Highly Specific and Sensitive Measurements of Human and Monkey

and accuracy, as well as stability and recovery, were found to be acceptable. ..... Wong , Ji Ma , Tiffany Dickerson , Colin Hall , Dan A Rock , T...
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Highly Specific and Sensitive Measurements of Human and Monkey Interleukin 21 Using Sequential Protein and Tryptic Peptide Immunoaffinity LC-MS/MS Joe Palandra,* Alyce Finelli, Ming Zhu, Jaime Masferrer, and Hendrik Neubert Pfizer Worldwide Research & Development, Andover, Massachusetts 01810, United States S Supporting Information *

ABSTRACT: A highly specific and sensitive immunoaffinity LC-MS/MS assay for quantification of human and cynomolgus monkey interleukin 21 (IL-21) was developed, qualified, and implemented. The workflow includes offline enrichment of IL-21 using an anti-IL-21 capture antibody, followed by isolation using magnetic beads, trypsin digestion, online enrichment of IL-21 derived tryptic peptides using antipeptide antibodies, and quantification using nanoflow LC-MS/MS. This assay was developed and qualified in human and cynomolgus monkey serum and tissues with a lower limit of quantitation of 0.78 pg/ mL based on the intact cytokine. Both intra- and interbatch precision and accuracy, as well as stability and recovery, were found to be acceptable. IL-21 was not detected in serum from normal healthy volunteers or from autoimmune disease patients. However, IL-21 levels were quantified in cynomolgus monkey spleen and colon tissue and normal and inflammatory bowel disease (IBD) human colon tissue as well as hyperplasia human tonsils.

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tional, and clinical biomarker investigations, it is important to develop confidence in the analytical platforms used for determination of IL-21 concentrations in serum of normal and disease patients. Furthermore, IL-21 concentrations in tissues have not yet been reported; however, measurements with high analytical confidence are needed in matrixes such as colon, spleen, and tonsils in order to establish the relationship between localized tissue concentrations and systemic circulation. The use of liquid chromatography tandem mass spectrometry (LC-MS/MS) has gained momentum lately as a means to quantify proteins from biological matrixes due to its selectivity advantages over traditional immunoanalytical methods.12 Although quantification at the intact protein level is a promising area, the predominant current practice is to measure enzymatically derived surrogate peptides.13,14 However, depending on the protein concentration, assay sensitivity remains a challenge without the use of immunoenrichment techniques or extensive fractionation prior to LC-MS/MS. One way this challenge can be met is with immunoaffinity enrichment of the protein of interest,15 where an antibody and a stationary phase, typically beads, are used to capture target proteins.16−18 An alternative approach involves capturing one or multiple surrogate peptides, after proteins have been enzymatically digested, via an

nterleukin-21 (IL-21) is an immuno-regulatory cytokine whose pleiotropic effects on the immune system have been extensively studied1,2 and which has been shown to have a key role in initiating and driving Th1 and Th17 helper cell mediated inflammatory conditions. This cytokine is exclusively produced by NK and activated CD4+ T cells, while IL-21 receptor is present on most cell types. Understanding the concentration of IL-21 in inflammatory and autoimmune processes is important to elucidate the possible role of this important cytokine in diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and inflammatory bowel disease (IBD) such as ulcerative colitis (UC) and Crohn’s disease (CD). Ligand binding assays (LBA) are commonly employed for measuring protein levels in biological matrixes, for example, using the enzyme-linked immunosorbent assay (ELISA) format for which numerous kits are commercially available for many known proteins, including IL-21. Reported serum levels of IL21 in both normal healthy donors and disease populations vary considerably in the literature ranging between 40 and 800 pg/ mL.3−8 It is well-known that cross-reactivity between cytokines may complicate ELISA analysis and that serum from autoimmune patients contains polyreactive autoantibodies, complement and other proteins that can interfere with ELISA assays.9,10 Thus, it is unclear if the differences previously observed can be explained by changes in population, differences in the employed assays, or interferences present in the biological samples.11 In order to support quantitative, transla© XXXX American Chemical Society

Received: March 5, 2013 Accepted: May 2, 2013

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Figure 1. Schematic of the IL-21 immunoaffinity LC-MS/MS assay workflow.

antipeptide antibody, using either an offline19 or online methodology.20−22 Akin to a methodology described recently,23 we have combined an offline IL-21 immunoaffinity technique with online high flow antipeptide antibody capture followed by nanoLC-MS/MS for IL-21 measurement. To this end, we describe in detail the development, qualification, and implementation of a novel assay for highly selective and sensitive quantification of IL-21 in serum and tissue matrixes.

IL-21. The remaining standards were prepared by 1:1 serial dilution of the 100 pg/mL STD7 using calibration matrix to yield 50.0 pg/mL STD6, 25.0 pg/mL STD5, 6.25 pg/mL STD4, 3.13 pg/mL STD3, 1.56 pg/mL STD2, and 0.78 pg/mL STD1. The 12.5 pg/mL standard was not used, leaving 7 calibration standards. Quality control (QC) samples were prepared by spiking human control serum (Sigma, St Louis, MO) with human recombinant IL-21 at concentrations of 3.5, 35, and 70 pg/mL and cynomolgus monkey colon tissue homogenate at concentrations of 35 and 70 pg/mL using WS. In addition, samples to assess IL-21 stability in matrix were prepared in human serum at 3.5 and 70 pg/mL and in cynomolgus colon tissue at 70 pg/mL using WS. Tissue Extraction. Tissue samples were pulverized by high mechanical impact using a cryoPREP Impactor (Covaris Inc., Woburn MA) to decrease sample heterogeneity. The samples were stored at −80 °C for multiple analyses or used immediately. Typically 25−100 mg of pulverized tissue was carefully transferred into a preweighed tube containing 0.6−1.0 mL of homogenization buffer (T-PER Tissue Protein Extraction Reagent; Pierce, Rockford, IL) and 1X Pierce Halt protease inhibitors (Thermo, Rockford, IL). The suspension was homogenized using a Bullet Blender (Next Advance, Averill Park, NY) using 0.5 mm zirconium oxide beads for 4−5 min. The samples were then centrifuged at approximately 10 000g for 10 min. The homogenates were carefully recovered to avoid as much contamination with lipid as possible (recentrifugation was typically necessary). The protein concentration of a small portion of homogenate (∼2 μL) was assayed with a NanoDrop (Thermo, Wilmington, DE) using absolute absorbance at 280 nm, while a 500 μL aliquot of the homogenate was assayed for IL-21 by immunoaffinity LC-MS/MS. IL-21 Immunoaffinity Capture and Magnetic Bead Processing. A 500 μL aliquot of plasma, serum, or tissue extract was combined with 300 μL of PBS buffer in a 2000 μL 96-deep well plate. To each well, 1.0 μg of biotin labeled monoclonal anti-IL-21 antibody was added. Samples were incubated overnight at 4 °C while shaking at 500 rpm. The following day, 20 μL of streptavidin magnetic beads were added to each well and the plate was incubated for 45 min at room temperature (RT) while shaking. The plate was then placed in a KingFisher Flex system (Thermo, San Jose, CA) at RT to remove the beads from the matrix and displace them in a wash buffer of PBS with 0.05% Tween-20. The protocol was repeated one more time for a second immunoaffinity enrichment step, beginning with incubation of a new aliquot of capture antibody for 2 h at RT rather than overnight at 4 °C. The combined magnetic bead suspensions were processed in 96-well format using a KingFisher Flex system.24 The protocol begins by removing the beads from the sample by binding it to the 96-

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MATERIALS AND METHODS Materials. Recombinant human IL-21 (3.16 mg/mL, 99% purity by SEC) was obtained from Pfizer Pharmaceuticals (Uniprot accession number Q9HBE4). Biotinylated antihuman IL-21 monoclonal capture antibody was obtained from eBioscience (San Diego, CA; clone eBio2B2-G20). Extended sequence stable isotope labeled (SIL) peptides contained trypsin cleavage sites and were custom synthesized by Thermo Biopolymers (Ulm, Germany). Sequences were HMIRMRQLIDI*VDQLKNYVNDL (I* = 7 Da mass shift; >97% purity; peptide identification number OR-279518-1) and QKHRLTCPSCDSYEK*KPPK (K* = 8 Da mass shift; >97% purity; peptide identification number FF303038-1). Polyclonal antibodies against surrogate IL-21 peptides were generated in rabbits (Lampire Biologicals, Pipersville, PA) and ligand affinity purified prior to use. The immunogen peptides (>95% purity by HPLC) were the tryptic IL-21 derived peptides QLIDIVDQLK and LTCPSCDSYEK conjugated to keyhole limpet hemocyanin (KLH) via an additional N-terminal cysteine (sequence CGGQLIDIVDQLK and CGGLTCPSCDSYEK; GG added as spacer; the internal C is carbamidomethylated cysteine). Dynabeads Streptavidin MyOne T1 were purchased from Invitrogen Life Technologies (Carlsbad, CA). Serum samples were provided by Pfizer (Cambridge, MA) and Bioreclamation (Long Island, NY). Flash frozen surgical specimen from UC, CD colon, and normal adjacent areas were purchased from Analytical Biological Services Inc. (Wilmington, DE) while human tonsil samples were purchased from BioOptions (Brea, CA). Preparation of Standards and Quality Controls. A stock solution of recombinant human IL-21 was received at a concentration of 3.16 mg/mL. Secondary stock solution and working standard (WS) solution were prepared at concentrations of 1 μg/mL and 10 ng/mL, respectively, using 1% bovine serum albumin (BSA) prepared in phosphate buffered saline (PBS) as a diluent. The highest serum calibration standard (STD 7) was prepared by adding 20 μL of WS solution to 1.980 mL of calibration matrix (prepared by mixing one part blank human serum to 3 parts PBS) to give a final concentration of 100 pg/mL. The human serum used for calibration matrix did not contain any measurable endogenous B

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Figure 2. Schematic of the online peptide immunoaffinity liquid chromatography configuration.

controlled the flow from the micropump for trap washing and equilibration while valve 3 controlled the analytical nanoflow to the trap and nanocolumn. Selecting tubing with appropriate inner diameter (ID) is critical for this LC configuration as high flow and nanoflow paths are combined. Connections to and from valve 1 had 250 μm ID tubing compatible with the higher flow rates through the antipeptide antibody column. The ID of connection tubing from valve 2 to valve 3 was narrowed to 100 μm ID and 20 cm length. The C18 trap cartridge in valve 3 was held with two 50 μm ID connection tubings of 7 cm length. During injection, the antipeptide antibody column was equilibrated with 25 mM ammonium formate (pH 7) at 0.3 mL/min (valve 1 in position 1 and valve 2 in position 2). The sample (95 μL) was loaded and washed for 1.5 min at the initial flow conditions; the antibody column was then washed from 1.5 to 2.2 min at 0.3 mL/min with 500 mM ammonium formate (pH 6) before switching to the elution buffer (0.4% TFA) from 2.9 to 5.3 min, also at 0.3 mL/min. During this time, valves 2 and 3 were switched to position 1, and the eluent was collected on a PepMap300 C18 trap cartridge (5 × 0.3 mm, 5 μm, 300 Å, Dionex). Following elution, valve 2 was switched to position 2 and the antibody column was washed with 0.75% formic acid and 3% isopropanol at 2.0 mL/min for 2.5 min. At the same time, valve 3 was switched to position 2 for the chromatographic separation to occur on a PepMap C18 RSLC nanocolumn (15 cm × 75 μm, 2 μm, 100 Å, Dionex) at a flow rate of 600 nL/min using buffers of 0.1% formic acid in 2% acetonitrile (solvent A) and 0.1% formic acid in 90% acetonitrile (solvent B). The gradient separation occurred from 2.3 to 9.8 min going from 10% to 45% B. In order to minimize carryover, the C18 trap column was washed with 1% formic acid, 25% isopropanol, and 50% acetonitrile at a flow rate of 400 μL/min for 1.2 min before re-equilibration with 0.1% TFA at 200 μL/min using the NCS loading pump. The total duty cycle of this LC method was just over 10 min. Finally, the efficiency of the peptide immunoaffinity step was frequently determined by bypassing the antipeptide antibody column in valve 1 (switched to position 2) using a digest of the

magnet head. The beads were then washed twice with 400 μL of 0.05% Tween-20 in PBS buffer, followed by a single wash with 400 μL of PBS and subsequent elution of the bound cytokine with 140 μL of 25 mM HCl. A summary of the immunoaffinity workflow is described in Figure 1. Reduction, Alkylation, and Digestion. The pH of the eluate was raised using 30 μL of 1 M Tris HCl, pH 8, followed by the addition of 5 fmol of each of the two SIL peptides (10 μL × 0.5 fmol/μL). Ten microliters of 35 mM DTT was added, and the sample was heated at 60 °C for 30 min. Samples were allowed to cool to RT for 10 min prior to the addition of 10 μL of 70 mM of iodoacetamide to each sample and incubation for 30 min in the dark. The samples were then digested overnight at 37 °C using 1 μg of modified sequencing grade trypsin (Roche, Indianapolis, IN). Liquid Chromatography Configuration. There are multiple factors to consider for successful use of an antipeptide antibody column such as temperature, loading flow rates, pH, and pressure. For example, the column temperature has to be high enough for efficient binding of the targeted peptide(s) to the antipeptide antibody while low enough to maintain longer term binding efficiency and integrity. For this purpose, 25−30 °C was found to be an optimal temperature in our hands. Due to the high binding capacity of the antipeptide antibody column, the flow rate during sample loading can typically be scaled between low microliter per min to 1 or 2 mL per minute and can be adjusted according to assay requirements, for example, relating to throughput and loading volume. Figure 2 outlines the liquid chromatography setup utilizing 3 valves and 3 pumps. A Dionex Ultimate 3000 was configured with a WPS-3000 autoinjector equipped with a 15 μL fused silica needle, 250 μL syringe, and a 200 μL loop. The system was also composed of a FLM-3300 flow manager consisting of a temperature controlled column compartment containing one 10-port valve (valve 1) and a quaternary micropump, a NCS3500 module composed of a binary high pressure nanopump, and a tertiary loading pump combined with a temperature controlled dual 6-port valve column compartment (valves 2 and 3). Valve 1 controlled flow through the antipeptide antibody column and to valve 2 contained in the NCS module. Valve 2 C

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alkylation, and digestion, and therefore these steps were included in the normalization of the workflow. An initial format of this assay used protein immunoenrichment with detection of IL-21 derived peptides employing a standard flow (0.2 mL/min) LC-MS/MS setup using a reverse phase trap (but no antipeptide antibody column). The assay at the time produced an approximate limit of quantitation (LOQ) of 100 pg/mL using 250 μL of serum. The assay was modified by introducing nanoLC to increase mass spectrometric sensitivity, which resulted in reduction of the LOQ to approximately 5 pg/mL using the same sample volume. The assay sensitivity was improved further by incorporating antipeptide antibody enrichment into the analytical workflow while maintaining analytical nanoflow rates. In addition, the starting serum volume was increased to 500 μL, which was practical for magnetic bead handling. These changes achieved a LOQ of approximately 1 pg/mL while using a LC duty cycle of about 10 min. The achieved sensitivity is shown in Figure 4a−e, which displays some extracted ion chromatograms of a blank and recombinant spikes at various concentrations. During assay development, it was determined that recovery of recombinant IL-21 spiked at low and high concentrations was approximately 90% for the first immunoenrichment step and an additional ∼10% for a second immunoenrichment cycle from the same sample (data not shown). A third protein immunoenrichment step did not provide any measurable IL-21 levels; therefore, it was concluded that combining two protein immunoenrichment would provide the complete IL-21 recovery needed for assay accuracy and sensitivity. Assay Qualification. A typical calibration curve obtained during assay qualification is shown in Figure 3. Summary

extended SIL peptide and comparison of the response with that obtained through the antipeptide antibody column23 Mass Spectrometry. The eluate from nanoflow chromatography was introduced into a nanospray III source (AB Sciex) containing a nanospray stainless steel emitter (50 mm × 30 μm ID, Proxeon, West Palm Beach, FL). The ionspray voltage was typically between 3500 and 4000 V, and nebulizer gas settings typically fell between 6 and 10 psi. Detection of IL-21 surrogate tryptic peptides was performed on an API5500Q mass spectrometer (AB Sciex, Toronto, Canada) by multiple reaction monitoring (MRM) in positive ion mode. The transitions monitored for peptide QLIDIVDQLK were 592.8 to 943.5 and 830.4 representing the doubly charged precursor ion and the y8 and y7 product ions, respectively. For the peptide LTCPSCDSYEK, the transitions were 680.3 to 1145.4 and 985.4 representing the doubly charged precursor ion and the y9 and y8 product ions, respectively. Dwell time was set at 100 ms; collision energy was set to 30 V, and Q1 and Q3 were operated in unit and low resolution, respectively (full width at halfmaximum [fwhm] was 0.7 and 1.0, respectively). Other relevant instrument settings were declustering potential of 80 V, entrance potential of 10 V, and collision cell exit potential of 15 V. AB Sciex Analyst software (Version 1.5.2) was used to acquire data and determine peak areas. Peak area ratios (PAR) of the IL-21 peak area to the SIL peptide peak area were calculated for the QLIDIVDQLK peptide using the transition to the y7 product ion and for the LTCPSCDSYEK peptide using the transition to the y8 product ion. Calibration curves were constructed using PAR of the calibration samples by applying a 1/(concentration) weighted nonlinear regression model using Labstats. All sample concentrations were then calculated from their PARs against their respective calibration curve. Qualification Outline. Assay qualification involved testing several key parameters to establish confidence in assay performance including accuracy, precision, and spike recovery as well as freeze/thaw and benchtop stability. The IL-21 calibrant concentrations were 100, 50, 25, 6.25, 3.13, 1.56, and 0.78 pg/mL. The calibration curve, including a zero standard containing no IL-21 spike, was analyzed in duplicate. QC samples fortified with 3.5 (low), 35 (medium), and 70 (high) pg/mL IL-21 in normal human serum or 35 and 70 pg/mL IL21 in cynomolgus colon homogenate were tested using 6 replicates at each of the spiked IL-21 levels in 3 separate batches run on different days. In addition, 3 freeze/thaw cycles and 6 h benchtop stability were tested by preparing 6 replicates of both low and high QC for serum and high QC for tissue homogenate. QLIDIVDQLK was used as the primary peptide for quantification purposes, and all results shown are from this peptide, unless otherwise stated; LTCPSCDSYEK was a secondary peptide used for confirmatory purposes only.

Figure 3. IL-21 calibration curve ranging from 0.78 to 100 pg/mL.

statistics of intra- and interbatch precision and accuracy of qualification samples is presented in Table 1. CVs (coefficients of variation) and accuracies of the spiked recombinant QCs were within 25% for human serum. Of note, endogenous IL-21 levels were undetectable in the normal human serum; therefore, no endogenous QC was used. Furthermore, cynomolgus monkey colon homogenate was used as a QC matrix as its composition was considered similar to the respective human matrix and larger volumes of human colon extract were unavailable for assay qualification purposes. However, IL-21 was confirmed to be undetectable in this particular homogenate; therefore, no endogenous tissue QC was used. Mean back-calculated calibration standards were within 15% of the nominal value at all levels and CVs were within 20%

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RESULTS AND DISCUSSION Assay Development. The human IL-21 surrogate peptides QLIDIVDQLK and LTCPSCDSYEK are proteotypic and conserved in cynomolgus monkey. Their positions in the mature human IL-21 amino acid sequence are shown in the Supporting Information. In addition, there are no reported or predicted post-translational modifications in these sequences. The respective SIL peptides contain trypsin cleavage sites and N- and C-terminal sequence extensions HMIRMR-QLIDIVDQLK-NYVNDL and QKHR-LTCPSCDSYEK-KPPK. These standards were added to the samples prior to reduction, D

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Table 1. Summary Statistics of Intra- and Interbatch Accuracy and Precision in Serum and Tissue human serum QC low IL-21 target conc. batch Rep1 Rep2 Rep3 Rep4 Rep5 Rep6

a

3.50 pg/mL 1

2

cynomolgus monkey colon extract

QC medium

QC high

35.0 pg/mL 3

1

2 30.3 34.1 21.3 31.7 36.1 38.3

2.88 4.23 4.26 3.74 3.34 3.84

3.81 4.13 3.73 2.04 2.88 2.88

5.02 3.96 3.49 3.31 4.76 3.83

39.2 36.9 35.8 35.2 34.3 33.0

mean SDa % CVb % REc

3.72 0.53 14.3 6.1

3.25 0.78 24.1 −7.3

4.06 0.69 16.9 16.0

35.7 2.2 6.0 2.1

mean SD % CV % RE n

3.67 0.72 19.6 5.0 18

34.6 5.0 14.6 −1.3 18

QC medium

70.0 pg/mL 3

1

2

QC high

35.0 pg/mL 3

43.7 64.6 76.7 74.4 37.9 74.4 67.0 59.5 36.2 73.4 74.9 60.9 35.9 73.6 64.5 67.8 36.5 62.7 69.4 55.7 25.7 75.1 45.4 44.3 Intrabatch Summary Statistics 32.0 36.0 70.6 66.3 60.4 6.0 5.8 5.5 11.2 10.3 18.7 16.2 7.8 17.0 17.1 −8.7 2.8 0.9 −5.3 −13.7 Interbatch Summary Statistics 65.8 9.8 14.9 −6.0 18

1

2

25.8 32.5 28.4 32.4 32.3 41.1

33.8 32.9 33.5 29.4 30.1 33.5

32.1 5.2 16.2 −8.3

32.2 1.9 6.0 −8.0

31.3 4.4 14.1 −10.6 18

70.0 pg/mL 3

1

2

28.2 29.2 25.7 39.8 30.6 24.1

67.1 67.7 75.4 70.0 77.9 72.9

74.0 77.9 76.9 69.1 71.1 62.1

55.2 55.6 65.5 44.6 54.0 45.6

3

29.6 5.5 18.7 −15.4

71.8 4.3 6.0 2.6

71.9 5.8 8.1 2.6

53.4 7.7 14.3 −23.7

65.7 10.6 16.2 −6.1 18

Standard deviation. bCoefficient of variation. cRelative error.

Measuring IL-21 Produced from Human T-cells. Purified human T-cells and human whole blood was stimulated to express elevated concentrations of IL-21 in order to assess the assays capability to bind endogenously produced cytokine. Purified T-cells were obtained using Leukopak from 4 donors and then purified using the RosetteSep negative selection kit (Stemcell technology, Vancouver Canada). After purification and dilution, the cells were incubated at 37 °C with anti-CD3/ anti-CD28 beads (Invitrogen, Carlsbad, CA). After 46 h, the cells were pelleted and the supernatant was removed. Analysis using the IL-21 LC-MS/MS assay demonstrated that endogenously produced IL-21 from four independent donors ranged from approximately 300 to 700 pg/mL. Separately, in order to stimulate cytokine release, 10 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, St. Louis, MO) and 500 ng/mL ionomycin (Sigma-Aldrich) was added to human whole blood from four donors and incubated for 4 and 24 h. Analysis of the processed plasma indicated the presence of IL-21 with levels ranging from approximately 1 to 150 pg/mL after 4 h of stimulation and increasing to above 1 ng/mL in some cases after 24 h of stimulation. These results reaffirm that endogenous IL-21 produced in human blood cells can be successfully captured and detected using this immunoaffinity LC-MS/MS workflow. Evaluating Stability in Human Whole Blood. Stability of recombinant and endogenously produced IL-21 in human whole blood was assessed to determine whether IL-21 levels were negatively impacted by possible protease activity or circulating binding proteins. Fresh heparinized human whole blood (within 15 min of collection), serum, and plasma were spiked with recombinant human IL-21 at 100 pg/mL and analyzed at 0, 4, and 24 h at RT. Samples were prepared in duplicate for each matrix tested. The 0 h samples were immediately processed along with a standard curve prepared in serum. For the human whole blood samples, the plasma portion was processed by first centrifuging the whole blood at 3000 rpm for 10 min and removing the plasma layer. After 4 and 24

(Supporting Information Table S1). IL-21 was found to be stable to up to three repeated freeze/thaw cycles of 6 replicates at two QC levels as well as on the benchtop for 6 h with 6 replicates at two QC levels with both CVs and accuracy results within 20% (Supporting Information Table S2). Additionally, carryover was assessed for IL-21 by injecting a blank immediately after the highest standard (ULOQ) and measuring the response in the blank relative to the lowest standard (LLOQ). The carryover as a percentage relative to the LLOQ was determined to be 0%. Biological Confidence in Measurement. In order to establish increased confidence in the IL-21 assay, the following experiments were performed to confirm the ability to measure in biological matrixes: (1) Screening of various IL-21 antibodies to investigate potential differences in their ability to bind recombinant versus endogenous IL-21; (2) detecting endogenously produced IL-21 through in vitro stimulation of purified human T-cells and human whole blood to show that the assay is capable of measuring endogenous IL-21 in addition to the recombinant protein; (3) evaluating IL-21 stability and recovery in human whole blood to explore if proteases or binding proteins are either degrading the cytokine or competing with its binding to the capture antibody. Antibody Screening. More than 20 commercial and inhouse antibodies were tested for their ability to bind IL-21 in this assay. A single amount of antibody per sample, i.e., 1 μg, was tested to bind recombinant and endogenous IL-21 in serum or plasma matrix from both healthy normal volunteers and disease populations. While several antibodies displayed good binding to recombinant IL-21 (Supporting Information Table S3), no endogenous IL-21 could be detected from any of the human serum samples. Ultimately, a mouse monoclonal antibody (Ebioscience, clone eBio2B2-G20) was selected because it captured recombinant IL-21 effectively and was readily available in biotinylated form. Lastly, this antibody has previously been used as a capture reagent in an ELISA for IL-21 quantification.4,8 E

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and high, approximately 5 and 75 pg/mL, respectively) in both serum and human whole blood matrixes, and IL-21 stability was assessed at 4 and 24 h at RT. In conclusion, recovery of endogenously produced IL-21 was high in all matrixes tested, indicating that IL-21 is stable in these matrixes (Table 2), providing additional confidence in the analytical procedure and evidence for stability of the peptide sequences detected in this assay. IL-21 in Cynomolgus and Human Serum, Plasma, and Tissues. Numerous serum and plasma samples from both cynomolgus monkey and human were tested for IL-21 levels using this assay. These samples included approximately two dozen normal cynomolgus serum and plasma, over one hundred normal human serum and plasma samples, and over one hundred selected serum samples from patients with RA, CD, UC, and SLE. Given that no IL-21 was detected in any of these samples using this immunoaffinity LC-MS/MS assay, it was concluded that circulating levels were below the limit of quantitation of 0.78 pg/mL both in human and in cynomolgus monkey serum and plasma, which contradicts previously published literature.3−7 In contrast to the absence of measurable concentrations in human and cynomolgus serum or plasma, IL-21 was detected in various cynomolgus and human tissues (example illustrated in Figure 4f). Quantified IL-21 concentrations in tissue extract were typically normalized by the weight of the tissue used. Cynomolgus tissue IL-21 levels ranged from approximately 20 to 120 pg/g in lymph nodes and approximately 10 to 80 pg/g in spleen (Table 3). Furthermore, IL-21 levels were also measured in human colon surgical resections from 3 CD patients, 4 UC patients, and 4 nonlesional colon (CD) samples as well as 15 human tonsils (lymphoid tissue hyperplasia)

h, the remaining samples were subsequently processed for IL21 analysis. Recovery for all matrixes was back-calculated against the serum standard curve. The results of the experiment are shown in Table 2 (spiked IL-21 only since no IL-21 was Table 2. Recovery of Spiked Recombinant and Spiked Endogenous IL-21 in Multiple Matrixesa recovery of 100 pg/mL recombinant IL-21 [%]

incubation time [h] 0 4 24

serum plasma 97 92 108

103 107 90

recovery of endogenously produced IL-21 [%]

whole blood

low spike serum

high spike serum

low spike whole blood

high spike whole blood

96 105 113

94 92 91

100 99 99

92 87 86

90 95 95

a

Low endogenous IL-21 spike: approximately 5 pg/mL; high endogenous IL-21 spike: approximately 75 pg/mL.

detected in the nonspiked samples). It was evident that recombinant IL-21 was stable in all matrixes for up to 24 h at RT, indicating the recovery of the recombinant protein was not negatively impacted by matrix components such as protease activity or any potential binding proteins or receptors that would interfere with the capture antibody in the assay. Furthermore, a similar experiment was performed using an IL-21 preparation produced by human T cell stimulation as described above, in order to address if the endogenous IL-21 has different stability and binding characteristics compared to recombinant IL-21. The IL-21 concentration in these stimulated T cell preparations was first determined and then subsequently diluted at two separate IL-21 concentrations (low

Figure 4. Extracted ion chromatograms for IL-21 peptide QLIDIVDQLK from (a) a zero standard containing no IL-21; calibration standards at (b) 0.78 pg/mL, (c) 1.56 pg/mL, (d) 3.13 pg/mL, and (e) 25 pg/mL human IL-21, and (f) a human colon extract containing 5381 pg/g IL-21. MRM transitions were recorded for the native peptide (top panel) Q1 592.8 [M + 2H]2+ to Q3 830.5 (y71+, blue) and 943.5 (y81+, red) and SIL peptide (bottom panel) Q1 596.3 [M + 2H]2+ to Q3 837.5 (y71+, blue) and 950.5 (y81+, red). F

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quantification of the range of tissue expression of the cytokine IL-21 and indicates that IL-21 is highly expressed in both tonsil and colon tissue especially in diseased tissue as shown by levels as high as 19 ng/g of IL-21 per gram weight of tissue.

Table 3. Cynomolgus Monkey and Human IL-21 Tissue Levels summary statistics sample ID cynomolgus monkey spleen cynomolgus monkey lymph node human tonsils (hyperplasia) human colon adjacent to disease human colon Crohn’s disease human colon ulcerative colitis

IL-21 tissue levels (pg of IL-21/g of tissue)

mean (pg/g)

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std deviation

10, 13, 15, 23, 35, 84

30

28

21, 22, 35, 44, 105, 122

58

44

402, 478, 478, 490, 627, 709, 710, 750, 779, 1309, 1436, 1779, 2123, 3124, 3157 83, 192, 889, 3342

1223

932

1127

1520

133, 2533, 5381

2682

2627

1182, 1989, 15925, 18940

9507

9235

CONCLUSIONS Low abundance proteins such as cytokines can be measured in biological matrixes using ligand binding assay techniques. However, lack of analytical specificity may hamper a ligand binding assay’s ability to measure with high confidence especially in fluids from patients with autoimmune diseases. We have shown that, by combining immunoaffinity techniques with LC-MS/MS, high measurement specificity can be achieved together with high analytical sensitivity that can match or outperform most standard ligand binding assay formats. The analysis of several hundred serum and plasma samples from healthy normal individuals and various disease patient populations demonstrates that IL-21 is not present above the qualified limit of quantification of 0.78 pg/mL. This was unexpected since several previous reports have indicated the presence of IL-21 in normal serum and the elevation in autoimmune diseases in the high pg/mL to low ng/mL range.3−7,25 Our data using high specificity mass spectrometric determination of IL-21 suggests that previous reports may have over-represented the IL-21 levels in normal or disease serum perhaps due to matrix interferences in the assay. In this study, several critical control experiments were performed to confirm that the immunoaffinity LC-MS/MS approach is reliable, for example, the addition of different concentrations of recombinant or human T cell-derived IL-21 to serum and blood and incubating them for up to 24 h. In all cases, recovery of the added cytokine was >95%, which validated this immunoaffinity LC-MS/MS assay. Furthermore, stimulation of time dependent IL-21 release from human T cells using PMA and ionophore further demonstrated the assays ability to measure endogenous IL-21. In contrast to serum, IL-21 levels were clearly detected in tissues. These new data suggest that IL-21 exerts its effect mainly in the inflamed tissues via activation of inflammatory cells that contain the IL-21 receptor. Given the pleiotropic function of IL-21, our data indicates that this cytokine contributes to the inflammation at the disease site in the tissue by its local effect on T cell, B cell, and NK cells.26 The sequential immunoaffinity LC-MS/MS approach presented herein together with previously published approaches for other low abundance proteins of interest, such as human beta nerve growth factor,23 now provides a selective and sensitive bioanalytical strategy for the development of similar assays supporting translational pharmacology and clinical biomarker investigations. Furthermore, as this sequential immunoaffinity LC-MS/MS approach is exquisitely suited for the measurement of soluble proteins in tissues, we recommend considering if tissue bioanalysis of the protein of interest can aid general assay development, for example, to potentially measure endogenous analyte in a tissue compartment either as a positive control or to assess local expression.

(Table 3). The human tissues showed average IL-21 levels in the single digit ng/g range. Although, IL-21 tissue quantification presented herein was achieved using the peptide QLIDIVDQLK (used for assay qualification and all analysis described above), the IL-21 surrogate peptide LTCPSCDSYEK was also recorded from tissue extracts and simultaneously used for confirmation purposes. Correlation between the two surrogate peptides demonstrated very good agreement as shown for all individual human colon and human tonsil IL-21 measurements (Figure 5), providing additional confidence in

Figure 5. Correlation of IL-21 tissue concentrations determined from the same measurement from human colon and tonsil tissues using two different IL-21 surrogate peptides.

the analytical measurement. The two surrogate peptides are located in different parts of the IL-21 amino acid sequence (see Supporting Information; one peptide originated from a part of the IL-21 sequence toward the N-terminus and the other toward the C terminus), therefore allowing the conclusion that detected IL-21 was minimally spanning amino acid residues 12 to 101. This data, for the first time, provides highly specific

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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. G

dx.doi.org/10.1021/ac4006765 | Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

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Article

AUTHOR INFORMATION

Corresponding Author

*Address: 1 Burtt Rd, Andover, MA 01810. Tel: (978) 2471758. E-mail: joe.palandra@pfizer.com. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to Yulia Vugmeyster, Anson Abraham, and John Douhan for guidance and useful discussions. The authors also thank Joe Gardner for raising the polyclonal antipeptide antibodies.

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REFERENCES

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