Absolute Quantification of Peptides by Isotope Dilution Liquid

Mar 14, 2013 - A detection limit (3 SD) of 7.2 fmol estimated for ID LC–ICPMS with a 10 μL injection volume from three procedure blanks was obtaine...
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Absolute Quantification of Peptides by Isotope Dilution Liquid Chromatography−Inductively Coupled Plasma Mass Spectrometry and Gas Chromatography/Mass Spectrometry Rui Liu,†,‡,§ Xiandeng Hou,‡ Yi Lv,‡ Margaret McCooeye,† Lu Yang,*,† and Zoltán Mester† †

Chemical Metrology, National Research Council Canada, Ottawa, Ontario, Canada, K1A 0R6 College of Chemistry, Sichuan University, Chengdu, Sichuan, 610064, China § College of Materials and Chemistry and Chemical Engineering, Chengdu University of Technology, Chengdu, Sichuan, 610059, China ‡

S Supporting Information *

ABSTRACT: Absolute quantitation of peptides/proteins in dilute calibration solutions used in various diagnostic settings is a major challenge. Here we report the absolute quantitation of peptides by non-species-specific isotope dilution liquid chromatography−inductively coupled plasma mass spectrometry (ID LC−ICPMS) based on stoichiometric Eu tagging. The method was validated by species-specific isotope dilution gas chromatography/mass spectrometry (GC/MS) analysis of constituent amino acids of the target peptide. Quantitative labeling of bradykinin peptide was accomplished with a commercially available 2′,2″-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetic acid (DOTA-NHS-ester) and subsequently tagged with Eu. A 151Eu-enriched spike was used for the non-species-specific ID LC−ICPMS determination of bradykinin. The non-species-specific ID LC−ICPMS method was cross-validated by a speciesspecific ID GC/MS approach, which is based on the determination of phenylalanine in bradykinin to derive the concentration of the peptide in the sample. The hydrolysis of the peptide into amino acids was achieved by microwave digestion with 4 M methanesulfonic acid, and derivatization of phenylalanine with methyl chloroformate (MCF) was performed prior to its detection by GC/MS based on a 13C-enriched phenylalanine spike. The accuracy of the method was confirmed at various concentration levels with a typical precision of better than 5% relative standard deviation (RSD) at 20 pmol for non-speciesspecific ID LC−ICPMS and 500 pmol for species-specific ID GC/MS. A detection limit (3 SD) of 7.2 fmol estimated for ID LC−ICPMS with a 10 μL injection volume from three procedure blanks was obtained for bradykinin, confirming the suitability of the method for the direct determination of peptides at trace levels. To the best of our knowledge, the proposed method is the first ICPMS peptide quantification strategy which employs an independent validation strategy using species-specific ID GC/MS for amino acid quantitation.

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analyte because the relationship between the quantity of protein or peptide and the signal intensity obtained in ESI-MS and MALDI-MS varies depending on ionization efficiency, molecular weight, etc.5 Production of high-purity authentic standards for all peptides of interest could be costly and difficult; thus, analytical methods that do not rely on peptide standards are attractive. Even when peptide or protein standards are produced the assessment of purity and assignment of amount fraction/concentrations is far from trivial. Inductively coupled plasma mass spectrometry (ICPMS) is a sensitive technique for the determination of a wide range of metals and several nonmetals.6−8 The advantages of ICPMS as an elemental detector include low detection limits (picogram

radykinin has been associated with allergic inflammation and the pathogenesis of allergic conditions.1 It binds to endothelial B1 and B2 receptors, exerts potent pharmacological and physiological effects such as decreased blood pressure and increased vascular permeability, and promotes classical symptoms of inflammation such as vasodilation, hyperthermia, edema, and pain. On the other hand, bradykinin has been shown to have potent beneficial antithrombogenic, antiproliferative, and antifibrogenic effects. Thus, the sensitive quantification of peptides (even purified ones), such as bradykinin, is of great importance for diagnostic and therapeutic purposes.2−4 Currently, molecular mass spectrometry based techniques, such as electrospray ionization mass spectrometry (ESI-MS) or matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), are the most powerful analytical tools available for protein and peptide quantification. Molecular mass spectrometry typically requires standards for each and every © 2013 American Chemical Society

Received: January 16, 2013 Accepted: March 14, 2013 Published: March 14, 2013 4087

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operating parameters of the ICPMS and HPLC are given in Table S1 (in the Supporting Information). The HPLC instrument was coupled with an electrospray mass spectrometer (ESI-MS) to identify the products of the labeling process and assess the efficiency of the procedure. The LTQ-DECA XP Plus ion trap MS manufactured by ThermoFinnigan (San Jose, CA) was operated in full scan mode, and samples were introduced using the HPLC method described above. An Excellence Plus electronic microbalance (XP205, Mettler Toledo) with readability of 0.01 mg was used to accurately weigh the peptides and eluent during a chromatography run to calculate the mass flow of the 151Eu-enriched spike working solution. A Hewlett-Packard HP 6890 GC (Agilent Technologies Canada Inc., Mississauga, ON, Canada) fitted with a HP 7683 automatic injector system was used for the determination of amino acids in peptides. Detection was achieved with an HP model 5973 mass-selective detector (MSD) operated at 70 eV. The control of components of the chromatographic system and the analysis of the chromatograms including identification and quantification of amino acids were performed with Enhanced Chemstation software. A DB-5MS column (30 m × 0.25 μm, Iso-Mass Scientific Inc., Calgary, Alberta, Canada) was used for chromatographic separation. A 1 μL sample in chloroform was injected in splitless mode. Helium carrier gas flow rate was 1.5 mL min−1, and injector temperature was 280 °C. The pressure was 17.6 psi. The oven temperature program started at 120 °C and was held for 2 min, then increased at 15 °C min−1 to 290 °C and held for 1 min. The MS ion source temperature was 250 °C, and transfer line temperature was 260 °C. Selected ion monitoring (SIM) mode was used for the determination of amino acids. The microwave irradiation hydrolysis system was a CEM DISCOVER-S microwave reactor equipped with an Explorer 48-position sampler handler and a digital camera. Synergy-D software was used to control and monitor temperature, power, and pressure in the device. The vessel design features a secure snap-on cap for ease of use. The CEM DISCOVER-S utilizes a magnetron, which operates at 2.45 GHz with a maximum output power of 300 W. A 10 mL Pyrex vessel was used for this analysis. Reagents and Solutions. The bradykinin peptide, DTPAA, and triethylammonium acetate buffer (TEAB) were purchased from Sigma-Aldrich (Ottawa, Ontario, Canada). DOTA-NHS-ester was from Macrocyclics Inc. (Richardson, TX, U.S.A.). Nitric and hydrochloric acids were purified inhouse prior to use by sub-boiling distillation of reagent grade feedstock in a quartz still. A 1000 μg g−1 natural abundance Eu stock solution was purchased from SCP Science (Baie D’Urfé, Québec, Canada). 151Eu-enriched stock solution at 853 μg g−1 was prepared by dissolving the solid 151Eu2O3 (Trace Sciences International, Richmond Hill, Ontario, Canada) in concentrated HNO3 at room temperature. The solution was gradually evaporated to dryness on a hot block, and the residue was dissolved in 0.5 mL of HNO3 and diluted with distilled water (DIW). The final concentration of 151Eu stock solution was verified by reverse spike isotope dilution using a natural abundance Eu standard. A working 151Eu spike solution of 4.27 ng g−1 was prepared gravimetrically in 2% HNO3 from the above stock solution. Environmental grade ammonium hydroxide was purchased from Anachemia Science (Montreal, Québec, Canada). High-

per milliliter level for most elements), large dynamic range, and simple spectral profiles for elements and isotopes, which make it directly applicable to peptide and protein quantification with an elemental tag.9−18 In addition, isotope dilution (ID) ICPMS may provide superior accuracy and precision compared to other calibration strategies.19,20 Given the known stoichiometric ratio of the tag element to the analyte protein/peptide, a tag element standard solution could be used directly for the quantitation of peptides and proteins instead of high-purity peptide and protein standards which are usually required by ESI-MS techniques. As a result, applications of these methods for the determination of biomolecules containing a naturally occurring heteroelement (such as S,21−23 P,24 Se,25 Fe,26 etc.) and biomolecules after labeling with a heteroelement (such as lanthanides,27−33 Ru,34 Hg,35,36 I,37 etc.) have increased significantly in the past decade. Protein and peptide labeling with heteroelements, especially lanthanides, enhances sensitivity in ICPMS. For example, a macrocyclic compound diethylenetriamine-N,N,N′,N″,N″-pentacetic dianhydride (DTPAA) is often applied as a low-cost, efficient biofunctional lanthanide chelating agent.29,31 The objective of this study was to develop an absolute quantitation method for the determination of peptides without the use of peptide standards. However, as observed in our preliminary studies, the commonly used peptide labeling reagent DTPAA can form bisadducts since DTPAA is a dualreactive labeling reagent. Therefore, monoreactive 2,2′,2″-(10(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetic acid (DOTA-NHS-ester) was investigated for peptide labeling and quantification in this study. Bradykinin was used as a model peptide. The 1:1 stoichiometric ratio of the Eu to bradykinin allowed the use of a generic inorganic Eu standard for quantitation. In order to validate the proposed method, microwave hydrolysis38−40 and species-specific ID gas chromatography/mass spectrometry (ID GC/MS) were applied to the quantification of the constituent amino acid (AA) content of bradykinin and the subsequent peptide amount fraction. The MS-based AA determination is considered to be the current state of the art by the measurement science community for peptide quantitation.41 To the best of our knowledge, the proposed method is the first validated, generic ICPMS-based peptide quantification strategy.

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EXPERIMENTAL SECTION Instrumentation. A PerkinElmer SCIEX ELAN 6000 (Concord, Ontario, Canada) quadrupole ICPMS (qICPMS) equipped with a Gem cross-flow nebulizer and a custom-made quartz sample injector tube (0.9 mm i.d.) were used. A doublepass Ryton spray chamber was mounted outside the torch box and maintained at room temperature. Optimization of the ELAN 6000 and implementation of dead time correction were performed as recommended by the manufacturer. An Agilent HPLC 1200 series (Agilent Technologies Canada Inc., Mississauga, Ontario, Canada) with a ZORBAX Eclipse Plus C18 column (2.1 mm × 150 mm, 5 μm, Agilent Technologies) was used for the separation of labeled peptides from labeling matrixes. The coupling of the LC to the ICPMS was accomplished by directing the eluent from the column to the ICPMS nebulizer through a 0.5 m length of PEEK tubing (0.13 mm i.d., 1.59 mm o.d.). The 151Eu working spike solution of 4.27 ng g−1 was continuously mixed with the eluent from the HPLC column via a three-way connector and was introduced to the nebulizer of the ICPMS via a peristaltic pump. The 4088

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purity water was generated using a Milli-Q-Advantage A10 system from Millipore Corporation (Saverne, Alsace, France). Chloroform (certified grade), pyridine (99.9% purity), and methanol (99.9% purity) were purchased from Fisher Scientific (Ottawa, Ontario, Canada). Methyl chloroformate (99% purity) and methanesulfonic acid (98% purity) were obtained from Sigma-Aldrich Canada (Oakville, Ontario, Canada). Phenylalanine (Phe) was obtained from Sigma-Aldrich Canada (Oakville, Ontario, Canada). A 3 μmol g−1 stock solution was prepared in 1% HCl (v/v) and kept in the refrigerator until used. Isotopically enriched 13C-labeled phenylalanine (13C-Phe) was purchased from Cambridge Isotope Laboratories (Andover, MA, U.S.A.). A 3 μmol g−1 stock solution of 13C-Phe was prepared in high-purity water and kept refrigerated until used. Sample Preparation Procedure and Labeling of Peptides for ID LC−ICPMS. In the first step, peptides were reacted with DOTA-NHS-ester. A 0.5 mL buffered peptide solution (50 mM TEAB, pH 8) was added to the 2 mg of solid DOTA-NHS-ester. Following 1 min of vortexing, the vials were incubated with shaking in the dark at room temperature for 1 h. The subsequent metal chelation step with Eu was performed at pH 6. An amount of 50 μL of the peptide−DOTA solution (50 mM TEAB, pH 8) was mixed with 2450 μL of Eu solution (30 μg g−1) at pH 6 and reacted for 1 h at room temperature. A 2fold molar excess with respect to the presence of DOTA was chosen for the added Eu solution. The bioconjugate solutions obtained were injected into the HPLC−ICPMS for analysis. Sample Preparation for Species-Specific ID GC/MS. The peptide solution or powder was accurately weighed in a 10 mL dry Pyrex microwave vessel. An appropriate amount of 13Clabeled phenylalanine was added to each sample to result in a ratio near 1 for reference to spike ions. Amounts of 2 mL of 1% (v/v) HCl and 0.75 mL of MSA were added. The sample was hydrolyzed in a closed vessel microwave irradiation system, similar to methods described previously.39,40 The microwave conditions used were 200 W, 150 °C, and 30 min holding time. After cooling, an appropriate amount of ammonium hydroxide was added to adjust the pH of the sample to about 5.5. The derivatization procedure used in the study followed that of an earlier study.39 After addition of 900 μL of pyridine−methanol (1:3 v/v) to each sample, 300 μL of methyl chloroformate (MCF) was slowly added. The sample was vortex-mixed for 1 min followed by addition of 1 mL of chloroform, and the vial was again vortex-mixed for 1 min. After the sample was centrifuged (2000 rpm, 10 min), the chloroform layer was transferred to a 2 mL vial (insert was 200 μL) for GC/MS analysis.

Figure 1. ESI-MS spectrum of bradykinin before (a) and after DOTANHS-ester labeling (b) and Eu labeling (c) in TEAB.

derivatization. Interestingly, two dominant peaks were found in the HPLC−ESI-MS chromatogram obtained from a derivatized bradykinin standard solution (Figure S2a in the Supporting Information). The resulting products were identified as bradykinin−DTPA (Supporting Information Figure S2b) and bradykinin−DTPA−bradykinin (Supporting Information Figure S2c) bisadduct, as confirmed by their respective mass spectra. Contrary to previous studies,29,31,42 the formation of bisadduct of bradykinin with DTPAA was observed, which could present a problem for ICPMS-based peptide quantification because of the skewed stoichiometry. DTPAA possesses two reactive groups, and thus it is difficult to control the reaction which could result in cross-linking of biomolecules and ultimately nonstoichiometric labeling.43 Alternatively, monoreactive DOTA-NHS-ester could be a better choice for the derivatization of bradykinin. The two steps of the labeling reaction were monitored by ESI-MS. In the first step, the reaction of DOTA-NHS-ester with the primary amine group is expected to produce a signal with a mass difference of m/z 386 with respect to the underivatized peptide. In the case of bradykinin (m/z 1061, Figure 1a), after labeling with a 10fold molar excess of DOTA-NHS-ester in TEAB at pH 8, the resultant ion was monitored at m/z 1447, as shown in Figure 1b. A signal at m/z 1183 in Figure 1a resulted from sodium

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RESULTS AND DISCUSSION Labeling of Bradykinin with Macrocyclic Compounds. In this study, bradykinin was selected as a model peptide since there is an interest in its determination. The schematic diagram of the labeling and subsequent determination of peptides by ICPMS and GC/MS are shown in Figure S1 (in the Supporting Information). DTPAA is an inexpensive and commercially available reagent which has been commonly employed for covalent derivatization of peptides and proteins. The reaction of DTPAA with proteins and peptides and subsequent chelation with rare earth elements have already been applied to the quantification of peptides and proteins using molecular mass spectrometry42 and ICPMS.29,31 In our preliminary experiments, the DTPAA was initially tested for bradykinin 4089

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Figure 3. Species-specific ID GC/MS for the quantification of bradykinin: (a) ID GC/MS TIC signal of 2 nmol of bradykinin; (b) extracted ID GC/MS spectra of Phe peak (time range 7.75−7.85 min).

Figure 2. Non-species-specific ID ICPMS for the quantification of bradykinin: (a) Eu signal intensity of 20 pmol bradykinin; (b) Eu mass flow of 20 pmol bradykinin.

Quantitation of Peptide Using External Calibration with HPLC−ICPMS. In order to test the developed method, bradykinin solutions at various concentration levels ranging from 0 to 0.5 μmol mL−1 were prepared and labeled with DOTA-NHS-ester. Signals increased proportionally as concentrations of peptide increased. The external calibration curve shows a good linear peak area response (Y = 4.5 × 104X + 40 000) in the concentration range tested, 2−100 pmol, with a correlation coefficient (r2) of 0.9953, demonstrating the feasibility of quantitative determination of bradykinin by external calibration using Eu elemental standard solutions. On the basis of the external calibration using bradykinin standard solutions, a concentration of 19.6 ± 1.0 pmol (1 SD, n = 3) was obtained in a test solution, in agreement with its gravimetrically prepared value of 20 pmol. The detection limit (LOD) for the external calibration HPLC−ICPMS technique was evaluated using three procedural blanks based on a 10 μL injection. A value of 6.5 fmol was estimated for bradykinin based on three times the standard deviation of the measured concentrations in procedural blanks. Quantitation of Peptide Using Non-Species-Specific Isotope Dilution HPLC−ICPMS. The postcolumn infusion non-species-specific ID approach could simplify calibration and eliminate effects encountered in ICPMS such as signal suppression and matrix effects. In addition, this calibration

buffer adducts. Since there was plenty of HPF6 in DOTA-NHSester, signals at m/z 1593 and 1739 observed in Figure 1b were most likely the adducts and bisadducts of HPF6 (m/z 146) of the labeled bradykinin, respectively. A signal corresponding to unreacted bradykinin (m/z 1061) was not detected after the reaction. Importantly, the bisadduct of two bradykinin molecules bound to one DOTA molecule was not detected. The second reaction step, the chelation with Eu, was performed at pH 6 with a 2-fold molar excess of europium with respect to the DOTA concentration present in the sample. Figure 1c shows the spectra of the reaction mixture in TEAB buffer which led to the formation of the expected bradykinin−DOTA−Eu product (signal at m/z 1597). The signal at m/z 1743 resulted from bradykinin−DOTA−Eu and HPF6 adduct. Signals at m/z 1107, m/z 1129, and m/z 1151 were from Eu−DOTA and buffer adducts. Again, a signal corresponding to unreacted bradykinin−DOTA (m/z 1447) was not detected. Clearly, the above results obtained from ESI-MS demonstrate a high specificity of the labeling with the use of DOTA-NHS-ester without formation of bisadducts of two peptides cross-linked via DOTA. Furthermore, the ESI-MS results indicate highly efficient labeling reactions (virtually 100% labeling efficiency based on ESI-MS evaluation).

Table 1. Results of Quantitative Analysis of Bradykinin, Reported as Absolute Amounts method

sample 1

sample 2

sample 3

sample 4

sample 5

by non-species-specific ID ICPMS, mean, pmol 1 SD, n = 3, pmol U, K = 2, pmol by species-specific ID GC/MS, 1 SD, n = 3, pmol

500 18 37 483 ± 24

810 37 75 812 ± 21

1101 22 47 1043 ± 40

1508 36 76 1479 ± 55

2018 79 161 1989 ± 83

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strategy only needs an inorganic standard (Eu in this case) without the need of high-purity peptide standard. However, it must be noted that this does not correct or compensate for any analyte loss during the sample preparation and chromatographic separation. This calibration approach assumes identical behavior in the ICP for the infused Eu spike and peptide-bound Eu tag which is a reasonable assumption in ICPMS.29,31 The concentration of the enriched 151Eu stock solution was determined by reverse isotope dilution with a natural abundance Eu standard using the following eq 1.44 Cy = Cz

mz Bxz R′n − Axz AWy m′ y A y − By R′n AWz

Eu-enriched spike solution at the beginning and the end of a chromatographic run. Eu signal intensity and Eu mass flow for non-species-specific ID ICPMS for the quantification of 20 pmol of bradykinin are shown in Figure 2b. Results obtained in five different solutions using non-species-specific ID ICPMS are summarized in Table 1. Precision of less than 5% was generally obtained with the ID calibration depending on concentrations. Method LOD using ID, evaluated from procedural blanks based on a 10 μL injection, was calculated to be 7.2 fmol, comparable or superior to reported non-species-specific ID detection limits using element tag labeling29,31,37 and naturally occurring S tag.21,22 Estimations of uncertainty for bradykinin concentrations were made in accordance with JCGM 100:28 (Evaluation of Measurement DataGuide to the Expression of Uncertainty in Measurement),47 using the following law of propagation of uncertainty:

(1)

where Cy is the analyte concentration (μg g−1) in the 151Euenriched spike, Cz is the concentration of natural abundance Eu standard (μg g−1), mz is the mass of natural abundance Eu standard solution used (g), m′y is the mass of enriched spike used to prepare the mixture of spike and natural abundance Eu standard solution (g), Ay is the abundance of the reference isotope (153Eu) in the spike, By is the abundance of the spike isotope (151Eu) in the spike, Axz is the abundance of the reference isotope in the sample or natural abundance standard, Bxz is the abundance of the spike isotope in the sample or natural abundance standard, R′n is the measured ratio of reference/spike isotopes (mass bias corrected) in the mixtures of spike and natural abundance standard, AWy is atomic weight of analyte in the enriched spike, and AWxz is atomic weight of analyte in the sample or natural standard. A concentration of 853 ± 8 μg g−1 (1 SD, n = 3) was obtained for 151Eu-enriched stock solution. A concentration of 4.27 ng g−1 151Eu working spike solution was prepared gravimetrically from the above stock solution and used for the postcolumn isotope dilution determination of peptide. In order to perform postcolumn non-species-specific ID for the quantitation of peptide, a 4.27 ng g−1 151Eu working spike solution was continuously mixed with the eluent from the column via a three-way connector to the nebulizer of the ICPMS. As shown in Figure 2a, both 151Eu and 153Eu signals were monitored in order to obtain the 151Eu/153Eu ratio to calculate the final concentrations of peptide in the test solutions. Equation 2 was used for ID in accordance with previous studies.45,46 MFx(t ) = MFy

A y − By R n(t ) AWxz Bxz R n(t ) − Axz AWy

N

uci 2(y) =

N−1 ⎛ ∂f ⎞2 2 ⎟ u (xi) + 2 ∑ ⎝ ∂xi ⎠ i=1

∑⎜ i=1

u(xj)r(i , j)

⎛ ∂f ⎞⎛ ∂f ⎞ ⎜ ⎟⎜⎜ ⎟⎟u(xi) j = i + 1 ⎝ ∂xi ⎠⎝ ∂xj ⎠ N

∑

(3)

where y = f(x1, x2, ..., xN). The partial derivatives ∂f/∂xi are often referred to as sensitivity coefficients, u(xi) and u(xj) are the standard uncertainties associated with the input parameters xi and xj, and r(i,j) is the correlation coefficient (−1 ≤ r(i,j) ≤1). The combined uncertainty of the grand mean, uC̅ , was obtained by combining the uncertainties of the individual estimates and the variations between these means as per recent guidelines from NIST.48 The following equation was used: uC̅ 2 = SD2 +

1 n2

∑ uc 2 i

(4)

where SD is the standard deviation of the n values and uci is the uncertainty of the individual measurand estimates, i = [1, ..., n]. Validation of Peptide Quantification by SpeciesSpecific Isotope Dilution GC/MS. The combined use of the microwave assisted hydrolysis (MAH) and GC/MS is a rapid and relatively low-cost method to assay the amino acids in peptides and proteins. The amount of a given peptide or protein can be subsequently obtained from the amount of any constituent amino acid, providing that the amino acid sequence of the peptide or protein is known. To validate the proposed procedure, five quantitatively prepared bradykinin samples were labeled and subjected to HPLC−ICPMS determination via 151 Eu spiking for non-species-specific ID and MAH GC/MS determination via 13C-labeled amino acid spiking for speciesspecific ID. The amino acid sequence of bradykinin is Arg-ProPro-Gly-Phe-Ser-Pro-Phe-Arg. In principle, any amino acid in the peptide can be used for GC/MS quantification. Phenylalanine (Phe) was chosen as the target amino acid because of its high sensitivity, stability, and baseline separation from other amino acids in GC/MS as observed in a previous study.40 The fragment (C10H10O2, M = 162) of methyl chloroformate derivatized Phe (C12H15NO4, M = 273) was selected for the quantification of Phe. The 13C-enriched Phe was used for ID GC/MS analysis. The natural isotope distribution was obtained from an isotope distribution calculator.49 Relative abundances of 89.229% and 9.865% for ions at m/z 162 and 163 in the sample and 0.892% and 88.436% in the spike were obtained and used for the quantitation. Figure 3 shows the

(2)

where MFx(t) is the sample mass flow at time t, MFy(t) is the 151 Eu-enriched spike mass flow at time t, and Rn(t) is the measured ratio of reference/spike isotopes (mass bias corrected) at time t. The mass bias correction factor was obtained from the IUPAC value of 153Eu/151Eu divided by the measured ratio of a 5 ng g−1 natural abundance Eu standard introduced via the three-way connector. The integration of the peak area of calculated sample mass flow MFx(t) gives the mass of Eu in an eluting peptide directly. Concentration of the peptide can be derived from the stoichiometric ratio (1:1 for bradykinin) of Eu to that peptide and the injection volume. Since it is a postcolumn ID, MFy can be easily calculated by the consumed mass of 151Eu-enriched working spike solution multiplying its concentration and dividing by the run time of the chromatogram, as reported in a recent study.46 The consumed mass was obtained by the difference in weights of 4091

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chromatography and mass spectrometry of species-specific ID GC/MS for the quantification of bradykinin. Results are summarized in Table 1. Concentrations obtained using nonspecies-specific ID ICPMS in all five test solutions are in good agreement with those using species-specific ID GC/MS, demonstrating the accuracy of the proposed method.

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CONCLUSIONS Derivatization of peptides with DOTA-NHS-ester and subsequent isotopic labeling with Eu has enabled LC ICPMS to become a powerful tool for the detection and quantitation of peptides. The proposed method has potential for the accurate and absolute quantitation of peptides using non-species-specific ID ICPMS without the need for pure peptide standards. Species-specific ID GC/MS can be an efficient validation method for the results obtained from the non-species-specific ID LC−ICPMS approach. It is noteworthy that the current ZORBAX LC column was able to sufficiently separate the labeled peptide from labeling matrixes in this proof-of-concept study. However, for complex sample matrix, more powerful peptide separation techniques may be needed.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the National Natural Science Foundation of China (Grant Nos. 20835003 and 21128006) for partially funding this study and the China Scholarship Council for financial support during R. Liu’s stay in Canada.

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