false positive protein identifications and eliminates low quality data from further analysis. The selected peptide is then fragmented, and the spectra of these product ions are collected by a MS/ MS scan [17]. The product ions from fragmentation of the precursor ion at the amide bond of the peptide backbone are classified as b-ions and y-ions. A b-ion is observed when the proton is retained by the N- 5 terminal fragment of the peptide, and a y-ion is observed with the retention of the proton by the C-terminal fragment of the peptide [18]. The fragmentation spectra is searched against species-specific computerized protein databases and used for peptide sequencing and protein identification. The data output from the database search yields protein identifications based on the detection of a tryptic peptide that is unique to the protein from which it was derived. A targeted MS method can be created for a specific protein or proteins of interest using the results from these global profiling studies. Identification of the protein of interest and the MS/ MS spectra generated from the fragmentation of the precursor ion are collected by the global studies and are used to set up a targeted Multiple Reaction Monitoring (MRM) assay using the same instrument [19]. The m/z values of the precursor ion and respective fragmentation y- and b-ions obtained from the global profiling studies are used to set up a targeted assay to measure only specific m/z values. Only these m/z values will be collected by the MS while the m/z values of precursor ions that do not fall within the set m/z window are filtered out. The MS peptide signal from the precursor ion can be integrated to obtain the area-under-curve value (AUC) which can be used to quantify the target protein. These MS methods were used to develop a targeted assay for the quantification of apoM in human and rodent serum. This assay spans multiple species to streamline the use of this assay between pre-clinical and clinical measurements. The resulting assay is versatile and quantitative for apoM and will provide a powerful tool to expand research in CHD and other diseases and provide a deeper understanding of the biology of apoM in many different applications. 6 Figure 1 Figure 1: Lipoprotein involvement in lipid transport The transport of lipid through the bloodstream to target cells is achieved by lipoproteins. This schematic of lipid transport was from Brewer, HB Jr., N Engl J Med 2004; 350:1491-1494, Apr 8, 2004. 7 EXPERIMENTAL SECTION Reagents Human serum was purchased from Biowhittaker (cat #14-491E and 14-402E) and Bioreclamation (cat #HMSRM). Rabbit serum was purchased from Biomeda (cat #MS008). Horse serum was from GIBCO (cat #16050-122). Rat serum was from Pel- freeze (cat #36125-3), Harlan (cat #4511) and Biomeda (cat #MS009) and mouse serum was from Lampire (cat #SI-1409). Dulbecco’s Phosphate Buffered Saline (PBS) was from Invitrogen (cat #10010-023). PHM-Liposorb was from CalBiochem (cat #524371). Ammonium bicarbonate (cat #A6141), DL-Dithiothreitol (cat #D9779), iodoacetamide (cat #A3221) and urea (cat #208884) were from Sigma. Modified trypsin was purchased from Promega (cat #V5280) and NP40 detergent was from Pierce (cat #28324). HPLC- grade 0.1% formic acid in water (cat #HB523-4) was from Fisher Scientific. HPLC- grade water (cat #365-4) and acetonitrile (cat #015-4) were from Burdick & Jackson. Formic acid was from J.T. Baker (cat #0129-01). Synthetic peptides were from Midwest Biotech (Fishers, IN) and 15 N-labeled human apolipoprotein A-IV was prepared in-house by purification of the recombinant protein from E.coli grown in a medium containing 15 N-urea. Mouse anti-apoM primary antibody was from BD Transduction Labs (cat #612333) and an ECC anti-mouse IgG-Horseradish peroxidase secondary antibody was from Amersham (cat #NA931V). ECL kit was from Amersham (cat #RPN303D). Human recombinant apoM from E.coli was grown in-house and given by Dr. Thomas Lee (Eli Lilly and Co.). 8 Sample Preparation for Global LC-MS/ MS Analysis of Human and Rat Serum Serum was enriched with lipoprotein-binding beads to selectively purify apolipoproteins from serum prior to digestion with trypsin. The digested proteins were separated by reverse-phase HPLC and analyzed by MS using a global profiling method to identify the entire protein content of each sample. Aliquots of human and rat serum were prepared with PHM-Liposorb in PBS to selectively remove lipoproteins from serum. A method of lipoprotein removal using Liposorb has been described previously [20] and was adapted for selective removal of apolipoproteins from serum prior to MS analysis by Dr. Bomie Han (Eli Lilly and Co.). Liposorb powder (one gram) was suspended in 50 mL PBS and filtered through a 250 µm filter. The flow-through was used as the Liposorb working stock solution (1 g/ 50 mL). Ten microliters (10 µL) of serum was diluted in 90 µL of PBS and incubated with 100 µL of Liposorb stock for 10 minutes at room temperature with shaking to keep Liposorb suspended. The samples were spun down to pellet the Liposorb and supernatant was removed. The pellet was washed three times with 500 µL of 100 mM ammonium bicarbonate (ABC) and digested overnight at 37°C with 1 µg of modified trypsin prior to MS analysis. Samples were filtered and 50 µL of 1 mL final volume (0.5 µL of serum) was analyzed by triple-play LC-MS/ MS (LTQ from Thermo) in global profiling studies. 9 Global LC-MS/ MS Analysis of Human and Rat Serum or Plasma Global profiling studies were used to evaluate the entire protein content of each sample using a triple-play method to collect spectral data from the fragmentation of unique peptides to identify each respective protein. The HPLC gradient was controlled using a Surveyor MS pump (Thermo) equipped with a sample loop and a Zorbax SB300- C18 (3.5 µm particle size) 1 mm x 50 mm reverse phase column (Agilent Technologies, cat #865630-902). The column was kept at 27°C and sample tray was 4°C during analysis. 50 µL of 1 mL total sample volume (0.5 µL of human or rat serum) was injected into a 100 µL sample loop using the partial loop method. The HPLC gradient ran for 142 minutes per injection and utilized a three buffer system [0.1% formic acid in H 2 O (Buffer A), 0.1% formic acid in 50% acetonitrile (Buffer B), and 0.1% formic acid in 80% acetonitrile (Buffer C)]. The gradient ran at a 50 µL per minute flow rate of: 90% A, 10% B from 0.0-5.0 min, then ran a sloped gradient from 90% A, 10% B to 5% A, 95% B from 5.0 to 125.0 min, then 100% C from 125.1-130.0 min, and 90% A, 10% B from 130.1 min-142.0 min. This gradient separated the peptides based on hydrophobicity. The peptides were eluted, ionized, and sprayed directly into an online MS for mass and charge state determination. Mass spectrometry data were collected in triple-play mode with three scans per peptide: centroid peptide full MS scan, profile zoom scan, and centroid fragment MS/ MS scan. Protein identification was performed using a Sequest and X! Tandem algorithm that combined the protein identifications output from each search [17] and these results were searched against a reverse database to confirm protein identifications. The p-value 10 of each tryptic peptide was used to determine the quality of the peptide identification to help avoid using false-positive identifications for method development. Sample Preparation for Targeted MS Analysis of ApoM A heavy isotope-labeled apolipoprotein standard was spiked into each sample at the beginning of sample preparation to normalize variations in protein recovery from serum that may occur during sample preparation or instrument analysis. Serum or plasma was enriched with lipoprotein-binding beads and the bound proteins were denatured with urea prior to digestion with trypsin in the presence of detergent. 15 N-labeled human apolipoprotein A-IV ( 15 N-Apo A-IV) was used as an internal standard (iSTD). The labeled protein was diluted into PBS and 300 µL (250ng) was spiked into each experimental and external calibration sample. Rabbit or horse serum was mixed with PBS (1:6) and used as a dilution matrix for human or mouse experimental samples, respectively. 10 µL of human or mouse serum was mixed with 140 µL of the dilution matrix to dilute the experimental sample in the background serum at a 1:2 ratio (3x dilution of the experimental sample). The total serum-to-PBS ratio was 1:4 to mimic the ratio of total serum-to-PBS in the calibration samples. 50 µL of the diluted sample was mixed into the internal standard solution. 200 µL of Liposorb was used per 10 µL of serum. Samples were incubated in Liposorb at 4°C for 20 minutes with shaking to keep Liposorb suspended. The Liposorb was spun down and supernatant aspirated. The Liposorb pellet was washed one time with 100 mM ABC. The washing step can also be done in a filter plate for higher throughput and will be described later. The Liposorb pellet was resuspended [...]... (fragmentation site, ion charge) AFLLTPR was from m/ z of 409.51 to m/ z of 599.39 (y5, M+ H+ ion), m/ z of 486.30 (y4, M+ H+ ion), and m/ z of 373.22 (y3, M+ H+ ion) FLLYNR was from m/ z of 413.50 to m/ z of 565.31 (y4, M+ H+ ion), m/ z of 452.23 (y3, M+ H+ ion), and m/ z of 678.39 (y5, M+ H+ ion) AFLVTPR was from m/ z 402.49 to m/ z of 585.37 (y5, M+ H+ ion), m/ z of 472.29 14 (y4, M+ H+ ion), and m/ z of 373.22 (y3, M+ H+... and Western Blot of Human ApoM Human serum and human recombinant apoM were prepared for SDS-PAGE and Western blot to evaluate the use of human recombinant apoM as a standard to quantify apoM in human serum Two sets of dilutions of the recombinant protein were made at 100 µg/mL, 20 µg/mL, and 4 µg/mL, one using PBS as the dilution matrix and the other using 12.5 mg/mL bovine serum albumin, BSA (Pierce,... LC-MS/ MS performs three scans per peptide: full MS scan, zoom scan, and MS/ MS scan The most abundant peptide present in the full MS scan was selected for zoom scan and then fragmented prior to full MS/ MS scan The zoom scan estimated the charge state of the peptide and the full MS/ MS scan collected spectra from the product ions generated in the fragmentation of the precursor ion (tryptic peptide) The. .. Post-digestion samples were filtered in the high-throughput format described above 50 µL of 500 µL final volume (1 µL of serum) was injected to the LTQ for measurement of apoM using the MRM assay 20 Human Clinical Study Samples for Human ApoM Measurement The conditions of the human serum samples used in this experiment have been described earlier [23] Briefly, serum samples were collected from participants... concentration measurements from a more conventional proteomic method, Western blot, in an orthogonal validation of the MS assay Aliquots of the same human serum samples containing different concentrations of apoM were analyzed using both methods The MS 23 measurement was performed using the urea-containing method as described above Serum samples were also prepared for SDS-PAGE Five microliters (5 µL) of human... from the MS source to the waste This helped maintain a cleaner instrument and did not interfere with the measurement of the target tryptic peptides in this method ApoM was not included in these experiments because they were performed earlier in method development of other apolipoproteins and prior to the development of this assay, but the affect of detergent on apoM recovery was evaluated during the. .. PBS as 11 the rest of the calibration samples The total volume of human or mouse serum was larger than the total volume of serum in the rest of the calibration samples Every other calibration sample (12 total) in the dilution series was used to make up one calibration set, called mouse calibration A (Cal-A) and human Cal-G, and the remaining 12 samples created another calibration set, called mouse validation... Each calibration sample was then mixed 1:4 with PBS to maintain the same fixed dilution in PBS as the experimental samples In the human and mouse calibration sets, two samples were prepared using a higher volume of total serum in PBS (without matrix) than the 100% serum-equivalent samples, to create 125% and 156% of human and mouse serumequivalents These two samples did not have the same fixed dilution... 300 µL (250 ng) of iSTD to each well Rabbit serum pre-mixed with PBS (1:6) was used as a background matrix for the serum samples and calibration standards The robot transferred 10 µL of serum samples into 140 µL of dilution matrix to dilute the experimental serum samples 1:2 (3x dilution) with the serum 17 matrix Samples were diluted to bring the apoM concentration within the range of the standard curve... human Val-H The rat serum calibration samples were prepared in the same manner, but without preparation of samples that contained greater-than-100% rat serum The rat serum calibration sets were rat Cal-A and Val-B Serial dilutions of synthetic apoM tryptic peptides were used as a calibration standard to measure the molar concentration of apoM in these human, mouse, and rat serum calibration samples The . integral part of cell membranes and in the synthesis of steroids, bile, and vitamin D. Cholesterol found in the human body comes from two sources: biosynthesis and diet [1]. Cholesterol is a hydrophobic