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Protein Quantitation Using Ru-NHS Ester Tagging and Isotope Dilution High-Pressure Liquid Chromatography−Inductively Coupled Plasma Mass Spectrometry Determination Rui Liu,†,‡ Yi Lv,‡ Xiandeng Hou,‡ Lu Yang,*,† and Zoltan Mester† †

Institute for National Measurement Standards, National Research Council Canada, Ottawa, Ontario, Canada, K1A 0R6 College of Chemistry, Sichuan University, Chengdu, Sichuan, 610064, P. R. China

‡

S Supporting Information *

ABSTRACT: An accurate, simple, and sensitive method for the direct determination of proteins by nonspecies specific isotope dilution and external calibration high-performance liquid chromatography−inductively coupled plasma mass spectrometry (HPLC−ICPMS) is described. The labeling of myoglobin (17 kDa), transferrin (77 kDa), and thyroglobulin (670 kDa) proteins was accomplished in a single-step reaction with a commercially available bis(2,2′bipyridine)-4′-methyl-4-carboxybipyridine-ruthenium N-succinimidyl ester-bis(hexafluorophosphate) (Ru-NHS ester). Using excess amounts of Ru-NHS ester compared to the protein concentration at optimized labeling conditions, constant ratios for Ru to proteins were obtained. Bioconjugate solutions containing both labeled and unlabeled proteins as well as excess Ru-NHS ester reagent were injected onto a size exclusion HPLC column for separation and ICPMS detection without any further treatment. A 99Ru enriched spike was used for nonspecies specific ID calibration. The accuracy of the method was confirmed at various concentration levels. An average recovery of 100% ± 3% (1 standard deviation (SD), n = 9) was obtained with a typical precision of better than 5% RSD at 100 μg mL−1 for nonspecies specific ID. Detection limits (3SD) of 1.6, 3.2, and 7.0 fmol estimated from three procedure blanks were obtained for myoglobin, transferrin, and thyroglobulin, respectively. These detection limits are suitable for the direct determination of intact proteins at trace levels. For simplicity, external calibration was also tested. Good linear correlation coefficients, 0.9901, 0.9921, and 0.9980 for myoglobin, transferrin, and thyroglobulin, respectively, were obtained. The measured concentrations of proteins in a solution were in good agreement with their volumetrically prepared values. To the best of our knowledge, this is the first application of nonspecies specific ID for the accurate and direct determination of proteins using a Ru-NHS ester labeling reagent.

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treated and control systems to produce a mass shift which allows direct comparison between treated and nontreated samples when they are analyzed simultaneously.7 Absolute quantification using isotopically labeled standard proteins or peptides is feasible; however, production costs could be prohibitive.7 Inorganic mass spectrometry, in particular, inductively coupled plasma mass spectrometry (ICPMS), can be an alternative for absolute protein quantification. ICPMS is characterized by high sensitivity, large dynamic range, low detection limits, and multielement capability.8−15 If two interference-free isotopes of a given element can be found, isotope dilution ICPMS (ID ICPMS), which generally provides superior accuracy and precision over other calibration strategies,16,17 can be considered. Furthermore, a single element standard could be used for the quantitation of peptides and proteins (either a native element such as S, P, or a heavy metal tag attached to the proteins/ peptides). When metal tagging is employed, it should be noted

roteomics, the analysis of large-scale proteins in a given cell, tissue, or organism, has grown rapidly in the last few decades due to its importance in understanding biological functions in life sciences, for the diagnosis of disease, and to support the development of new drugs.1−4 Quantitative determination of proteins (even purified ones) is a challenge in analytical chemistry. The classic two-dimensional gel electrophoresis-based method has been widely used for protein separation and quantification. However, this technique suffers from significant limitations including low sensitivity, bias against categories of proteins, and poor dynamic range.3 In the last 2 decades, molecular mass spectrometry-based methods such as electrospray ionization-mass spectrometry (ESI-MS) or matrixassisted laser desorption ionization-mass spectrometry (MALDIMS) have become major techniques for the identification and characterization of peptides and proteins.5 Molecular mass spectrometry typically requires standards for each and every analyte because the relationship between the quantity of protein or peptide and the signal intensity obtained in ESI-MS and MALDI-MS varies due to ionization efficiency, molecular weight, etc.6 Relative quantitation in proteomics is usually based on differential stable isotope labeling (2H, 13C, 15N, or 18O) of © 2012 American Chemical Society

Received: November 25, 2011 Accepted: February 10, 2012 Published: February 10, 2012 2769

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electrochemiluminescence detection.41 Proteins can be labeled with the Ru-NHS ester in a one-step reaction in an hour.41 In principle, this Ru-NHS ester can be used for all protein determinations since the NHS ester reacts efficiently with primary amino groups (−NH2) forming an amide bond. The objective of this study was to evaluate the use of a RuNHS ester for the labeling of proteins with post column nonspecies specific isotope dilution HPLC−ICPMS for the sensitive, accurate, and precise determination of proteins. For this purpose, myoglobin (Myo, 17 kDa), transferrin (Tra, 77 kDa), and thyroglobulin (Thy, 670 kDa), covering a range of sizes of proteins, were used as model compounds. To the best of our knowledge, this is the first report of the application of nonspecies specific ID calibration for the direct determination of intact proteins using ICPMS with the Ru-NHS ester for protein labeling.

that although an element standard can be used for protein quantification, the stoichiometric ratio of the element to the analyte has to be known. In addition, baseline separation of analytes should be achieved prior to ICPMS detection. ICP and ESI mass spectrometry are becoming complementary techniques. Structural information is preferably obtained by means of ESI- or MALDI-MS, whereas ICPMS is ideal to detect and quantify elements in proteins, even at very low concentrations. As a result, ICPMS has been applied to the screening of proteins that contain ICPMS-detectable elements to provide quantitative information on metals and metalloids in proteins.18−23 However, quantitation of proteins based on those naturally occurring elements which can be detected by ICPMS such as phosphorus, selenium, and metals (e.g., copper, zinc, and iron) has rather limited application. Sulfur is present in the majority of proteins, but due to its high ionization energy and spectral interferences, the detection limit for S by ICPMS is quite poor.24 Problems exist for Se and P determination as well. The use of highresolution, collision or reaction cell ICPMS instruments can overcome spectral interferences to some degree; however, detection limits in the range of 1−100 μg mL−1 can only be obtained for most proteins using ICPMS sulfur detection.24 Alternatively, chemical derivatization of proteins with compounds containing metals such as lanthanides25−31 of Eu, Lu, and Ce or other elements such as In,32 Hg,33−35 and I,36,37 which have good sensitivity and low detection limits in ICPMS, may be considered. Accordingly, various types of tags have been developed to label peptides and proteins. For the introduction of metallic tags into the proteins, use of bifunctional metal chelating agents including diethylenetriaminepentaacetate (DTPA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and diethylenetriamine-N,N,N′,N″,N″-pentacetic dianhydride (DTPAA), which can also bind to functional groups such as amino or sulphydryl groups in peptides or proteins, have become increasingly popular.38,39 Although many metals may be used for these bifunctional chelating reagents, the most frequently used are the rare earth elements since they respond well in ICPMS. DTPA is an inexpensive and commercially available reagent. DTPA-based tags allow the choice of different metals and can be bound to amino groups for peptide and protein labeling with two-step reactions (i.e., reaction of DTPA with amino groups followed by the introduction of the metal39). DOTA-based tags have a specially designed link and a reactive group for different functionalities in proteins.39 Most commonly applied are the maleimidoethylacetamide (MMA) group for specific thiol labeling as well as isothiocyanates (SCN) for the labeling of amino groups. Once a lanthanide-DOTA tag is produced, it can be used to label peptides or proteins in a one step reaction within 2−12 h depending on the size of the target protein and the complexity of the sample.38−40 Despite the progress in this field, only limited publications25,26,30,31,33−36 have addressed quantitative protein analyses using ICPMS detection using the above approaches. Recently, nonspecies specific isotope dilution coupled with LC−ICPMS has been applied to the direct determination of proteins30,34,35 to improve precision. However, efforts are still needed to develop simple, highthroughput methods for labeling relatively large intact proteins in order to achieve quantitative determination using ICPMS. Recently, a commercially available bis(2,2′-bipyridine)-4′-methyl-4-carboxybipyridine-ruthenium N-succinimidyl ester-bis(hexafluorophosphate) (Ru-NHS ester) has been successfully applied to the labeling of proteins for their quantitation by

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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 id) 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. For comparison studies, sector field (SF)-ICPMS, Thermo Fisher Element2 (Bremen, Germany), equipped with a Scott-type double-pass glass spray chamber and a PFA self-aspirating nebulizer (Elemental Scientific, Omaha, NE) at medium resolution was used in this work. A plug-in quartz torch with a sapphire injector and a Pt guard electrode were used. Optimization of the Element2 was performed as recommended by the manufacturer. The operating parameters of the ICPMS and HPLC are given in Table 1. An Agilent HPLC 1200 series (Agilent Technologies Canada Inc., Mississauga, Ontario, Canada) with a Superdex 200 10/300 GL (GE Healthcare Bio-Sciences Ltd.) size exclusion column with a mass range of 10−600 kDa were used for the separation of proteins. The coupling of the LC to the ICPMS was accomplished by directing the eluent from the column to the nebulizer of the ICPMS through a 0.5 m length of PEEK tubing (0.13 mm i.d., 1.59 mm o.d.). The 99Ru working spike solution of 10 ng mL−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 at 1.0 mL min−1 via a peristaltic pump. An Excellence Plus electronic microbalance (XP205, Mettler Toledo) with readability of 0.01 mg was used to accurately weigh the proteins and eluent during a chromatography run to calculate the mass flow of the 99Ru enriched spike working solution. Reagents and Solutions. Nitric and hydrochloric acids were purified in-house by sub-boiling distillation of reagent grade feedstock in a quartz still prior to use. High-purity deionized water (DIW) was obtained from a NanoPure mixed bed ion exchange system fed with reverse osmosis domestic feedwater (Barnstead/Thermolyne Corp, Iowa). Bis(2,2′-bipyridine)-4′methyl-4-carboxybipyridine-ruthenium N-succinimidyl ester-bis(hexafluorophosphate) (Ru-NHS ester), myoglobin (Myo, SwissProt P68082, 17 kDa, 19 lysine residues), transferrin (Tra, SwissProt P02787, 77 kDa, 58 lysine residues), and thyroglobulin (Thy, SwissProt P01267, 670 kDa, 74 lysine residues) were purchased from Sigma-Aldrich (Ottawa, ON, Canada). The phosphate buffer saline (PBS), dimethylsulfoxide (DMSO), and 2770

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Table 1. HPLC and ICPMS Operating Conditions Agilent HPLC 1200 series Superdex 200 10/300 GL size exclusion column (10 mm i.d. × 300 mm length) 200 mM ammonium acetate buffer (pH 7.2) 100% A for 40 min 0.75 mL min−1 20 μL 0.75 L min−1 Element2

column mobile phase A isocratic elution flow rate injection volume flow rate ELAN6000 rf power plasma Ar gas flow rate auxiliary Ar gas flow rate sampler cone (nickel) skimmer cone (nickel) lens voltage scanning mode points per peak dwell time sweeps per reading readings per replicate number of replicates dead time resolution data acquisition

1400 W 15.0 L min−1 0.75 L min−1 1.00 mm 0.88 mm 12.75 V peak hopping 1 40 ms 1 5000 1 50 ns

1300 W 15.0 L min−1 0.98 L min−1 1.1 mm 0.8 mm extraction, −2000 V; focus, −920 V; x deflection, −2.75 V; y deflection, −0.63 V; shape, 101 V

17 ns medium resultion E-scan, 4000 passes, 5% mass window, 0.0050 s sample time,

A volume of 25 μL of Ru-NHS ester solution was added to each 1 mL of protein solution. Following vortexing, the vials were incubated with shaking in the dark at room temperature for 60 min. A volume of 20 μL of 2 M glycine was added to each vial, and they were incubated at room temperature for 10 min to terminate the reactions. The bioconjugate solutions obtained were injected into the HPLC−ICPMS for analysis. Solutions of three proteins at concentrations ranging of 100− 500 μg mL−1 were used as test solutions for nonspecies specific ID HPLC−ICPMS measurements and labeled as described above. Similarly, three procedural blanks were prepared without adding proteins. It should be noted that the NHS-ester reactive group is susceptible to hydrolysis. The Ru-NHS ester solution should be prepared fresh, and any unused solution should be discarded. Low labeling efficiency was observed with the use of an old Ru-NHS ester solution which had been stored at −20 °C.

glycine were all obtained from Sigma-Aldrich (Ottawa, ON, Canada). Individual stock solutions of Myo, Tra, and Thy at 5000 μg mL−1 were prepared by volumetric dissolution of their solid materials in 0.01 M PBS, pH 7.8. Environmental grade ammonium hydroxide was purchased from Anachemia Science (Montreal, Quebec, Canada). A 1000 μg g−1 natural abundance stock Ru solution was purchased from SCP Science (Baie D’Urfé, Quebec, Canada). Unless otherwise stated, all other reagents used in this study were at least of analytical grade and obtained from Sigma-Aldrich (Ottawa, ON, Canada). A 200 mM ammonium acetate (mobile phase) was prepared by quantitative dissolution of 15.4 g of solid ammonium acetate (Certified, Thermo Fisher Scientific, Ottawa, ON, Canada) in 1 L of DIW. Enriched 99Ru in metal powder form (99.11%) was purchased from Trace Sciences International (Richmond Hill, Ontario, Canada). A 99Ru stock solution of 200 μg g−1 was prepared by dissolution of 99Ru metal powder in sodium peroxide according to a previously reported procedure.42 Briefly, 20 mg of 99Ru metal powder was mixed with 500 mg of sodium peroxide in a zirconium crucible and heated to 650 °C and held for 10 min. Following the addition of 10 mL of 25% HCl, the mixture was heated to 110 °C and held for 30 min. After filtering out residues, the solution was diluted to 100 mL with 10% HCl. The final concentration of 99Ru stock solution was verified by reverse spike isotope dilution using a natural abundance Ru standard. A working 99Ru spike solution of 10 ng mL−1 was prepared by volumetric dilution of the enriched spike in 2% HNO3. Sample Preparation Procedure. The labeling of proteins with Ru-NHS ester was performed in accordance with a previously reported procedure41 with slight modifications. In brief, the myoglobin (17 kDa), transferrin (77 kDa), and thyroglobulin (670 kDa) stock solutions were serial diluted with 0.01 M PBS (azide and nucleopilic reagent free) to 1 mL volume, pH 7.8. Immediately prior to use, a suitable amount of Ru-NHS ester was dissolved in DMSO to prepare a 10 mg mL−1 solution.

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RESULTS AND DISCUSSION

Protein Labeling. As noted earlier, the development a highly sensitive and simple method for the determination of proteins by ICPMS is needed. In this study, a commercially available Ru-NHS ester was used to label three test proteins. The low ionization energy of Ru and the availability of interference free isotopes, in combination with the fact that no Ru exists in proteins and peptides, makes Ru an ideal tagging element for the determination of proteins by ICPMS. Proteins typically have many sites for labeling, including the primary amines in the side chain of lysine residues and the N-terminus of each polypeptide. Because of its easy handling, stable labeling, and high sensitivity, Ru-NHS ester has become one of the most successful commercialized labeling reagents for electrochemiluminescence immunoassay43 since it was first developed in 1991.44 In contrast to the free sulfhydryl group modification used in previous protein absolute quantification ICPMS methods,25,28,30 Ru-NHS ester labels primary amines regardless of peptide classes, which 2771

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Figure 1. Schematic diagram of the labeling and detection of three proteins.

were investigated for the separation of the three proteins Myo, Tra, and Thy. Ammonium acetate buffer instead of the more commonly used sodium acetate buffer was chosen as the HPLC−ICPMS mobile phase in this study to avoid sodium salt deposition on the cones of the ICPMS. The effect of ammonium acetate concentration on the retention of proteins was examined. In this investigation, buffer concentrations ranging from 10 to 300 mM at pH 7.2 were studied. Peak shape improved when buffer concentration increased to 200 mM, and no further improvement was observed at higher concentration. The 200 mM ammonium acetate was chosen for all subsequent experiments. No obvious carbon deposition on the cones of ICPMS was observed when the instrument was run for an 8 h working day at this concentration of mobile phase. The effect of the pH of the ammonium acetate buffer was also investigated. Different pH in a range of 6−8 of 200 mM ammonium acetate solutions adjusted with the use of either ammonium hydroxide solution or acetic acid, were tested in this study. No significant effect was observed on peak shape and peak height under tested conditions. A pH of 7.2 was chosen for all subsequent experiments as having the best buffer capacity of the ammonium acetate solution. The protein peaks were identified by retention time matching. In addition, a SF-ICPMS was also used at medium resolution to separate polyatomic interferences in order to monitor isotopes of S and Fe, naturally occurring elements in proteins such as Tra to further confirm the identity of the labeled proteins. As shown in Figure 2, in addition to Ru signals, S and Fe peaks were evident for Ru labeled Tra. Although both Fe and S isotopes can be monitored with the SF-ICPMS under medium resolution, intensities of Fe and S isotopes were much lower than those of Ru. This observation clearly confirms that use of Ru-NHS ester labeling reagent for the determination of proteins by ICPMS is superior to the use of naturally occurring elements in proteins. Quantitation of Proteins Using External Calibration with HPLC−ICPMS. As noted earlier, since no significant amount of Pd was found during testing of protein solutions, there was no need for correction for isobaric interference from 102Pd on 102 Ru. In order to validate the developed method, solutions of

theoretically makes this approach capable of quantifying all proteins. The schematic diagram of the protein labeling using Ru-NHS ester and ICPMS detection is illustrated in Figure 1. The procedure is simple and consists of only a single step reaction. Buffer pH has a significant effect on labeling efficiency. As suggested by the manufacturer, in pH 7−9 buffers, NHS ester reacts efficiently with primary amino groups (−NH2) by nucleophilic attack, forming an amide bond and releasing the NHS group. Our preliminary results confirmed high derivatization efficiencies at buffer pH 7.4−8.4 in accordance to manufacturer’s suggestion. Derivatization efficiency decreased at higher or lower pH. Thus, pH 7.8 was chosen for all subsequent experiments. The influence of the amount of Ru-NHS ester used in labeling of proteins was investigated in order to achieve optimal labeling. Varying amounts (0, 1, 2.5, 5, 10, 25, and 50 μL) of Ru-NHS ester (10 mg mL−1 in DMSO) were added to 1 mg mL−1 aliquots of transferrin and labeled as described in the Experimental Section above. 102Pd is a potential interference for 102Ru determination; however, no significant amount of Pd was found in the protein solutions tested, thus the abundance of 102Ru was monitored for maximum sensitivity for ICPMS measurements. The solutions were injected into the HPLC− ICPMS, and peak areas of the signal associated with 102Ru in labeled transferrin increased as the volume of Ru-NHS ester increased from 0 to 25 μL. There was no increase in peak areas when the volume of Ru-NHS ester used was greater than 25 μL. The same limit was also observed for 1 mg mL−1 myoglobin and 1 mg mL−1 thyroglobulin, respectively. Thus 25 μL of 10 mg mL−1 (250 μg) Ru-NHS ester was used for the subsequent study of protein labeling and quantification. Clearly with the use of an excess amount of Ru-NHS ester labeling reagent, constant stoichiometric ratios can be reached for the quantitative determination of proteins. Separation of Proteins by Size Exclusion Chromatography. Size exclusion chromatography (SEC) is an established method for separation of the proteins studied.20 Buffer ionic strength and pH, which could affect the retention of proteins, 2772

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must be understood that this does not correct/compensate for any issues associated with the sample preparation and chromatographic separation. This calibration approach also assumes identical behavior in the ICP for the infused Ru spike and protein bound Ru tag. The concentration of the enriched 99Ru stock solution was determined by reverse isotope dilution with a natural abundance Ru standard.46 A concentration of 194 ± 4 μg g−1 (1SD, n = 3) was obtained for 99Ru enriched stock solution. A concentration of 10 ng mL−1 99Ru working spike solution was prepared volumetrically from the above stock solution and used for the postcolumn isotope dilution determination of proteins. In order to perform postcolumn nonspecies specific ID for the quantitation of proteins, the 10 ng mL−1 99Ru 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 4a, both 102Ru and 99Ru signals were monitored in

Figure 2. Segments of chromatograms of a 100 μg mL−1 transferrin sample by (a) qICPMS and (b) SF-ICPMS.

three proteins (Thy, Tra, and Myo) were prepared at various concentration levels ranging from 10 to 1000 μg mL−1 and labeled with Ru-NHS ester using the optimum experimental conditions. As shown in Figure 3, signal increased proportion-

Figure 3. HPLC−ICPMS chromatograms obtained for Thy, Tra, and Myo standard solutions. (The molar ratio for Thy, Tra, and Myo is 1:8.7:39).

ally as concentrations of proteins increased. The double peaks observed for Thy might be due to the presence of Thy aggregates, as was observed in a previous study.45 Only the second largest peak was used and integrated for peak area to obtain a calibration curve and quantitation of Thy in test solutions. Indeed, external calibration curves for Thy, Tra, and Myo all show good linear responses in the ranges tested, 10−1000 μg mL−1. Correlation coefficients of 0.9901, 0.9921, and 0.9980 were obtained for Thy, Tra, and Myo, respectively. On the basis of the calibration curves, concentrations of 102 ± 6, 98.9 ± 5.1, and 101 ± 5 μg mL−1 (1SD, n = 3) for Thy, Tra, and Myo, respectively, were obtained for a test mixture, which is in good agreement with their volumetric value of 100 μg mL−1 each. The detection limits (LOD) for the external calibration HPLC−ICPMS technique were evaluated using three procedural blanks. Values of 0.050 μg mL−1 (0.075 pmol mL−1, 1.5 fmol), 0.013 μg mL−1 (0.17 pmol mL−1, 3.4 fmol), and 0.007 μg mL−1 (0.42 pmol mL−1, 8.4 fmol) were estimated for Thy, Tra, and Myo, respectively, based on 3 times the standard deviation of the measured concentrations in procedural blanks. The difference in detection limits for three proteins may be due to the number of Ru tags in each protein. Quantitation of Proteins Using Nonspecies Specific Isotope Dilution with HPLC−ICPMS. The postcolumn infusion nonspecies specific ID approach could simplify calibration and eliminate issues (suppressions, etc.) associated with the detection portion of the measurement system. However, it

Figure 4. Nonspecies specific HPLC−ICPMS chromatograms of 200 μg mL−1 Thy, Tra, and Myo: (a) Ru signal intensity and (b) Ru mass flow.

order to obtain the 102Ru/99Ru ratio to calculate the final concentrations of proteins in the test solutions. Equation 1 was used for ID in accordance to previous studies.47,48 MFx(t ) = MFy(t )

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

(1)

where MFx(t) is the sample mass flow at time t; MFy(t) is the 99 Ru enriched spike mass flow at time t; 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 102Ru/99Ru divided by the measured ratio of a 5 ng mL−1 natural abundance Ru standard introduced via the three way connector. The integration of the peak area of calculated sample mass flow MFx(t) gives the mass of Ru in an eluting protein. Concentration of the protein can be derived from the stoichiometric ratio of Ru to that protein and the injection 2773

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Table 2. Quantitative Analysis of Proteins by Ru labeling and ID-ICPMS, μg mL−1 (1SD, n = 3)

expected volumetric values, μg mL−1

sample

thyroglobulin

transferrin

myoglobin

thyroglobulin

transferrin

myoglobin

1 2 3

102 ± 5 191 ± 8 499 ± 10

211 ± 10 487 ± 18 114 ± 5

523 ± 18 106 ± 5 209 ± 10

104 197 486

207 500 109

504 109 212

concentration. Method LODs using ID, evaluated from procedural blanks, were calculated to be 0.054 μg mL−1 (0.081 pmol mL−1, 1.6 fmol), 0.012 μg mL−1 (0.16 pmol mL−1, 3.2 fmol), and 0.006 μg mL−1 (0.35 pmol mL−1, 7.0 fmol) for Thy, Tra, and Myo, respectively. These LODs are comparable or superior to detection limits reported in the literature using nonspecies specific ID with element tag labeling25,30,31 and naturally occurring S tags.20,24

volume. Since it is a postcolumn ID, MFy(t) can be easily calculated by using the consumed mass of 99Ru enriched working spike solution divided by its density, then multiplied by its concentration and divided by the run time of the chromatogram, as was reported recently.48 The consumed mass was obtained by the difference in weights of the 99Ru enriched spike solution at the beginning and at the end of a chromatographic run. With the calculated MFy(t), the Ru mass flow chromatogram for the three labeled proteins was thus constructed, as shown in Figure 4b. Molecular MS can be a powerful technique for the study of stoichiometric ratios of bioconjugates. Initial studies with the use of ESI-MS to obtain a stoichiometric ratio of Ru to protein (bovine serum albumin, for an example) were attempted. However, proteins are complex biomacromolecules with complex spatial structures, and only part of the surface amino groups could be available for the NHS ester tagging. As shown in Figure S1a,b in the Supporting Information, the tagging was incomplete and varied from protein to protein. The labeled proteins displayed massive peaks and made it impossible to obtain exact stoichiometric ratios from the ESI-MS spectrum. To avert this difficulty, the stoichiometric ratio of Ru to a particular protein was thus calculated by using eq 2 based on an injection of labeled 200 μg mL−1 protein standard solution into the HPLC−ICPMS:

SR =

nRu n protein

=

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CONCLUSIONS An accurate, simple, and sensitive method for the direct determination of Thy, Tra, and Myo proteins using nonspecies specific ID and external calibration HPLC−ICPMS is described. Concentrations of three proteins measured in three test solutions using the 99Ru enriched spike for nonspecies specific ID or by external calibration are in a good agreement with their volumetric values. This is the first application incorporating a commercially available Ru-NHS ester reagent with a simple one step reaction for quantitative labeling of proteins and using a 99 Ru enriched spike for nonspecies specific ID for the direct determination of intact proteins by HPLC−ICPMS. Sufficiently low detection limits (1.6−7.0 fmol) for proteins for the direct and absolute quantitation of proteins at trace level were obtained. It is noteworthy that SEC has relatively low separation power. In this proof of concept study, proteins which have large molecular weight differences were efficiently separated. However, for complex sample matrix, more powerful protein separation techniques, such as reverse phase HPLC and capillary electrophoresis may be needed.

mRu MRu

C proteinVinjection

(2)

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where SR is the stoichiometric ratio of Ru to protein in the labeled product, mRu is the measured total mass of Ru corresponding to the labeled protein peak in a run obtained by the nonspecies specific ID HPLC−ICPMS, MRu is the atomic weight of natural abundance Ru, Cprotein is the concentration of protein volumetrically prepared, and Vinjection is the injection volume (20 μL) for the chromatographic run. Values of 14, 4.2, and 1.7 were obtained for stoichiometric ratios of Ru to Thy, Tra, and Myo, respectively, comparable to early results.41 The obtained stoichiometric ratios indicate incomplete labeling of three proteins. However, it was found that the stoichiometric ratios of the Ru tag to each protein were maintained as constant at various protein concentrations under chosen experimental conditions. This observation suggests that quantitation of proteins can still be obtained by using these stoichiometric ratios. To validate the proposed procedure, three quantitatively prepared mixtures of Thy, Tra,0 and Myo were labeled and subjected to HPLC−ICPMS determination via 99Ru spike for nonspecies specific ID using eq 1. Results are summarized in Table 2, and concentrations obtained for Thy, Tra, and Myo using nonspecies specific ID in all three test solutions are in good agreement with their volumetric values. The average accuracy of 100 ± 3% (1SD, n = 9) was obtained based on nine measured concentrations. Precision in the range of 2.0−4.9% was generally obtained with the ID calibration depending on

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. The authors thank M. McCooeye for editing this manuscript.

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REFERENCES

(1) Wilkins, M. R.; Pasquali, C.; Appel, R. D.; Ou, K.; Golaz, O.; Sanchez, J. C.; Yan, J. X.; Gooley, A. A.; Hughes, G.; HumpherySmith, I.; Williams, K. L.; Hochstrasser, D. F. Nat. Biotechnol. 1996, 14, 61−65.

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Analytical Chemistry

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dx.doi.org/10.1021/ac203141d | Anal. Chem. 2012, 84, 2769−2775