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Strategy and Its Implications of Protein Bioanalysis Utilizing High-Resolution Mass Spectrometric Detection of Intact Protein Qian Ruan,† Qin C. Ji,*,‡ Mark E. Arnold,‡ W. Griffith Humphreys,† and Mingshe Zhu† †
Biotransformation and ‡Bioanalytical Sciences, Bristol-Myers Squibb Comapny, Route 206 and Province Line Road, Princeton, New Jersey 08543, United States
bS Supporting Information ABSTRACT: Currently, mass spectrometry-based protein bioanalysis is primarily achieved through monitoring the representative peptide(s) resulting from analyte protein digestion. However, this approach is often incapable of differentiating the measurement of protein analyte from its post-translational modifications (PTMs) and/or potential biotransformation (BTX) products. This disadvantage can be overcome by direct measurement of the intact protein analytes. Selected reaction monitoring (SRM) on triple quadrupole mass spectrometers has been used for the direct measurement of intact protein. However, the fragmentation efficiency though the SRM process could be limited in many cases, especially for high molecular weight proteins. In this study, we present a new strategy of intact protein bioanalysis by high-resolution (HR) full scan mass spectrometry using human lysozyme as a model protein. An HR linear ion-trap/ Orbitrap mass spectrometer was used for detection. A composite of isotopic peaks from one or multiple charge states can be isolated from the background and used to improve the signal-to-noise ratio. The acquired data were processed by summing extracted ion chromatograms (EIC) of the 10 most intense isotopic ions of octuply protonated lysozyme. Quantitation of the plasma lysozyme was conducted by utilizing high resolving power and an EIC window fitting to the protein molecular weight. An assay with a linear dynamic range from 0.5 to 500 μg/mL was developed with good accuracy and precision. The assay was successfully employed for monitoring the level of endogenous lysozyme and a potential PTM in human plasma. The current instrumentation limitations and potential advantages of this approach for the bioanalysis of large proteins are discussed.
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oncentration determination of analytes such as biomarker molecules and drug substances and their related compounds in biological matrixes, termed as “bioanalysis”, is a critical part of drug discovery and development. Although ligand-binding assays are still the main platform for the bioanalysis of protein and peptide analytes, liquid chromatographymass spectrometry (LCMS) assays play increasingly more important roles as a complementary platform.13 Two primary strategies used in the LCMS bioanalysis of proteins in biological matrices are (1) protein quantitation through mass spectrometric detection of a representative peptide segment(s) generated from enzymatic digestion or chemical cleavage47 and (2) protein quantification through direct mass spectrometric detection of intact proteins.8,9 As shown in Figure 1, while the strategy through measurement of a representative peptide segment(s) has gained increasing acceptance, this “bottom-up” approach has a major limitation that the peptide(s) may not accurately represent the protein analyte. For example, it cannot differentiate a protein analyte and its products derived from post-translational modification (PTM) and/or in vivo biotransformation (BTX) processes if unchanged peptide segments are monitored. This disadvantage could be overcome by the second strategy by direct measurement of the intact protein analyte. To apply this “top-down” approach, selected reaction monitoring (SRM) using triple-quadrupole mass spectrometers has been developed.8,9 Although SRM detection provides excellent r 2011 American Chemical Society
selectivity and duty-cycle, it also has some inherent disadvantages in the fragmentation-dependent MS detection. As the molecular weight of a protein increases, the fragmentation efficiency of the protein through the SRM process often decreases.10 Even when the protein is fragmented, the charges are more evenly distributed into many fragments rather than concentrated onto a few major fragments like small molecules. Furthermore, the electrospray ion source commonly used on triple-quadrupole MS tends to ionize a protein into different charge states. SRM can only detect the one particular fragment transition from a precursor ion at a specific mass to charge ratio, while the majority of the ions in all the other charge states cannot be utilized at the same moment. Full scan MS detection is independent of the fragmentation efficiency of a protein. In addition, if protein ions in several charge states have similar intensities, full scan MS with a nonionfiltering type mass spectrometer allows summation of these ion signals, thus improving the analyte signal intensity. In addition, full scan MS can provide additional qualitative and quantitative information of protein analyte related components in each individual sample, allowing the flexibility to reprocess acquired data for additional investigations, such as quantitation of Received: June 18, 2011 Accepted: October 4, 2011 Published: October 04, 2011 8937
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Figure 1. Protein bioanalysis strategy.
unpredicted PTMs in patient samples. One common drawback of the full scan MS approach is the lower selectivity caused by higher background or interferences as compared to that seen in the SRM approach. Recently, there have been significant advances in the capabilities of high-resolution mass spectrometry (HR-MS) instrumentation.11,12 HR-MS can provide additional advantages in resolving each isotopic ion from the background so that higher selectivity can be achieved. Furthermore, when used for identification of protein modifications present in various in vivo samples, the high accuracy offered by HR-MS can be critical to the mass assignment, e.g., differentiating oxidation (+15.9949 Da) and methylation (+14.0156 Da) as well as determining oxidation/reduction states of disulfide bonds. Here, we report our first exploratory use of full scan HR-MS for bioanalysis of an intact protein. Human lysozyme, a 15 kDa glycoside hydrolase13,14 circulating in plasma, was used as a model protein to demonstrate the feasibility and potential advantages of this approach. Lysozyme has four disulfide bonds, and their integrity is a critical indication of normal functionality.15 The HR-MS capability in isotopic resolution and accurate mass determination of a protein analyte is indispensable for the identification of the normal structure of the protein based on exact mass deviations, such as lack or presence of expected PTMs or amino acid substitutions. The assay development for direct LCMS analysis of the intact human lysozyme, such as sample preparation and optimization of chromatographic separation, are also discussed, and the ability of this assay to quantify the protein in human plasma is demonstrated.
’ EXPERIMENTAL DETAILS Chemical, Reagents, Materials, and Apparatus. Chemicals and Reagents. Human milk lysozyme, chicken egg yolk lysozyme,
acetic acid, trifluoroacetic acid (TFA), and ACS grade ammonium acetate were purchased from Sigma-Aldrich (St. Louis, MO). HPLC grade acetonitrile and methanol were obtained from Thermo Fisher Scientific Co. (Waltham, MA). Human and monkey K2EDTA plasma were obtained from Bioreclamation (Hicksville, NY). Materials and Apparatus. Plates used for sample collection and injection were Costar Brand 96-well assay blocks from VWR
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International (Bridgewater, NJ). Oasis HLB 30 mg solid phase extraction (SPE) plates were purchased from Waters (Milford, MA). The SPE manifold for a Tomtec Quadra 96 model 320 robotic liquid handler (Tomtec, Hamden, CT) was used for SPE processing. An SPE Dry microplate sample concentrator (Biotage AB, Uppsala, Sweden) was used for solvent evaporation of SPE eluents. LCMS Equipment. All sample analyses were performed on an LCMS system that consisted of Shimadzu LC-20AD HPLC pumps (Shimadzu Corporation, Kyoto, Japan), a LEAP autosampler (LEAP Technologies, Carrboro, NC), an Xbridge BEH300 C4, 50 mm 2.0 mm, 3.5 μm column (Waters, Milford, MA) with a Sidewinder column heater (Restek, Bellefonte, PA) and an LTQ/Orbitrap Classic mass spectrometer (Thermo Fisher Scientific, Waltham, MA). HPLC was performed at a constant flow rate of 400 μL/min using a binary solvent system. Mobile phase A was composed of 0.1% TFA and 0.5% acetic acid in HPLC grade H2O; mobile phase B was 0.1% TFA and 0.5% acetic acid in acetonitrile. The HPLC gradient started with 5% B, linearly increased to 40% B in 4 min, continued to 50% B in 4 min, increased to 100% B in 0.5 min, and maintained at 100% B for 0.4 min prior to column re-equilibration. The LTQ/Orbitrap settings were as follows: sheath gas at 60, auxiliary gas at 30, sweep gas at 5, spray voltage at 5 kV, tube lens at 100 V, automatic gain control (AGC) at 5 105, max injection time at 300 ms, and resolving power of 100 000 at 400 m/z for all analyses. During method development, MS full scans were carried out using a mass range of 2004000 m/z. For protein quantitation of the human plasma samples, the MS full scan range was narrowed down to 15801850 m/z. Sample Preparation. Preparation of Calibration Standards and Quality Control (QC) Samples. Stock solutions containing 100 mg/mL human lysozyme (analyte) and 100 μg/mL chicken lysozyme (internal standard (IS)) were prepared in water. An aliquot of 20 μL human lysozyme stock solution was added into 3.98 mL of control monkey plasma to make a plasma working solution (500 μg/mL). Two sets of eight standard levels (0.5, 1.00, 2.50, 10.0, 50.0, 100, 200, and 500 μg/mL) were prepared by successively diluting the working solution into control monkey plasma. Four replicates of five QC levels (0.5, 1.5, 12.0, 100, and 160 μg/mL) were prepared by adding an appropriate volume of the working solution into control monkey plasma. Solid Phase Extraction. Aliquots of 400 μL of QCs, standards, or human plasma samples were mixed with 80 μL of chicken lysozyme (IS, 100 μg/mL) and 400 μL of buffer (50 mM NH4OAc, 0.2% TFA in H2O). The mixture was vortexed and then centrifuged at 1500g for 10 min. The supernatant was separated and subjected to SPE cleanup. Each well in a SPE plate was conditioned by 1 mL of methanol followed by 1 mL of 0.1% TFA in H2O. After plate conditioning, 800 μL of each sample mixture was loaded into the plate. Each well on the plate was washed with 1 mL of 1% TFA in ACN/H2O (15/85 v/v) and eluted with 1 mL of 1% TFA in ACN/H2O (70/30 v/v). Samples were dried under a stream of N2 for 2 h at room temperature and reconstituted in 150 μL of 0.1% TFA and 0.5% acetic acid in 5% ACN/H2O. After vortexing and centrifugation, the supernatants were transferred to injection vials for LCMS analysis. Each injection contained 30 μL of the reconstituted samples. Data Processing. The acquired data were processed by summing the extracted ion chromatogram (EIC) of the most intensive isotopic ions of octuply protonated lysozymes. For 8938
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Figure 2. Mass spectra of human and chicken lysozymes: (A) full scan spectrum of human and chicken lysozymes in multiple charge states, (B) full scan spectrum of human lysozyme and PTM carrying eight charges in plasma, (C) enlarged spectrum of human lysozyme carrying eight charges, and (D) enlarged spectrum of human dehydrated lysozyme carrying eight charges.
chicken lysozyme (IS), the most intensive isotopic ions were m/z 1788.6280, 1788.7540, 1788.8810, 1789.0060, 1789.1300, 1789.2550, 1789.3800, 1789.5060, 1789.6300, and 1789.7580; for human lysozyme (analyte), m/z 1836.9140, 1837.0400, 1837.1590, 1837.2890, 1837.4160, 1837.5560, 1837.6670, 1837.7920, 1837.9150, and 1838.0410; for dehydrated human lyzozyme, m/z 1834.9910, 1835.0320, 1835.1610, 1835.2850, 1835.4040, 1835.5350, and 1835.6580. The EIC window for quantitation was set at (10 ppm. Peak areas were calculated using QualBrowser software (Thermo Fisher Scientific, Waltham, MA). The standard curve was fitted to a linear regression model with a 1/x2 weighting factor using in-house software. All statistic parameters were calculated by Microsoft Excel. The exact molecular weight of the most abundant isotope was determined by Protein Prospector (University of California, San Fransisco).
’ RESULTS AND DISCUSSION High-Resolution Mass Spectrometry Detection. When a strategy of MS detection is used for protein bioanalysis, HR-MS has unique advantages over low-resolution approaches. In a commonly used ESI source, proteins can be protonated into multiple charge states (Figure 2A). The ion at each charge state consists of a cluster of isotopic peaks (Figure 2B,C), which cannot be resolved in low-resolution mass spectrometry (Figure 3A,B).
If the resolving power increases enough to resolve these peaks, only the MS response of the resolved peaks will be used as the signal for quantitation while the MS response from background interference between these peaks will be excluded (Figure 3D). As a result, the assay sensitivity (signal-to-noise ratio) for quantitation will be enhanced. This is especially important for most biological samples, such as plasma or serum samples, which contain large amounts of interfering components. As shown in Figure 4, the interference of background ions in plasma spreads over the entire mass range of the analyte protein’s isotopic peaks of interest. Utilization of HR-MS in the analysis of biological samples provides an opportunity for minimizing such interference. In order to obtain effective improvement in selectivity and sensitivity using this approach, three critical interrelated factors need to be considered: mass spectrometer resolving power, molecular weight of the protein analyte, and the EIC window for quantitation. We propose here that the value of the resolving power needs to be at least 4 times the molecular weight of the protein analyte, and the EIC window should be set around the peak width of each isotopic ion. The following is the rationale of this consideration using the 15 kDa human lysozyme as an example. The full-scan mass spectrum of an ionized lysozyme should present every two adjacent isotopic peaks one Dalton apart, which is ∼60 ppm (1 Da/15 kDa) apart regardless of the charge states. When the resolving power of 10k (full width at 8939
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Analytical Chemistry half-maximum (FWHM): 1/10k = 100 ppm, baseline peak width = 200 ppm) was employed on the low-resolving power linear ion trap MS (LTQ) using the zoom/ultrazoom scan function, the mass spectrometer was unable to resolve the isotopic peaks from the baseline (Figure 3B). When the resolving power was increased on an HR-MS to 30k (twice the protein’s molecular weight), baseline separation of the isotopic peaks was just barely achieved (FWHM, 1/30k ≈ 30 ppm, baseline peak width ≈ 60 ppm (Figure 3C)). However, if all isotopic peak signals were used for quantitation, EIC windows of these peaks would all be joined
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together, suggesting no advantage compared to using one EIC window to cover the whole cluster of isotopic ions in the lowresolution approach. When the resolving power further increased to 4 times of the protein’s molecular weight (60k), a gap of one peak width in the background emerged between adjacent isotopic peaks (FWHM, 1/60k ≈ 15 ppm, baseline peak width ≈ peak gap width ≈ 30 ppm (Figure 3D)) and enabled clear separation of the background from the signal of lysozyme. If the EIC window was narrowed down to one peak width accordingly, interference signal present in the gaps could be removed without sacrificing the analyte’s peak signal. Taken together, it is recommended to use the following equation to obtain resolving power that allows adequate separation of isotopic peaks for HR-MS quantitation of an intact protein: R ¼ kM
Figure 3. Effect of resolving power on separation of isotopic peaks of octuply charged lysozymes: (A) resolving power of 2k, (B) resolving power of 10k, (C) resolving power of 30k, and (D) resolving power of 60k.
where M represents the molecular weight (Da) of the protein of interest; k represents the factor of isotopic peak separation (k = 2 indicates baseline separation; k g 4 is recommended for selective quantitation of an intact protein); and R represents the resolving power that needs to be obtained for the ions used for quantitation. When the practical R is determined, the EIC window can be set to around the base peak width of the isotopic ion (2/R) to allow maximum signal from the analyte with minimal signal from the background interference. With the application of this equation to the human lysozyme (M = 15 kDa), the expected practical resolving power (R) for sensitive quantitation (k = 4) is 15 000 4 = 60 000. Since the resolving power of the Orbitrap declines proportional to m/z0.5, the highest resolving power setting of 100 000 at 400 m/z was utilized to ensure the practical solution of ∼60 000 for the octuply charged lysozymes at m/z 18001850 (analytes and IS (Figure 3D)). The FWHM of the isotopic ions was thus calculated as 1/60 000 ≈ 15 ppm (baseline peak width ≈ 30 ppm). As demonstrated in Figure 5AC, when the EIC window for human
Figure 4. LCMS spectra comparison of lysozyme and matrix interference in human plasma: (A and B) full scan MS of lysozyme and (C and D) full scan MS and enlarged full scan MS of interference peaks eluted after lysozyme. 8940
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Figure 5. Effect of EIC window on assay selectivity and sensitivity: (A) EIC window, (0.5 m/z; (B) EIC window, (30 ppm; (C) EIC window, (10 ppm; (D) EIC window, (1 ppm.
Figure 6. Effect of HPLC column temperatures (T) on the chromatography: (A) T = 30 °C, (B) T = 40 °C, (C) T = 50 °C, (D) T = 60 °C, and (E) T = 70 °C.
lysozyme was narrowed down from (0.5 m/z to (10 ppm, the peak intensity ratio of the analyte over the interference increased 5 times. Further reduction of the extraction window from (10 ppm to (1 ppm caused reductions in the peak intensity of both analyte and interference and thus did not significantly improve the analyte/interference ratio (Figure 5C,D). Combined with consideration of minimizing the matrix interference in plasma samples, the EIC window was set to a little less than the baseline peak width (at (10 ppm). The example of lysozyme quantitation demonstrates improvement of the signal-to-noise ratio using the isotopic cluster peaks from one charge state (z = 8). Full scan mass spectrometry detection enables the capability in summing signals of isotopic cluster ions from multiple charged states. The ion selection for quantitation depends on the distribution of signal intensity in different charged states. In this case, the protonated lysozyme was concentrated at the z = 8 charge state in the optimized ionization conditions. The isotopic peaks from the other charged states were
