Measuring H218O Tracer Incorporation on a QQQ-MS Platform

Article pubs.acs.org/jpr

Measuring H218O Tracer Incorporation on a QQQ-MS Platform Provides a Rapid, Transferable Screening Tool for Relative Protein Synthesis James P. Conway, Douglas G. Johns, Sheng-Ping Wang, Nykia D. Walker, Thomas A. McAvoy, Haihong Zhou, Xuemei Zhao, Stephen F. Previs, Thomas P. Roddy, Brian K. Hubbard, Nathan A. Yates, and Ronald C. Hendrickson*,† Merck Research Laboratories, 126 East Lincoln Avenue, Rahway, New Jersey 07065, United States S Supporting Information *

ABSTRACT: Intracellular proteins are in a state of flux, continually being degraded into amino acids and resynthesized into new proteins. The rate of this biochemical recycling process varies across proteins and is emerging as an important consideration in drug discovery and development. Here, we developed a triple-stage quadrupole mass spectrometry assay based on product ion measurements at unit resolution and H218O stable tracer incorporation to measure relative protein synthesis rates. As proof of concept, we selected to measure the relative in vivo synthesis rate of ApoB100, an apolipoprotein where elevated levels are associated with an increased risk of coronary heart disease, in plasma-isolated very low density lipoprotein (VLDL) and low density lipoprotein (LDL) in a mouse in vivo model. In addition, serial time points were acquired to measure the relative in vivo synthesis rate of mouse LDL ApoB100 in response to vehicle, microsomal triacylglycerol transfer protein (MTP) inhibitor, and site-1 protease inhibitor, two potential therapeutic targets to reduce plasma ApoB100 levels at 2 and 6 h post-tracer-injection. The combination of H218O tracer with the triple quadrupole mass spectrometry platform creates an assay that is relatively quick and inexpensive to transfer across different biological model systems, serving as an ideal rapid screening tool for relative protein synthesis in response to treatment. KEYWORDS: labeled water, protein synthesis, triple quadrupole mass spectrometry, ApoB100, SRM

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liquid-scintillation counting.7 Short radio-isotope half-lives and safety concerns led to the development of stable isotopes in the protein turnover assays.8 However, these early protocols often measured stable isotope derivatives of metabolites as a tracer for enzyme turnover. Developed on a GC−MS platform, these assays required significant sample preparation. Turnover assays that measure stable isotope incorporation by LC−MS offer a sensitive and inexpensive option with relatively short development and analysis time. In addition, LC−MS assays based on the incorporation of a labeled tracer into unique amino sequences provides the ability to measure synthesis rate or turnover of individual peptides and proteins. Stable isotopes can be administered in in vivo protein measurement assays through direct infusion, diet, or drinking water. In all cases, the label needs to equilibrate quickly, and the plasma levels reflect that of the intracellular pool. Direct infusion of a labeled amino acid, such as 13C6-leucine, produces a well-defined mass shift between unlabeled “light” and labeled “heavy” peptides. However, the intravenous administration of the isotope is not as convenient as oral ingestion in the clinical

INTRODUCTION The classical view that structural proteins are static and that dietary proteins are used only as fuel has been replaced with an appreciation that intracellular proteins are in a steady state of flux, continually degraded into amino acids and resynthesized into newly made proteins. This recycling process is called protein turnover or accretion and is a balance between protein synthesis and degradation.1 Intracellular proteins are turning over extensively, whereby individual proteins degrade at vastly different rates, with half-lives ranging from a few minutes to many days to the most stable intracellular protein, hemoglobin, which has a normal lifetime of approximately 120 days.2 Proteolysis of proteins is a highly complex, tightly regulated process that is individually controlled for many proteins.3 For example, in the ubiquitin-proteosome system, a protein is tagged with ubiquitin or polyubiquitin, which leads to energydependent targeted degradation in the 26S proteosome complex.4−6 Understanding protein turnover is emerging as an important consideration in drug development. Protein turnover measurements have advanced with the development of LC−MS assays of stable isotope incorporation. Initial assays employed radioactive isotopes that could be incorporated during new protein synthesis and assayed by © 2012 American Chemical Society

Received: August 8, 2011 Published: January 30, 2012 1591

dx.doi.org/10.1021/pr2007494 | J. Proteome Res. 2012, 11, 1591−1597

Journal of Proteome Research

Article

setting. Isotope-labeled drinking water, deuterated (2H2O) or heavy-oxygen (H218O) labeled water, provides an ease of use advantage over intravenous administration, provided that rapid equilibration occurs between the plasma pool and the tissue of interest and suitable levels of stable isotope can be incorporated. 9−11 The m/z spectra of a peptide that incorporates 2H or 18O will show an increased signal intensity in the second (M1) or third (M2) isotope peak, respectively, over the naturally occurring isotopic distribution. The labeled (M1 or M2) to monoisotopic (M0) signal intensity ratio, M1/ M0 or M2/M0, can be measured over time on a LC−MS platform to quantify the rate of tracer incorporation that occurs with new protein synthesis. The relative ease of administration, sample preparation, and analysis have made “heavy water” LC− MS-based assays a promising method for protein synthesis rate measurements or for protein turnover measurements.11 In this study, using specific selected reaction monitoring (SRM) transitions, we show that 18O-label incorporation is readily measurable on a triple quadrupole mass spectrometer operating at unit resolution. The selectivity and sensitivity of the method is demonstrated for a mouse ApoB100-specific peptide present in lipoprotein particles isolated from plasma. Different strategies were used to test whether the method could readily differentiate relative syntesis rates of ApoB100 in plasma. For example, we contrasted the synthesis of isolated VLDL and LDL, lipoprotein particles that have known and distinct ApoB100 protein production rates, and studied mice under different treatments that were expected to modulate ApoB100 (e.g., control, siRNA, MTP, and S1P). The application of this assay as a rapid screening tool for relative protein synthesis rate is demonstrated.

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from canine plasma using the same size exclusion and trypsin digestion procedure, which served as a background matrix during LC−MS method development and quality control sample generation. 18

O-Labeling of Plasma Water

The 18O-labeling of plasma water was determined using GC− MS as described by Brunengraber et al.12 Briefly, 5 μL of plasma is reacted with PCl5 to generate phosphoric acid, 150 μL of TMS−diazomethane (Sigma) is then added to generate the trimethylphosphate. The solution is evaporated under stream of nitrogen and then dissolved in 150 μL of chloroform. Samples were analyzed using an Agilent 5973 MS coupled to a 6890 GC oven fitted with an Agilent DB5-MS column, 30 m × 250 μm × 0.15 μm, the oven was initially set at 100 °C and then programmed to increase at 35 °C per min to 250 °C, helium carrier flow was set at 1.0 mL × min−1 (2 μL of sample was injected using a 40:1 split), trimethylphosphate eluted at ∼1.9 min, and the mass spectrometer was set to perform selected ion monitoring of m/z 140 and 142 (10 ms dwell time per ion) in the electron impact ionization mode. Mass Spectrometry Method Development

A selective reaction monitoring (SRM) method was developed to measure changes in isotopic ratio. ApoB100 tryptic peptides were screened to determine the best candidate for this assay on the basis of sequence (i.e., no amino acid identity to other murine tryptic peptides, including ApoB48), elution time, precursor and product ion intensity. A mouse ApoB100-specific peptide (INWEEEAASR) was selected because of the presence of a high intensity y8+ ion in the product ion spectrum, which maximized the potential sites for 18O incorporation. This peptide was monitored by the following SRM transitions: 602.8 → 977.4 (M0, monoisotopic peak), 603.8 → 979.4 (M2, 18Olabeled peak). A peptide from the N-terminal portion of ApoB that is common to both ApoB48 and ApoB100 (LSLEDTPK) is described in the Supporting Information, Table S1. Instrument method parameters were optimized through direct infusion of synthetic INWEEEAASR peptide. Specifically, collision energy was set to 20 V, and tube lens was set to 128 V to maximize signal intensity. Q1 and Q3 were each set to 0.7 m/z. Scan width was set to 0.01 m/z, and scan time was set to 0.1 s. Synthetic INWEEEAASR peptide was spiked into canine plasma LDL prepared as described above (0.01, 0.1, 5, 10, 50, 100, 500, and 1000 nM) to determine the accuracy and precision of the isotopic ratio measurement in relation to monoisotopic peak intensity. Monoisotopic peak intensities greater than 105 resulted in M2/M0 measurements with a precision of ≤5% (n = 5 technical replicates). As a requirement, M2/M0 values were calculated exclusively for time courses that were entirely greater than 105 monoisotopic peak intensity.

MATERIALS AND METHODS

Animal Model and Sample Preparation

Human CETP hemizygous Tg/LDLr ± male mice were exposed to various treatment conditions (n = 6 mice per treatment group). For siRNA treatments, mice were treated with SSB (single strand binding protein) siRNA (3 mg/kg) or PBS control (dosed at same volume as siRNAs) via tail vein injection. For small molecule treatments, mice were treated with either an MTP inhibitor (50 mg/kg, p.o. administration, T-1 h before tracer) or a S1P inhibitor (30 mg/kg, i.p. injection, T-1 h before tracer). One hour following the final inhibitor treatment, all treatment groups were intraperitoneally injected with 20 mL/kg of 18O-labeled water. Blood (20 μL) was collected via tail nick at 0 h, and at 0.5, 1, 2, 4, and 6 h following tracer injection. EDTA plasma was pooled to create 80 μL samples for each time point and treatment group. VLDL and LDL lipoprotein particles were isolated by size exclusion chromatography using a single Superose 6 column (GE Healthcare; Piscataway, NJ) and separated in PBS + 1 mM EDTA at 0.2 mL/min, and 1 mL fractions were collected. VLDL- and LDL-containing fractions were identified using a colorometric assay for total cholesterol (Wako). Pooled lipoprotein fractions were concentrated using Agilent Spin Concentrators (5 kDa MWCO; Agilent; Santa Clara, CA), reduced (10 mM DTT; 45 min at 60 °C), and alkylated (15 mM iodoacetamide; 30 min at room temperature). Samples were trypsin-digested at 37 °C for 18 h (1:50 trypsin/protein ratio) using sequencing-grade trypsin (Promega; Madison, WI), lyophilized to dryness, and resuspended in 0.1% formic acid to a concentration of 1 μg/μL. LDL was biochemically isolated

LC−MS/MS Study Sample Analysis

LC−MS/MS was performed on a Thermo Quantum triplestage quadrupole (QQQ) mass spectrometer equipped with an Ion Max API source (Thermo Fisher Scientific, Waltham, MA). The LC system consisted of an Agilent 1100 capillary pump and an Agilent 1200 microplate autosampler (Agilent; Santa Clara, CA). Samples were loaded for 3 min at 8 μL/min onto a 15 cm reverse phase capillary column with a 320 μm inner diameter (Part #MC-15-C18WSS-320EU; CVC Technologies, Fontana, CA). The capillary column particle width was 3 μm, and the pore size was 300 Å. Microcapillary chromatography was achieved at 8 μL/min by applying a binary linear gradient 1592



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Figure 1. M2/M0 ratio measurement from triple quadrupole MS and MS/MS data. (A) Simulated mass spectra (MS) of the naturally occurring M2/M0 ratio for the peptide INWEEEAASR [(M + 2H)2+ ions at m/z = 602.8]. Isotopic peaks are not resolved, thus preventing the measurement of M0 and M2. (B) Simulated tandem mass spectra (MS/MS) of the same peptide but measuring the singly charged (M + H)1+ y8 product ions at m/z = 977.4 (Q3 resolution is set to 0.7 amu fwhm), demonstrating partially resolved M0 and M2. (C) Predicted M2/M0 ratio as a function of increasing 18O label incorporation.

dependent on 18O concentration that remains constant following an initial introduction.

of 0−50% solvent B for 12 min (solvent A, 0.1% formic acid (HCOOH); solvent B, acetonitrile (MeCN)). A QC sample was designed by spiking 10 nM synthetic peptide INWEEEAASR into the biochemically isolated canine plasma LDL. This selected mouse peptide is not present in the canine proteome. For LC−MS/MS acquisition, samples were ordered into treatment group brackets, and the QC sample was run after each treatment group bracket. Each sample was injected at a volume of 5 μL, representing approximately 16 μL of initial mouse plasma.

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RESULTS AND DISCUSSION Studies of protein synthesis require confidence in the precursor labeling and reliable measurements of the product labeling. We consider each of these points. First, experiments that rely on the administration of labeled water assume that a subject will rapidly generate labeled amino acids; this requires that the rate of entry of water into a cell and equilibration with free amino acids is faster than the rate at which amino acids are incorporated into a newly made protein. Earlier studies by Dietschy and colleagues clearly demonstrated that labeled water [(3H)water] is equilibrated in total body water within ∼1 h following a bolus injection and persists for hours. 13 Importantly, Dietschy et al. showed that the specific activity for tissue water in multiple tissues such as liver, intestine, kidney, and spleen was equal to the specific acivity of plasma water at all time points measured, including the initial 5 min time point. The authors conclude that the specific activity of intracellular[3H] labeled water equals the specific activity of plasma water at all time points, including the earliest time point that the tissues were sampled. We assume the enrichment of tissue water is equivalent to plasma water on the basis of the published findings of Dietschy et al. Second, the use of heavy water as a tracer for protein labeling is anticipated to result in small changes in peptide isotopic ratios that could be detected by mass spectrometry (MS).

Isotope Calculations and Data Analysis

Experimental peak areas were calculated uniformly in Thermo Xcalibur by applying a processing method to the entire data set. Integration was performed on the most intense chromatographic peak within a 30 s window centered on the expected retention time of 14.3 min. Experimental M2/M0 was calculated from product ion chromatographic peaks by dividing the third isotopic peak (M2) area (18O peak) by the monoisotopic peak area (M0). Theoretical isotope spectra were plotted in the QualBrowser application of Thermo Xcalibur v2.0.7 (Thermo Scientific) to determine theoretical peak area ratios at natural isotopic abundance and with added 18 O. Prism v. 5.03 (GraphPad Software; La Jolla, CA) was used to apply a pseudo-first-order nonlinear curve fit to the time course data. The applied algorithm assumes a plateau followed by one phase association kinetics, since incorporation is 1593



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Stable isotope tracer incorporation results in a shift in the isotope distribution of peptides that can be measured by MS analysis of precursor and/or product ions.7,14,15 We chose to develop an SRM method to measure these shifts in product ions because we anticipated that small changes in isotope ratios would need to be measured with high selectivity. To investigate the measurement of 18O-label incorporation on a triple-stage quadrupole mass spectrometer (QQQ-MS) operating at unit resolution, theoretical mass spectra of the ApoB100 peptide INWEEAASR were plotted as a proof-of-concept. We selected ApoB100 because it is an important protein in pharmaceutical development. Elevated levels of ApoB are associated with an increased risk of coronary heart disease. Therapeutic drugs designed to modify ApoB production and/or adjust clearance may have potential health benefits. As the tryptic peptide is observed predominately in the +2 charge state, shown in Figure 1A is the calculated isotopic distribution of the (M + 2H)2+ precursor mass measurements. The monoisotopic (M0) and 18 O-isotope peak (M2) are not resolved; thus, the discrete measurement of M2/M0 can not be made. The major strength of measuring product ions generated by the tandem mass spectrometry method is that it provides selectivity based on the primary amino acid sequence of the peptide. On a triple-stage quadrupole mass analyzer, tandem MS analysis is performed by isolating a precursor mass in Q1. Precursor ions are transferred to Q2 and induced to fragment by collision-activated dissociation predominately at the various amide linkages in the peptide molecule. Product ions are transferred to Q3 and separated by mass-to-charge ratio (m/z). A specific combination of precursor ion mass and product ion mass is called a SRM transition. Figure 1B shows a theoretical calculation of the y8+ product ions. Note that the M0 and M2 peaks are readily resolved. Figure 1C shows the expected M2/ M0 ratio with increasing concentration of 18O-label, assuming all amino acids have an equal probability of incorporating a single 18 O. Given a fixed precision of the QQQ-MS measurement, a minimum detectable change in M2/M0 can be determined. For the purpose of this study, we conservatively set the minimum detectable change in M2/M0 equal to five times the standard deviation of the experimentally determined M2/M0 measurement. The data in Figure 1C relates a change in M2/M0 with a required %-label incorporation of 18O. For example, an increase in 18O-label incorporation from 0 to 5% will increase the M2/M0 measurement from 0.15 to 0.20 (ΔM2/M0 = 0.05). To measure a change in M2/M0 at 5% 18 O, the standard deviation of M2/M0 can be no more than 0.01. To establish that the approach can measure a small % change in isotope ratio with high selectivity, a synthetic peptide was used to determine the accuracy and precision of the M2/M0 measurement on the QQQ-MS platform. The murine ApoB100 synthetic peptide (INWEEEAASR) was added at concentrations of 10 pM to 1 μM to a background matrix of canine plasma−isolated LDL. The selected murine peptide sequence in ApoB100 is not present in the canine genome; thus, there is no interfering endogenous peptide in the plasma background. Monoisotopic signal intensity and the M2/M0 ratio for the y8+ product ions are listed in Table 1 for each peptide concentration. As monoisotopic signal intensity increases, the accuracy of the ratio measurement improves by trending toward the theoretical value of M2/M0. The precision of the ratio measurement also improves with increasing monoisotopic signal intensity, as indicated by the decrease in measurement

Table 1. Accuracy and Precision of the M2/M0 Ratio Measurements as a Function of Monoisotopic Signal Intensitya peptide concentration (nM) 0.01 0.1 5 10 50 100 500 1000

mean M0 signal intensity

mean M2 signal intensity

mean M2/M0 accuracy (% deviation from theoretical)

× × × × × × × ×

1.4 × 103 1.3 × 103 3.5 × 104 4.7 × 104 2.9 × 105 3.8 × 105 1.4 × 106 3.3 × 106

− − 12 5.6 2.3 2.3 0.8 0.8

2.1 2.9 1.8 3.4 1.9 2.2 8.5 1.9

103 103 105 105 106 106 106 107

mean M2/M0 ± SD − − 0.18 0.15 0.16 0.16 0.16 0.16

± ± ± ± ± ±

0.007 0.007 0.007 0.003 0.001 0.001

a

Synthetic peptide INWEEEAASR, unique to mouse ApoB100, was spiked into a non-mouse plasma LDL background at concentrations of 10 pM to 1 μM. Samples were analyzed by LC−MS on a triple quadrupole mass spectrometer in SRM mode as described in the Materials and Methods to determine the accuracy and precision of the M2/M0 ratio measurement (n = 5 injections per sample). M0 and M2 values represent mean signal intensities at each peptide concentration. M2/M0 represents the mean ratio of peak areas from the extracted ion chromatograms. Values represented by (−) correspond to M2 signal intensities that could not be distinguished from background noise.

error. At monoisotopic signal intensities greater than 105, the minimum detectable change in M2/M0 is greater than or equal to 0.035. The results of this validation experiment provided minimum signal intensity thresholds and minimum detectable change in M2/M0 that were applied to the following in vivo mouse experiments. To further explore the method to measure relative protein synthesis rates, we decided to measure ApoB100 isolated from the lipoprotein particles, VLDL and LDL. Over 98% of VLDL apoB100 is synthesized in the liver.16 In addition, the in vivo protein production rates for ApoB100 are well studied. In humans, for example, the VLDL and LDL ApoB100 fractional production rate has been measured using a primed-constant infusion of isotopically labeled essential amino acids [5,5,5,-2H3]-leucine, [4,4,4,-2H3]-valine, and [6,6-2H2,1,2-13C2]-lysine. The VLDL ApoB-100 fractional production rate is 3.1 pools per day (or 0.54% enrichment/ hour) and LDL ApoB-100 fractional production rate is 0.03 pools/day (0.05% enrichment/hour).17 First, we performed a pilot experiment to measure the 18O-labeling of plasma water to confirm rapid distribution and maintenance of steady state. Consistent with the literature, we observed a rapid distribution of H218O following a bolus injection in mice (Figure 2a) and that the 18O-labeling of water was maintained at a steady-state for ∼8 h. Next, incorporation rates of H218O into ApoB100 isolated from plasma derived VLDL and LDL were compared. Figure 2b shows the isotopic ratio (M2/M0) for the y8+ product ions plotted as a function of time in VLDL and LDL isolated from PBS-treated mice. These results show that ApoB100 in VLDL incorporates 18O-tracer at a faster rate in comparison to ApoB100 in LDL, consistent with published studies that show that plasma VLDL is produced at a faster rate than plasma LDL.17−19 An increase in lipoprotein particle ApoB100 18O-label incorporation should be directly related to the presence of newly synthesized ApoB100 in the lipoprotein particle, and by extension, the production of new VLDL or LDL. 1594



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distribution expected to occur naturally, represented by a M2/M0 ratio of 0.16. As new protein is synthesized, 18O tracer is incorporated, and the M2/M0 ratio increases over this initial value. A significant change in M2/M0 is defined by the technical variability of the assay, which was characterized in the validation experiments and used to establish the minimum detectable change in M2/M0 as 0.035 in these experiments, as described above. Increases in 18O-label incorporation were observed in the PBS and SSB siRNA treated mice at 2 and 6 h postinjection. S1P inhibitor treated samples showed no 18Oincorporation at 2 h postinjection but showed an initial increase in 18O-incorporation at 8 h post-tracer-injection. Following MTP inhibitor treatment, the M2/M0 ratio does not change from the theoretical initial value, indicating no measurable 18Olabel incorporation through 8 h. Peak areas and M2/M0 for all time points and treatment groups used to measure INWEEEAASR are available in the Supporting Information, Table S2. The synthesis and assembly of VLDL in the liver involves several enzymes or lipid transfer proteins (including MTP), which regulate the synthesis of triacylglycerol and lipidation of apolipoprotein B. In this study, two inhibitors were used to reduce ApoB production and secretion into the plasma by suppressing its maturation and lipidation via pharmacological inhibition of MTP or S1P. MTP is predominantly expressed in the hepatocytes and enterocytes, and it transfers neutral lipids (triglycerides, phospholipids, cholesterol esters) to nascent ApoB as a rate-limiting step in the assembly of ApoB containing lipoproteins such as VLDL in the liver and chylomicrometers in the intestine. Mutations leading to loss of MTP activity are linked to familial abetalipoproteinemia, in which the affected individuals have undetectable levels of ApoB in the plasma. Therefore, the observation of no 18O label incorporation is consistent with an effective “shutting down” of ApoB secretion via inhibition of MTP. Site 1 protease belongs to the proprotein convertase family and represents the first step in regulation of lipid metabolism and cholesterol homeostasis via cleavage of the sterol regulatory element-binding proteins (SREBP-1 and SREBP-2). SREBP-1 and -2 activate expression of enzymes involved in the synthesis and catabolism of sterols, lipids, and the LDL receptor. The site 1 protease inhibitor used in the current study was previously shown to inhibit synthesis of both cholesterol and fatty acids in cultured hepatocytes and in mice.20,21 In this study, 18O-label incorporation into apoB100 was inhibited, albeit transiently, which supports the notion that suppression of lipid and cholesterol synthetic pathways results in reduction of apoB100 secretion. The transient nature of this response is consistent with reports of rapid clearance of this compound in rodents.20 Different mass spectrometers including quadrupole-time-offlight (Q-TOF), ion trap, and Fourier transform instruments can be used to measure stable isotope label incorporation into peptides and proteins.15,22,23 In this study, however, a triplestage quadrupole mass spectrometer was used to provide a selective and sensitive measurement. Selectivity based on the primary amino acid sequence of a peptide can be obtained by measuring specific product ions that are generated by collisionactivated dissociation of selected precursor m/z. A consideration associated with selected reaction monitoring, however, given that these experiments involve tryptic peptides, is the preference for high mass y-ion series product ions that span the majority of the precursor peptide. Sometimes a trade-off between choosing maximum signal intensity of the specific

Figure 2. 18O-labeling measurements of plasma water at multiple time points and incorporation rate of 18O into ApoB100 differs in LDL and VLDL. (a) Mice were given an IP bolus at t = 0, and plasma was drawn various time points and measured for 18O incorporation of plasma water as described in Materials and Methods. (b) A single peptide specific to mouse ApoB-100 (INWEEEAASR) was analyzed in LDL (○) and VLDL (□) at 0, 0.5, 1, 2, 4, and 6 h post-bolus injection of 20 mL/kg of 18O-labeled water. Incorporation rate was fit to a pseudofirst-order nonlinear equation (LDL, k = 0.27, R2 =0.981; VLDL, k = 0.66, R2 = 0.998).

To demonstrate the practical application of this assay as a rapid screening tool for relative protein synthesis, an in vivo experiment was designed to measure 18O-label incorporation in response to different treatment conditions. Figure 3 evaluates

Figure 3. Measurement of relative ApoB100 synthesis can be applied as a screening tool for compound treatment. LDL ApoB100 incorporation of 18O was compared across four treatment conditions (PBS (vehicle), SSB siRNA, S1P inhibitor, MTP inhibitor) at 2 h (empty bars) and 6 h (filled bars) post-tracer-injection. The naturally occurring M2/M0 ratio is represented by the dashed line (---).

relative ApoB100 synthesis at 2 and 6 h following H218O injection in mice previously treated with PBS, SSB siRNA, a MTP inhibitor, or a S1P inhibitor. Mice treated with PBS represent the control group in this study. Isotopic ratio measurements are shown with reference to the isotopic 1595



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Present Address

product ion and the number of amino acids included in the product ion measurements is required. Tools that utilize predictive algorithms in combination with spectrum database repositiories24,25 and automated software tools that make empirical determinations of the optimum SRM transitions26 are becoming widely available. In biology, peptide sequences are oftentimes conserved across species, and thus the same mass spectrometry assay for quantitation may be used in different species. In cases where the protein sequence identity diverges across species or organisms, if the genome of the given organism has been determined, the SRM transition can be easily modified to determine if a mass spectrometry assay can be made to measure that protein in that specific species. To modify the protein measurement methods described here from one species, such as mouse, to another preclinical model such as hamsters, guinea pigs, rabbits, or nonhuman primates, a short development turn-around time on the order of two to three weeks is required and can be achieved at low cost. This flexibility provided by the mass-spectrometry-based measurement removes a previous bottleneck where specific antibody reagents may need to be developed for each species and specific antibody reagents may require a considerable lead time of months to years. It may be worth noting that a number of factors may influence the rate of in vivo label equilibration (age, lean vs obese, etc.), and thus, to make comparisons between groups that differ in these factors, one may need to confirm that the necessary assumptions hold true within the particular experimental design.

†

Memorial Sloan-Kettering Cancer Center, New York, New York 10065, United States. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank our colleagues Alan B. Sachs and Andy Plump for the helpful discussions and support.

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CONCLUSION A triple-stage quadrupole mass spectrometry based assay based on product ion measurements at unit resolution and H218O stable tracer incorporation was developed to measure relative protein synthesis rates. The results demonstrate that this assay successfully measures relative ApoB100 synthesis in unique lipoprotein particle fractions and in response to treatment conditions. We anticipate that this type of assay can be generalized to hundreds or thousands of other proteins. Although the combination of H218O tracer with the triple quadrupole mass spectrometry platform creates an assay that is relatively quick and inexpensive to transfer across different biological model systems, serving as an ideal rapid screening tool for relative protein synthesis in response to treatment, investigators should consider the relative rates of protein synthesis vs tracer distribution. Clearly, studies of ApoB100 meet certain criteria. As demonstrated, production of ApoB100 does not appear to be influenced by tracer equilibration under the conditions used in this study. Broad application of this method to other proteins may be limited in cases where the rates of protein synthesis approach or exceed the rates of amino acid labeling.

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ASSOCIATED CONTENT

S Supporting Information *

Supporting Tables S1 and S2. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

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