Immuno–MS Based Targeted Proteomics: Highly Specific, Sensitive

Article pubs.acs.org/ac

Immuno−MS Based Targeted Proteomics: Highly Specific, Sensitive, and Reproducible Human Chorionic Gonadotropin Determination for Clinical Diagnostics and Doping Analysis Hanne Lund,† Karoline Løvsletten,† Elisabeth Paus,‡ Trine Grønhaug Halvorsen,† and Léon Reubsaet*,† †

Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo, Oslo, Norway Central Laboratory, Radiumhospitalet, Rikshospitalet, Oslo University Hospital, Oslo, Norway

‡

S Supporting Information *

ABSTRACT: The human chorionic gonadotropin (hCG) proteins constitute a diverse group of molecules that displays biomarker value in pregnancy detection and cancer diagnostics, as well as in doping analysis. For the quantification of hCGβ and qualitative differentiation between other hCG variants in a selective, sensitive, and reproducible manner, the targeted proteomics approach based on mass spectrometric (MS) selected reaction monitoring (SRM) detection was exploited. By optimizing immunoaffinity extraction using monoclonal antibodies coated to magnetic beads, access was granted for the MS to the low-abundance target proteins, ensuring proper sensitivity with limits of detection (LODs) of 2 and 5 IU/L, respectively, for urine and serum samples. Validation according to key elements and recommendations defined by the European Medicines Agency in Guideline on Validation of Bioanalytical Methods was performed. For both matrixes this demonstrated good within-day precision results (within 20% for the lowest concentration, and within 15% for the medium and high concentration), good accuracy results (within 15% for all concentrations), and proper linearity, >0.997 for serum and of 0.999 for urine, in the concentration range up to 5000 IU/L. The method’s application in clinical diagnostics was tested on samples from a pregnant woman and from patients previously diagnosed with testicular cancer. For doping analysis, samples from one man having received injection of the hCG-containing pharmaceutical Pregnyl were analyzed. The method proved to be quantitatively accurate with indisputable identification specificity, reducing risks of false positive and false negative results. The successfully validated method advocates thus for more extended use of MS in routine analysis.

I

has shown potential as a sample preparation strategy to grant access for the mass spectrometers to the low-abundance plasma proteins.6,8 This must be further explored in order to implement the potential of MS-based detection of proteins into use in a clinical setting. A much used and well-established protein biomarker is human chorionic gonadotropin (hCG). hCG is a placental hormone which is mainly produced during the full course of pregnancy, thus being the biomarker used for pregnancy detection.9,10 Several disease-related conditions also produce hCG, such as gestational trophoblastic diseases10−12 (disorders of pregnancy) and nontrophoblastic neoplasms11,13 (i.e., germ cell tumors). Together with Down syndrome screening,14 this constitutes the major clinical use of hCG as a biomarker. Additionally, hCG is included in the World Anti-Doping Agency’s (WADA) list of prohibited substances for which male athletes are tested while in competition. This misuse of hCG is related to the pharmacological effect of hCG following injection of hCG pharmaceuticals; the hCG hormone antagonizes the down-regulation of the body’s own testosterone production

n recent years, mass spectrometry (MS) has become the most powerful tool in clinical proteomics1 and is increasingly being used for the quantification of metabolites in clinical diagnostics.2 This has resulted in a growing interest for exploring the potential of MS to make the transition from a discovery tool to a clinical diagnostic tool,1,3,4 and in 2009 targeted proteomics analysis by MS was selected as “Method to Watch” by Nature Methods.5 The strategy of MS-based targeted proteomics often involves the enzymatic conversion of proteins into their constituent peptides. This way the detection of a unique structural component of the target protein, referred to as a signature peptide, can replace the far more complicated MS detection of intact proteins.6 By using a triple-quadrupole detector, MS detection in the selected reaction monitoring (SRM) mode can be performed, enabling near-absolute structural specificity, high sensitivity, and reproducibility. This principle has been demonstrated by several research groups.3,7 However, not many of these methods have been validated according to existing guidelines, although validation is the foundation for implementation of novel methods into routine analysis. This is likely due to challenges related to the complexity of the vast proteome and other interfering components of the matrixes, in addition to the variability linked to the conversion of proteins to peptides by tryptic digestion. Selective immunoextraction prior to MS detection © 2012 American Chemical Society

Received: June 18, 2012 Accepted: August 15, 2012 Published: August 15, 2012 7926

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following the misuse of anabolic steroids.15 Today, detection of hCG is based on immunometric methods for both clinical use and doping analysis. However, hCG detection is strongly associated with the problems of interassay variation inevitably linked to the immunoassays and is thus an interesting candidate for detection by MS-based targeted proteomics. Another reason for the use of MS is the fact that it allows determination of altered forms of hCG. Although referred to as one, hCG is not a single molecule. It is a heterogeneous molecule that can be diverted into variants having different cell origins in the placenta and different biological effects. Once released from the cell, biological processes occur that further complicates the structure differences of the hCG variants. Depending on the biological process in progress, certain variants of the hCG molecule might be produced to a larger extent than the other variants.10,16,17 The specific differentiation between these hCG variants that together constitutes the immunoassay-based “total” hCG response can, therefore, provide additional diagnostic information to the clinician. The regular or intact hCG variant is a highly glycosylated protein (37.5 kDa) which constitutes two subunits that are noncovalently linked: the hCGα subunit (92 amino acids, 14 kDa) and the hCGβ subunit (145 amino acids, 23.5 kDa). The hCGα subunit is identical to that of the other pituitary glycoprotein hormones (the luteinizing hormone, follicle-stimulating hormone, and thyroid-stimulating hormone), whereas the hCGβ subunit is unique to hCG.18 However, differences in the structure of the hCGβ subunit of the hCG molecule might occur, resulting in variants of the hCG molecule. In short, this variation can, i.e., be the dissociation of the heterodimers into free subunits, or it can manifest as nicked variants, degradation variants, and hyperglycosylated variants.16,19,20 For more detailed information on hCG structure variation reference is made to the expert review on the matter by Cole.16 These variants are all regarded as hCG molecules. Proper MS detection is founded on the establishment of adequate signature peptides representative of all these hCG molecules.6,21 In this work a tailor-made and adequately validated targeted MS-based method for immunoextraction, detection, and quantification of the hCG molecule including the differentiation between the hCG molecule and its structural variants, all in one single run, will be presented. Its applicability on realistic samples will be demonstrated. To our knowledge, this is the first described method on targeted MS-based quantitative determination of the hCG molecules at very low concentrations, applicable for use in clinical diagnostics and doping analysis.

of ion-exchanged water and transferred to Protein LoBind Eppendorf tubes from Eppendorf AG (Hamburg, Germany). Working solutions were made by diluting the stock solution to a concentration of 50 IU/mL by ion-exchanged water and were further aliquoted to adequate volumes and frozen to −32 °C. Working solutions originating from the same stock solution were used throughout the entire validation due to the possible variation in different batches of the Pregnyl ampules. Working solutions were thawed and used to spike both serum and urine to desired concentrations. Trypsin Solution for Tryptic Digestion. All trypsin solutions were freshly made within 1 h prior to use. A work solution of 1 mg/mL was made and further diluted to desired concentration. Internal Standard and Quantitative Analysis. The internal standard AQUA peptide is a synthetic analogue of the signature peptide βT5 of the unmodified hCG specific βsubunit (which can originate from intact hCG and the free hCGβ subunit) and is referred to as IS or isβT5 (Supporting Information Figure S-1). This peptide contains an isotopically labeled arginine residue at the carboxy-terminal of the peptide, which generates a mass shift of +10 Da. This allows relative comparison of chromatograms from different analyses through the normalization of the generated signal response influenced by the intra- and interday variability produced by the MS. This IS does, however, not correct any variability due to immunocapture or tryptic digestion since it is added to the sample straight before liquid chromatography−tandem mass spectrometry (LC−MS/MS) analysis. For detailed information on preparation of internal standard solutions, this can be found in the Supporting Information. Quantitative analysis of the hCGβ subunit (present in both intact hCG and as free hCGβ subunit) is achieved by dividing the signal intensity of the βT5 signature peptide by that of the internal standard. This value was plotted against hCG concentrations (expressed in IU) from standards prepared using Pregnyl. The other hCG isoforms were detected (identified) if present, but no quantitative measurements were assigned. Realistic Samples. Serum samples from cancer patients were obtained from the Central Laboratory, Norwegian Radium Hospital, Oslo University Hospital (Oslo, Norway). Serum and urine samples from a pregnant woman were voluntarily donated to the project. Serum and urine samples from one subject having received one single injection of the hCG-containing pharmaceutical Pregnyl were voluntarily donated to the project. All samples were kept in the freezer at −32 °C until use. Immunocapture Sample Cleanup. Antibody Specificity. The monoclonal antibody (mAb) E27 was chosen based on its selectivity characteristics, which were demonstrated and established using the first WHO reference reagents for hCG and five related molecules, as presented in the work by Berger et al.19 This work shows that E27 is selective for the extraction of the following hCG variants: intact hCG heterodimer, free hCGβ subunit, nicked hCG heterodimer (47/48), nicked free hCGβ subunit (47/48), nicked hCG heterodimer (44/45), nicked free hCGβ subunit (44/45), and hCGβ core fragment.19 In the annotated reference the mAb E27 is referred to as mAb 271. Coupling of Antibodies to Magnetizable Particle. Coupling was performed essentially as previously described,22 using 20 mg of antibody to 1 g of Dynabeads (Dynabeads M280 tosylactivated, Life Technology, Invitrogen Dynal, Oslo Nor-

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EXPERIMENTAL SECTION Chemicals. The pharmaceutical formulation Pregnyl was distributed by Organon (Oss, The Netherlands). Anti-hCG (monoclonal antibody E27) was supplied by the Central Laboratory, Norwegian Radium Hospital, Oslo University Hospital (Oslo, Norway). The internal standard AQUA (absolute quantification) peptide, trypsin (TPCK treated, from bovine pancreas), 1,4-dithiothretiol (DTT), iodoacetic acid (IAA), and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). All other chemicals used were of analytical grade. Human serum from healthy subjects was obtained from Oslo University Hospital, Ullevaal (Oslo, Norway). Solutions. hCG Stock Solution and Working Solutions. One ampule of Pregnyl (5000 IU hCG) was dissolved in 1 mL 7927

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Table 1. Table Summarizing the Final MS Parameters for the SRM-Based Detection of the Signature Peptides Representative of the Various hCG Proteins signature peptide

precursor ion m/z value

fragment ion m/z valuea

fragment ion designation

retention timeb

scan segments

segment durationb

segment CID energyc

scan events per segmentd

hCGα subunite nicked hCGβ subunit 47/48

αT2 nβT5

409.3 765.7

1 2

0.0−12.0 12.1−14.4

20 30

2 3

nβT5′

914.7

14.78

3

14.5−15.5

30

6

hCGβ subunit

βT5

964.2

15.00

3

14.5−15.5

30

6

internal standard β subunit

isβT5

969.3

15.01

3

14.5−15.5

30

6

hCGβ core fragment

cfβT9

955.7

y5 y8 y11+2 y8+2 y8 y11 y8 y11 y8 y11 b8 y6

11.32 14.19

nicked hCGβ subunit 44/45

583.4 1036.3 659.2 519.1 1036.3 1317.8 1036.3 1317.8 1046.3 1327.8 924.1 740.0

15.83

4

15.6−17.0

30

2

hCG variant

a

The respective signature peptides can have between one and three transitions from the same parent ion incorporated in the SRM detection program. These transitions generate one peak which is the sum of the signal intensity of the respective fragment ions. bIn minutes. cIn percent. dIn q3. eThe internal standard α-subunit is included in the signature peptide list to have a complete set of SRM transitions for all hCG-related variants.

way). This resulted in approximately 15 μg of immobilized antibody/mg of beads (magnetizable particles). For improved orientation the antibody was exposed to acid conditions by adding hydrochloric acid (HCl) to pH 2.5 prior to incubation for 1 h at 0 °C, before coupling at pH 9.5. The solution was diluted to 10 mg of beads/mL using phosphate buffer saline (pH 7.4) (PBS). Immunoaffinity Extraction of hCG Using Beads. The immunoaffinity extraction procedure is summarized in Figure S-2 in the Supporting Information and was carried out as follows: (1) 500 μL of PBS containing 0.005% Tween 20 was added to the empty Eppendorf tubes; (2) 20 μL of beads coated with mAb E27 was added; (3) the tubes were shaken on the vortex mixer, quickly centrifuged, placed in the magnetic rack (DynaMag-2 from Invitrogen, Carlsbad, CA, U.S.A.), and the solution was removed; (4) 1 mL of sample (serum or urine) was added, and the tubes were placed in the HulaMixer (Invitrogen) for 1 h (to facilitate antigen−antibody interaction); (5) the tubes were placed in the magnetic rack to collect the beads. (6) The following wash solutions were added (one at a time), and for every wash solution added the tubes were shaken on the vortex mixer, quickly centrifuged, placed in the magnetic rack: (i) 500 μL of PBS containing 0.05% Tween 20; (ii) 500 μL of PBS; (iii) 500 μL of 10 mM Tris−HCl (pH 7.4). Tryptic Digestion Procedure. A volume of 200 μL of 50 mM freshly prepared ammonium bicarbonate (ABC) buffer was added to the tubes containing the beads before adding 6 μL of freshly prepared 100 mM DTT. The samples were shaken, placed in the rack of the thermomixer, and subjected to vibration at 95 °C for 20 min. The samples were then set to cool to room temperature. Afterward a volume of 12 μL of freshly prepared 800 mM IAA was added, and the solutions were placed in the dark for 15 min. Tryptic digestion on captured hCG from serum samples was carried out by adding 500 ng of freshly prepared trypsin in solution (50 μL of 10 μg/ mL), whereas for the urine samples 100 ng of trypsin was added (10 μL of 10 μL/mL). All samples were placed in the thermomixer and shaken lightly for 18 h at 37 °C. Finally, 200 μL of the sample was subjected to solid-phase extraction (SPE). Detailed information on the production of homemade SPE tips can be found in the Supporting Information.

Solid-Phase Extraction. The tips were activated using 100 μL of 100% MeCN and washed with 100 μL of 20 mM formic acid prior to use. The amount of sample applied was 200 μL. After a washing step (100 μL of 20 mM formic acid) the peptides were eluted using 60 μL of 80% MeCN in 0.1% TFA. The eluate was evaporated to dryness under a stream of N2 gas at 60 °C, reconstituted in 40 μL of 20 mM formic acid and 10 μL IS (50 pmol/mL), and analyzed by LC−MS/MS. Liquid Chromatography−Mass Spectrometry System. LC−MS Triple Quadrupole. The Dionex UltiMate 3000 chromatographic system consisted of an autosampler, two pumps, and a flow manager, all Dionex (Sunnyvale, CA, U.S.A.). The processing was managed through Chromeleon software, version 6.80 SR6. The triple-quadrupole MS detector (TSQ) was a Thermo Scientific TSQ Quantum Access detector. Data acquisition and processing were carried out using Xcalibur software, version 2.0.7 SP1. Chromatography. Chromatographic separation was carried out on a Biobasic-C8 column from Thermo Scientific (Rockford, IL, U.S.A.) with average pore size of 300 Å, particle diameter of 5 μm, and column dimensions of 50 mm × 1 mm i.d. The mobile phases consisted of A, 20 mM formic acid and MeCN (95:5, v/v), and B, 20 mM formic acid and MeCN (5:95, v/v). A linear gradient was run from 0% to 40% mobile phase B in 8 min, followed by a linear gradient from 40% to 46% mobile phase B the next 4 min. The elution strength was further increased to 95% mobile phase B within 0.1 min, kept constant for 2 min, and returned to starting conditions within 1 min. The column was regenerated for at least 10 column volumes. Flow rate was set to 50 μL/min; the injection volume was 40 μL. Mass Spectrometric Detection. The electrospray ionization (ESI) source was operated in the positive ionization mode, with spray voltage of 4000 V, sheath gas (nitrogen) pressure of 10 units, drying gas (nitrogen) between 5 and 10 units, and aux gas pressure of 0. The capillary temperature was 270 °C. Experiments were performed in the SRM mode, using predefined specific m/z values for hCG signature peptides. The signal response is generated based on the sum of all monitored fragment ions of the respective signature peptides. These values are shown where appropriate. The collision gas (argon) pressure in Q2 was 1.7 mTorr. Scan time was set to 0.5 7928

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Figure 1. (A) Signals from the selected fragment ions of the signature peptide βT5 of the hCGβ subunit and the corresponding isotopically labeled internal standard, isβT5. The amino acid sequence for both peptides is shown, with the y-fragment cleavage sites emphasized. The isotopically labeled arginine residue at the end of the sequence of the IS is marked in red. (B) Chromatogram showing the peaks of the different hCG signature peptides and their respective retention times. The signal intensity is normalized, and the corresponding hCG variants are given for each signature peptide.

s/scan. Table 1 summarizes the four different scan segments used in the validated SRM method: the time range of each scan segment, the signature peptides which are detected in the current scan segment, and the respective collision-induced fragmentation (CID) energy. Method Validation. The method was validated according to key elements and recommendations defined by EMEA (European Medicines Agency), Guideline on Validation of Bioanalytical Methods.23 The validation results are related to signal responses from the signature peptide of the unmodified hCGβ subunit (both intact hCG as well as the free hCGβ subunit). The intact hCG molecule (and to a small extent the free hCGβ subunit) relates to the biological effect represented by the international units concentration of the pharmaceutical used as hCG source (Pregnyl). The other hCG variants also present in Pregnyl (and thereby in all validation samples) do not contribute to this activity and, thus, do not contribute to the concentration determination. Recovery. The determination of the recovery (R) was based on the signal response (S) generated by the extraction of a given concentration of hCG spiked to the current matrix, divided by the signal response generated by an unextracted sample representing the 100% recovery. The sample representing the 100% recovery was itself a sample of protein hCG that had to be trypsinated into peptides prior to dilution to desired concentration.

R=

S hCG(extracted and trypsinated) × 100% S hCG(unextracted and trypsinated)

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RESULTS AND DISCUSSION Our previous work demonstrated the immuno−MS principle through immunoaffinity extraction of the target hCG molecules by mAbs immobilized to the walls of 96 wells format microtiter plates, followed by tryptic digestion, LC separation, and MS detection of the unique hCG signature peptides. The detection was performed by a single-quadrupole detector in the single ion monitoring (SIM) mode.6 Although suitable for demonstration of principle, this method managed a limit of detection (LOD) of 100 IU/L, was not adequately reproducible, and could with this not meet the requirements for sensitivity. On the basis of this, a magnetic beads based immuno−LC−MS/MS method was developed on a triple-quadrupole detector in order to extend the sensitivity and increase the specificity of the detection. hCG Identification by Tailor-Made hCG SRM Design. In the Supporting Information Figure S-1 shows the amino acid sequence of the signature peptides and the internal standard (IS). The IS is the synthetic peptide analogue to the signature peptide βT5, with an isotopically labeled arginine residue at the carboxy-terminal at the end of the peptide (referred to as 7929

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Table 2. Table Summarizing the Validation Parameters Obtained for Both Serum and Urine Samples validation parameter

linearity (r2) n=5

LOD (IU/L) n=5

LLOQ (IU/L) n=5

serum

>0.997

5

10

urine

0.999

2

5

validation parameter matrix 10 IU/L 100 IU/L 1000 IU/L

within-day precision (%) n = 5 serum 19 17 4

urine 9 12 10

selectivity (matrix) n = 3 no interfering signal from blank matrix no interfering signal from blank matrix between-day precision (%) n=5 serum 13 11 7

urine 14 30 19

isβT5). As shown in Figure 1A, this generates a mass shift of +10 Da for the IS. In order to create an MS-based detection that ensured proper strength of the analytical evidence, two or more diagnostic fragment ions were included in the SRM transitions for each signature peptide. These fragment ions had to generate a constant ratio in every analysis. This fragment ion ratio is demonstrated in the mass spectra in Figure 1A, where the ratio of the y8 and y11 fragment ion of the signature peptide βT5 and the corresponding isotopically labeled synthetic analogue (the IS) is shown. The abundance ratio y8/y11 of the fragment ions is 0.47 (RSD of 4%, n = 20), and this ratio shall always be present whenever these peptides are identified. Finally, by including the dimension of retention time, absolute identification of the target hCG proteins could be made based upon the tailor-made SRM design. This optimization of the selectivity and sensitivity resulted in the MS parameters and selected precursor and fragment ions as summarized in Table 1. The resulting chromatogram is presented in Figure 1B. This chromatogram visualizes the detection and differentiation between seven diagnostic peptides in one single run. The single-quadrupole detector used in the previous work managed an LOD of 100 IU/L.6 When combined with the earlier described sample cleanup using the 96 wells plate format for immunoextraction, the selective and specific nature of the triple-quadrupole detector enabled detection of hCG proteins down to 20 IU/L (data not shown). Although improved, further extension of the LODs and lower limits of quantitation (LLOQs) was necessary since the reference range for serum and urine hCG concentrations in men and nonpregnant woman is as low as 0.7−5.4 IU/L.10 MS-based assays can potentially be extended into this concentration range if these low-abundant hCG proteins can be enriched prior to LC−MS/ MS analysis. For this reason, the sample preparation strategy based on immunoaffinity extraction had to be further explored. Reduction of Proteome Complexity: Immunocapture of Target Proteins by Antibodies Immobilized on Magnetic Beads. The attempts to validate the method based on mAbs immobilized in microtiter plates lead to inadequate LODs, LLOQs, intra- and interday precision, accuracy, and linearity (data not shown). We therefore hypothesized that the use of antibodies coated to magnetic beads instead of wells, through the extraction of target substances from a larger volume, would enable the necessary LODs and LLOQs. Different sample volumes were tested, 0.5, 1, and 2 mL, with desired sensitivity accomplished at the 1 mL sample volume. When it comes to the amount of beads used, 20 μL of beads solution (10 mg of beads/mL) gave linearity for the concentration range up to 5000 IU/L. For samples

selectivity (internal standard) n=3 no interfering signal from internal standard no interfering signal from internal standard accuracy (%) n = 5 serum 95 87 99

urine 104 98 100

matrix effects no signal suppression/ enhancement n = 3 no signal suppression/ enhancement n = 6 recovery (%) n = 3 serum 39 43 32

urine 65 57 57

containing hCG in concentrations above this, dilution will be necessary prior to analysis. When combined with the triple-quadrupole detector, the LOD in serum samples has been extended from 20 IU/L (using wells) to 5 IU/L using mAbs coated to beads, and from 10 IU/ L (using wells) to 2 IU/L (using beads) for urine samples. Furthermore, the linear concentration range has been increased from 2000 IU/L (using single-quadrupole detector and wells)6 up to 5000 IU/L (using triple-quadrupole detector and beads). The capacity of the mAbs can, if desirable, be further increased by using a higher amount of beads for extraction of the sample. Method Validation According to EMEA Guidelines. The validation parameters and results are summarized in Table 2. Linearity, Sensitivity, and Concentration Range. First, the concentration range was tested with matrix spiked to at least five different concentrations for both serum and urine. We observed that the linearity was >0.997 for serum in the concentration range from 10 to 5000 IU/L (for seven different concentrations) and 0.999 for urine in the same concentration range (for five different concentrations). Furthermore, the LODs for serum and urine were estimated from a signal-tonoise (S/N) ratio of 3:1 (n = 5). For serum the LOD was 5 IU/ L, and for urine 2 IU/L. The experimental LLOQs were defined as the concentration at which variation of less than 20% was obtained. For serum this was 10 IU/L, and for urine 5 IU/ L. This harmonizes quite well with the reference concentration area for serum and urine hCG in men and nonpregnant women.10 Precision, Accuracy, and Recovery. The precision and accuracy (n = 5) and recovery (n = 3) were tested at hCG levels at 10, 100, and 1000 IU/L. The intraday precision attested low within-batch variations for both matrixes, with relative standard deviation (RSD) of less than 20% for the lowest concentration. According to the recommendations of the EMEA the interday precision demonstrating the between-batch variation was acceptable for the serum samples, but not for the urine samples, where the medium- and high-concentration samples did not lie within 15% RSD. This can, however, be circumvented by making a standard curve for quantification purposes on a daily basis. Furthermore, the accuracy results were well within the 15% RSD suggested by the EMEA guidelines, for all concentrations of both the serum and urine samples. Finally, the recovery of the method was tested, and although not very high (approximately 40% for serum and 60% for urine), these recoveries were able to generate the desired LODs and LLOQs. By adding the IS straight before the LC− MS/MS analysis, the only variation it is able to correct for is the variability generated by the LC−MS/MS. However, the 7930

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imately 200 and 20 000 IU/L. The level of hCG resulting from the injection of a single dose of Pregnyl containing 5000 IU hCG was determined to be 139 IU/L, 1 day after injection. The hCG level in the urine sample was slightly higher than in the serum sample. This is likely due to the enrichment of hCG that occurs when collecting morning urine. In addition to the hCGα and β-subunits, the two nicked variants (hCGβ n44/45 and hCGβ n47/48) and the urine metabolite (hCGβ core fragment) were detected in the urine samples but not in the serum samples. According to the literature the degradation variants are mostly observed in urine since they are rapidly removed from serum due to their lack of biological activity. This rate of removal through the kidneys is also dependent on molecular size and degradation taking place in the kidneys.10 For this targeted MS-based method to make the transition from clinical prospect to bedside reality, a thorough validation of the method was required. For proteins in complex matrixes this is not straightforward when the sample preparation procedure involves the conversion of proteins into their constituent peptides. Tryptic digestion has proven to be rather difficult to standardize in a reproducible manner and is as such one of the bottlenecks in both top-down and bottom-up proteomics. Due to these issues not many, if any, targeted MS-based methods for protein determination have been successfully fully validated. However, the precision results shown in Table 2 indicate that the enzymatic cleaving of proteins into peptides can in fact be standardized to meet the criteria of bioanalytical method validation. This is further supported by the accuracy results. The multiplexing and differentiating power of the mass spectrometry was vastly demonstrated when both the intact hCG molecule and corresponding hCG metabolites were detected. The qualitative determination of the various hCG isoforms comes complementary to the quantification of the hCGβ subunit. If produced by certain hCG-related cancer conditions and other hCG-related diseases, the MS-based detection of different hCG isoforms might make a contribution to other diagnostic tools. Additionally, the absolute protein identification based on SRM detection reduces the risk of false positive responses, and the sample preparation strategy based on a selective antibody capable of recognizing and isolating all known hCG variants reduces the risk of false negative responses. All in all this advocates more extended use of MS in routine analysis. In doping analysis, absolute identification by mass spectrometry is the indisputable analytical evidence that illegal administration has taken place. State-of-the-art technology is expected in the analytical methods used as to ensure no false positive or false negative responses. hCG is included in WADA’s list over prohibited substances and is currently being screened for by using conventional immunometric assays. A method based on MS detection for this macromolecule can be useful as a confirmatory method when hCG has been detected in the primary screening phase using immunoassays.

linearity, precision, and accuracy results demonstrate the method’s robustness despite the lack of an adequate isotopically labeled hCG protein as IS. Selectivity and Matrix Effects. Blank serum and urine samples were analyzed both with and without internal standard (n = 3). No interfering peaks were observed from either of the biological matrixes or the internal standard, which assures no false positive responses. Additionally, urine samples (n = 6) and serum samples (n = 6) from different individuals were analyzed, with further regard to specificity. No interfering peaks were observed in either sample. Finally, continuously infusion of signature peptide directly into the MS, generating a constant signal, was applied during the analysis of blank matrix samples (n = 6). No matrix components interfered with the signal response, which demonstrates optimal signal detection in the MS. Stability. Experiments assessing the stability of one, two, and three freeze and thaw cycles, as well as benchtop stability and stability of prepared samples kept in the autosampler for 12, 24, and 48 h, respectively, were performed for both serum and urine samples. No decrease in signal was observed for either experiment or matrix (data not shown). These stability experiments were performed on a short-term basis; for longterm stability data on the hCG molecule and its variants, reference is made to the work by Lempiäinen et al.24 Pregnyl as Standard. This method was validated using the pharmaceutical formulation Pregnyl as standard. This can be regarded as challenging since this hCG source contains an unknown amount of hCG degradation variants in addition to the intact hCG molecule. However, the method aims at generating quantitative analysis of the hCGβ subunit and, additionally, detecting and identifying all other degradation variants that might also be present in a sample. The use of a complex hCG mixture as the one found in Pregnyl generates a more challenging and plausible hCG composition, allowing the method to perform under realistic conditions. As such, this contributes to the demonstration of the robustness of the method, when the obtained validation results are within the required range. By using a standard containing nothing but the intact hCG molecule, the discriminating and differentiating abilities of the MS, and thus the method, will be missed. Application of Method on Realistic Samples. Quantification of intact hCG and differentiation between various hCG isoforms were performed on sera and urine samples of a pregnant woman, patients previously diagnosed with testicular cancer, and of a man having received injection of Pregnyl. The results for all the various hCG-containing samples are summarized in Table S-1 in the Supporting Information. The hCG concentration of the pregnancy serum sample was estimated to approximately 2.6 × 104 IU/L, which is well in accordance with the hCG levels at 21 weeks of pregnancy.10 The corresponding urine sample had an hCG concentration of 6280 IU/L. However, what could be observed in urine but not in serum is the main urine degradation variant, the hCGβ core fragment. This is part of the well-known degradation and excretion of the hCG molecule.10 For the cancer patient serum samples no other hCG isoforms were observed other than the hCGα and hCGβ subunits, resulting from either the intact hCG molecule or the free hCGβ subunit. Although various hCG variants can be produced and detected in serum during the development of certain types of tumors, the intact hCG molecule is often the dominant form.10 The concentration of this endogenously produced hCG varied between approx-

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CONCLUSION This is, to our knowledge, the first paper on hCG detection by the targeted proteomics approach that has been fully and successfully validated to demonstrate satisfying sensitivity and reproducibility and that has proved to be quantitatively accurate with indisputable identification specificity through the use of highly specific SRM detection. Screening samples by using the conventional immunoassays followed by a confirmatory analysis 7931

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Analytical Chemistry

Article

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using this differentiating method could be a starting point for the implementation into routine laboratories. Additionally, the technique can be further extended to comprise the isolation of other target proteins by the combination of different antibodies prior to MS detection, this in the same analysis, thus resulting in a cost-efficient multiplexing assay. Finally, the crossfertilization of the complementary techniques of immunoextraction and MS detection represents a powerful method synergy that can lift targeted proteomics from the limited perspective as a discovery tool into the world of routine analysis.

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ASSOCIATED CONTENT

* Supporting Information S

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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REFERENCES

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dx.doi.org/10.1021/ac301418f | Anal. Chem. 2012, 84, 7926−7932