Development of High-Throughput Chemical Extraction Techniques

Nov 18, 2009 - Development of High-Throughput Chemical Extraction Techniques and Quantitative HPLC-MS/MS (SRM) Assays for Clinically Relevant Plasma P...
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Development of High-Throughput Chemical Extraction Techniques and Quantitative HPLC-MS/MS (SRM) Assays for Clinically Relevant Plasma Proteins Chris Barton,†,# Richard G. Kay,*,†,# Wolfgang Gentzer,‡ Frank Vitzthum,‡ and Steve Pleasance† Quotient Bioresearch Ltd, Newmarket Road, Fordham, Cambridgeshire, CB7 5WW, United Kingdom, and Siemens Healthcare Diagnostics Products GmbH, Emil-von-Behring-Strasse 76, 35041 Marburg, Germany Received July 23, 2009

The clinical application of targeted plasma protein analysis by selective reaction monitoring of peptides using LC-MS/MS requires the development of robust, inexpensive protein extraction techniques with the potential for high-throughput applications. We present the development of a novel mixed-mode solid phase extraction (SPE) technique for the removal of high abundance and high molecular weight proteins from plasma. This technique, coupled with fused-core HPLC-MS/MS analysis is compared to a previously developed extraction method to study a range of proteins in plasma, including routinely measured biomarkers of growth hormone action. To further validate this technique, it was used for the quantification of insulin-like growth factor I (IGF-I) levels and compared to a state-of-the-art immunoassay on a fully automated analyzer. Clinical reference materials were applied for method development to allow for further interlaboratory comparisons. The LC-MS/MS approach quantified IGF-I in plasma with an accuracy that is within the guidelines for macromolecular assays in a regulated laboratory environment. Furthermore, IGF-I levels determined using the SPE and ACN methods with LC-MS/MS analysis correlated well with the immunoassay results. This demonstrates the applicability of mixed-mode SPE coupled with fused-core HPLC-MS/MS to quantify plasma proteins with results suitable for clinical applications. Keywords: Plasma • Standard Human Plasma • Clinical Reference Material • SPE • ACN Depletion • SRM • uHPLC-MS/MS • Quantitation • IGF-I

Introduction The application of LC-MS/MS to plasma protein quantitation by targeting proteotypic peptide surrogates is a fast evolving technique and has recently demonstrated the ability to quantify proteins in the low nanogram per milliliter (ng/mL) levels.1 This technique enables highly multiplexed analyses,2 rapid assay development,3 and specificity that is based on physicochemical properties of the peptide and not on epitope recognition.4 In addition, absolute quantitation by stable isotope dilution LCMS/MS can represent a reference measurement procedure which allows measurements of highest metrological quality.5 However, due to the complexity of the human plasma proteome and the broad concentration range of plasma proteins,6 a major problem of LC-MS/MS approaches is the inability to directly detect low-abundance proteins in a complex matrix. For this application, there is a need to deplete high-abundance proteins prior to analysis. Although current depletion techniques have demonstrated high sensitivity, they use complex, expensive, and low-throughput immuno-depletion methods,1,7 which may be difficult to apply in a high-throughput clinical * To whom correspondence should be addressed. E-mail: richard.kay@ quotientbioresearch.com. † Quotient Bioresearch Ltd. # Authors contributed equally. ‡ Siemens Healthcare Diagnostics Products GmbH. 10.1021/pr900658d

 2010 American Chemical Society

environment. There is, therefore, a need for a range of simple, robust, high-throughput, and cost-effective plasma protein depletion techniques, which can be applied in routine sample analysis. An example for a specific clinical need is the quantification of biomarkers used for diagnosis and therapeutic monitoring of growth hormone (GH) deficiency and excess.8,9 Clinical diagnosis of GH-related diseases is complicated by the pulsatile nature of plasma GH concentrations. Therefore, downstream biomarkers such as insulin-like growth factor I (IGF-I) and IGFII as well as proteins that modulate the effects of the GH/IGF-I axis, including IGF binding protein II (IGFBP-II) and IGFBPIII, are measured in clinical practice as surrogate biomarkers, as they have more consistent plasma levels over time. Further, the measurement of GH-related biomarkers is important for detecting GH abuse in the field of competitive sports, where it is believed to enhance athletic performance.10 In clinical laboratories, biomarkers like IGF-I are usually quantified with high accuracy by clinical analyzers that apply reliable immunoassay technologies.11 Most clinical analyzer apply test formats that determine a single analyte per assay.12 LC-MS/MS based protein quantitation offers significant gains in analyte multiplexing usage over immunoassay approaches. However, for these gains to be realized in a typical clinical laboratory, there is a need for robust and reproducible anaJournal of Proteome Research 2010, 9, 333–340 333 Published on Web 11/18/2009

research articles lytics, including preanalytics, specifically for reliable protein extraction methods that also allow for automation to increase precision and enable efficient sample throughput.13 These requirements are essential to allow the smooth transition from a research environment to routine sample analysis in a typical clinical laboratory.12 The development of an acetonitrile (ACN) precipitation method, in conjunction with an SRM based LC-MS/MS analysis, has demonstrated the efficient removal of high abundance and high molecular weight serum proteins while being able to accurately detect medium-abundance proteins like IGF-I.14 Furthermore, advances in LC instrumentation and reversed phase column chemistries have enabled the high-throughput quantitation of serum proteins and demonstrated accuracy closely matching that seen with a clinical analyzer.3 Coupling these two approaches enabled the quantitation of mediumabundance serum proteins in a large sample cohort using a 5-min LC-MS/MS analysis, and demonstrated good comparison to ELISA-based approaches.15 The technique is, however, dependent upon a single extraction methodology, which demonstrated protein-specific enrichment, and relied upon the use of ultra high-pressure capable instrumentation for rapid peptide chromatography. Thus, it was worthwhile to assess, develop and optimize alternative preanalytical extraction techniques and LC-MS/MS approaches that are more suitable for routine clinical LC-MS/MS applications. Like ACN precipitation, solid phase extraction (SPE) is a common technique in small molecule bioanalysis, and is being considered for clinical protein analysis.16 Recent developments in size exclusion-based SPE-sorbents have demonstrated the ability to reduce ion suppression in small molecule LC-MS/ MS analyses. Novel size exclusion material prevents large proteins from entering the pores of the solid phase while allowing small molecules to be retained on the reversed phase material. Compatibility of such mixed-mode, that is, size exclusion and reverse phase, SPE applications for clinical LCMS/MS analyses is yet another crucial preanalytical feature that needs to be addressed. To increase the accessibility of uHPLC-like performance, newly developed reversed phase chromatographic materials have demonstrated high-resolution separation similar to that obtained using sub-2 µm particle packing material without the need for UPLC-compatible pumps. The fused-core particle consists of a solid silica 1.7 µm particle with a thin coating (0.5 µm) of porous C18 reversed phase material.17 The lower diffusion distance in the particle enables the material to perform high-resolution chromatography, while the larger particle size significantly reduces the required backpressure allowing improved chromatography with a standard HPLC pump. To our knowledge, this chromatographic material has not been applied to the separation of proteotypic peptides from protein digests. To address the needs for preanalytical extraction techniques and their compatibility to clinically relevant LC-MS/MS settings, we set out to develop a novel protein extraction technique based on mixed-mode SPE, combined with rapid high resolution HPLC. To ensure comparability of these results between laboratories, standard human plasma (SHP) was used as a reference material to compare the new SPE protein extraction technique against an existing ACN depletion technique and raw plasma digests. SHP is commercially available in a lyophilized form making it an attractive material for the assessment, development, optimization, and comparison of various meth334

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Barton et al. ods and techniques in different laboratories including extraction methods.5 As a demonstration of the techniques’ quantitative ability, the two extraction techniques were then used to generate extraction and analysis methods capable of reliably quantifying IGF-I with high accuracy.

Experimental Procedures Chemicals. Acetonitrile and methanol (LC grade) was purchased from Romil (Cambridge, U.K.); water was produced by an option 4 water purifier (Elga, High Wycombe, U.K.). Dithiothreitol, iodoacetamide, ammonium bicarbonate, and formic acid were purchased from Sigma Aldrich (Poole, U.K.). Acetic acid was purchased from BDH, (Poole, U.K.). rhIGF-I was purchased from the National Institute for Biological Standards and Control (NIBSC, Potters Bar, U.K.). The stable isotope labeled internal standard (IS) peptide analogue of the IGF-I T1 tryptic peptide GPETLCGAELVDALQFV(13C515N1)CGDR was synthesized by Cambridge Research Biochemicals (Billingham, U.K.). The peptide’s homogeneity was evaluated by LC-MS, its sequence was confirmed by LC-MS/MS and its purity calculated using elemental analysis. The 96 well Varian Plexa SPE plate was purchased from Kinesis (St. Neots, U.K.). Trypsin gold was purchased from Promega (Southampton, U.K.). Pooled human plasma was purchased from Matrix Biologicals (Hull, U.K.), and the Standard Human Plasma (Lot 503208) was from Siemens Healthcare Diagnostics Products GmbH (Marburg, Germany). Acetonitrile Precipitation Method. For the initial method comparison experiment, plasma proteins were extracted in 1.5 mL Eppendorf Lo-Bind tubes (Cambridge, U.K.), while for the quantitative experiment, precipitation was performed in 2 mL 96 well plates. ACN precipitation was performed by the addition of 325 µL of a 2.25:1 ACN/water mixture to 50 µL plasma, and vortexed to mix. The ACN precipitation mixture contained the IGF-I T1 internal standard peptide at a final concentration of 300 ng/mL. Precipitated proteins were pelleted by centrifugation at 3000g for 10 min, and the supernatant was removed and evaporated to dryness in a rotary evaporator (Genevac, Ipswich, U.K.). Plexa SPE Extraction Method. Prior to extraction, 50 µL of plasma was mixed with 950 µL of either (1) 50 mM ammonium bicarbonate (neutral conditions), (2) 0.1% formic acid (acidic conditions), or (3) 0.3% ammonia (basic conditions) and incubated for 10 min at room temperature (RT). The IGF-I T1 internal standard peptide was added to all the loading solutions at a concentration of 150 ng/mL. Bond Elut Plexa mixed mode SPE cartridges (Varian, CA) were conditioned with 1 mL of methanol and 1 mL of water prior to addition of 1 mL of diluted plasma. The SPE cartridge was then washed with 1 mL of 5% ACN, and proteins were eluted with two sequential additions of 0.25 mL of 75% ACN with 0.1% formic acid (v/v). The extract was then dried down overnight in a rotary evaporator. Total plasma protein concentration in the extract was measured using the Bradford assay. Reduction, Alkylation and Digestion of Extracted Serum. Dried sample extract was reconstituted into 50 µL of 10 mM DTT and incubated at 60 °C for 60 min. Samples were allowed to cool to RT before the addition of 10 µL of a 100 mM iodoacetamide solution and incubated at RT in the dark for 30 min. Samples were exposed to light for 30 min before addition of 7.5 µL of a 100 µg/mL trypsin solution in 50 mM acetic acid (representing a 20:1 and 100:1 ratio of trypsin to protein, for the ACN and SPE extraction methods, respectively). Digestion

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High-Throughput Chemical Extraction Techniques Table 1. SRMs Used for Proteotypic Peptide Analysis of Target Proteins peptide details protein

proteotypic peptide

M1

M3

time

figure

source

APOC3 ALB CO3 A2MG HPT TRFE A1AT APOA1 IGG HEMO APOB CERU APOA2 A1AG1 APOC1 CRP APOA4 APOE TTHY RET4 IGF-I A IGF-I IS APOD APO F SHBG LCAT SAA IGF-II IGFBPIII

DALSSVQESQVAQQAR LVNEVTEFAK IPIEDGSGEVVLSR VGFYESDVMGR VTSIQDWVQK SASDLTWDNLK LYHSEAFTVNFGDTEEAK DYVSQFEGSALGK GPSVFPLAPSSK NFPSPVDAAFR TEVIPPLIENR GPEEEHLGILGPVIWAEVGDTIR EPCVESLVSQYFQTVTDYGK YVGGQEHFAHLLILR TPDVSSALDK GYSIFSYATK LGEVNTYAGDLQK SELEEQLTPVAEETR AADDTWEPFASGK YWGVASFLQK GPETLCGAELVDALQFVCGDR GPETLCGAELVDALQFVCGDR NILTSNNIDVK SGVQQLIQYYQDQK LPLVPALDGCLR SSGLVSNAPGVQIR FFGHGAEDSLADQAANEWGR GIVEECCFR YGQPLPGYTTK

858.9 575.4 735.9 630.3 602.3 625.3 686.7 700.8 593.8 610.8 640.8 829.8 1175.6 585.0 516.8 568.9 704.4 865.91 697.8 599.6 769.7 771.7 615.4 849.4 662.4 692.4 726.6 585.2 612.8

1144.6 694.4 903.5 793.4 803.4 776.4 749.3 1023.4 699.4 959.5 838.4 860.4 1436.6 835.6 834.3 716.4 631.4 801.4 921.4 693.4 881.4 887.4 1003.5 1085.5 901.5 941.5 931.4 771.2 876.5

1.52 1.79 1.89 1.90 1.90 1.95 1.95 2.08 2.13 2.20 2.22 3.22 3.31 2.10 1.44 1.25 1.57 1.80 2.00 2.61 3.15 3.15 1.74 2.17 2.67 1.70 1.99 1.54 1.48

A A B B A A B A A A B C A B A C B B B A C B A B C C C B B

Published Transition4 Published Transition4 MRMaid MRMaid MRMaid MRMaid MRMaid Published Transition3 MRMaid MRMaid/Published Transition4 Published Transition4 MRMaid Published Transition3 MRMaid Published Transition4 Published Transition28 Published Transition3 Published Transition3 MRMaid/Published Transition4 Published Transition4 Published Transition14 Published Transition14 Published Transition3 Published Transition3 In house Published Transition14 Published Transition3 In house Published Transition29

was performed overnight at 37 °C and quenched by the addition of 15 µL of a 1% formic acid solution in water (v/v). HPLC Separation of Tryptically Digested Plasma Extracts. HPLC separation was performed on an Ultimate 3000 system (Dionex) using an Ascentis Express C18 100 × 2.1 mm 2.7 µm particle column (Supelco). Solvents used for the analysis were A ) 0.1% formic acid in water (v/v) and B ) 0.1% formic acid in ACN (v/v). Sample (20 µL) was injected onto the column at an initial condition of 2% B rising to 35% after 3 min (curve 4). The column was then washed at 100% B for 1 min before returning to starting conditions for 1 min, totalling 5 min for the entire analysis. A constant flow rate was set at 0.5 mL/min and the column compartment was set at a constant value of 50 °C. MS/MS Analysis. The analysis of peptides eluting from the column was performed using a Sciex 4000 QTrap system operating in SRM mode, with the Turboionspray source in positive mode. The source temperature was set at 550 °C, a voltage of 5.5 kV, gases 1 and 2 at 60 psi and curtain gas at 30 psi. The SRM transitions used for the method comparison and quantitative experiments are displayed in Table 1. Peak areas from the LC-MS/MS analysis of the IGF-I standard curve and quality control (QC) samples were calculated using the Intelliquan algorithm in Analyst 1.4.1 (Sciex, Ontario, Canada). Generation of IGF-I Standard Curve and Quality Control Samples. A standard addition curve was generated by initially spiking a pooled plasma sample with rhIGF-I to give a concentration of 2000 ng/mL. The sample was then serially diluted with pooled plasma to generate concentrations of 1000, 500, 250, 125, 62.5, and 31.3 ng/mL. The pooled plasma used to generate the standard curves was also aliquoted to be

analyzed as a blank sample, which increased the number of calibration standards to 8. Three 50 µL aliquots of each calibration standard were extracted and analyzed using the ACN and basic SPE approaches and two aliquots for the Immulite assay. QC samples were generated at concentrations of 1000, 500, 250, and 125 ng/mL. Six 50 µL aliquots of each QC sample were analyzed by the ACN and basic SPE approaches and two for the Immulite assay. Eight 50 µL aliquots of SHP were analyzed by the ACN and basic SPE approaches, and three by Immulite. The stable isotope labeled internal standard peptide was added to each sample (to a final quantity of 300 ng/mL) prior to extraction to account for ion suppression effects in the source. A total of 3 replicates of each calibration standard, and six of each of the QC samples were extracted using ACN and basic SPE methodologies in 96 well plate formats. Samples were then analyzed on the LC-MS/MS system using the same LC gradient as described. The MS/MS method was altered such that only the SRMs for albumin, Apolipoproteins A1, A2, C3, IGFBP-III, IGF-I, IS peptide, plasma retinol binding protein, serum amyloid A and sex hormone binding globulin were monitored. This analysis involved two periods, where the method was split at t ) 2.2 min, so that more data points over each peak could be recorded to achieve quantitative information of higher quality. Immulite IGF-I Analysis. To have an appropriate benchmark, Siemens Immulite IGF-I analysis was performed. In a comparison study of five commercially available IGF-I assays, Siemens Immulite IGF-1 analysis has been reported to be the most precise method.11 In the reported setting, within-day coefficients of variation (CVs) were 1.7% in serum and 2.2% in Journal of Proteome Research • Vol. 9, No. 1, 2010 335

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Barton et al.

control material. Between-day CVs ranged from 1.9 to 3.5%. According to the manufacturer’s package insert, within-run CVs of the Immulite 2000 IGF-I assay are in the 2.3-3.9% range, total CVs are in the 4.7-8.1% range, and recoveries are reported to be in the 94-107% range. These data are in agreement with the ones observed in this study (data not shown). In view of ample data and its reliable application in routine clinical chemistry, a detailed analysis of variation and recovery for Immulite IGF-I analysis was not undertaken here as it would have been beyond the scope of this study. The Immulite IGF-I assay is a solid-phase, two-site chemiluminescent immunometric assay. The solid phase is represented by a bead that is coated with antibodies to specifically capture the analyte IGF-I. Reagent applied contains alkaline phosphatase that is conjugated to another antibody. Upon incubation, a sandwich complex is formed. Unbound enzyme conjugate is removed by a centrifugational wash. The addition of chemiluminescent substrate leads to the generation of a signal that is proportional to the concentration of IGF-I. Of note, Immulite 2000 IGF-I determination is approved for serum and heparinized plasma only. Commutability of results derived from other specimens has not been shown.

Results and Discussion Development of a 5-min LC-MS/MS Protein Analysis Method. To monitor a range of proteins in plasma, an LC-MS/ MS analysis method was first created. The list of proteins of interest was generated by considering proteins of various physicochemical properties in the nanogram per milliliter (ng/ mL) to milligram per milliliter (mg/mL) concentration range. Transitions specific to proteotypic peptides from 28 plasma proteins and a stable isotope labeled IGF-I T1 tryptic peptide were identified using a combination of existing published material, in-house developed SRMs and those suggested using the online SRM design software (MRMaid18). These were further developed, optimized and validated as described previously,2 and are summarized in Table 1. To maintain a highly multiplexed protein analysis, only one transition per peptide was analyzed. For detailed protein characterization and quantification, there is a case for using both multiple transitions and isotopically labeled internal standard peptides for analysis. However, when monitoring a single peptide for multiplexed SRM analysis, a single, well-characterized and suitably defined transition (with no chromatographic interferences) is often sufficient,3,15 and also allows for adequate MS duty cycle time. The single SRM transition targeting the IGF-I T1 peptide that was used in this, and previous studies, has demonstrated good analytical characteristics, suggesting that in this case the single SRM approach is fit-for-purpose.15 We next performed an experiment to study the use of fusedcore LC-MS/MS for peptide separation in human plasma. A rapid 5-min SRM-based LC-MS/MS analysis method was developed to monitor the target peptides using HPLC as opposed to the previously used uHPLC.3,19 SRM traces from the two analyses from an SPE plasma extract loaded using basic conditions are displayed in Figure 1. The data were split into three panels representing high-, medium-, and low-abundance peptides. The source of each of the peptide peaks in Figure 1 can be identified through its retention time, as stated in Table 1. The peak shapes of the proteotypic peptides of the 28 plasma proteins and the stable isotope labeled internal standard peptide demonstrated very good chromatographic properties, with all peak widths being 6 s or less at baseline. Further, the 336

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Figure 1. Extracted ion chromatograms from a 5-min LC-MS/MS analysis for 28 proteotypic peptides and one IS peptide. Peptides from the LC-MS/MS analyses are displayed in three panels relating to their relative abundance. Panel A contains 11 peptides, panel B contains 12 peptides, and panel C has 6 peptides. The corresponding identities of the peptides in each panel are noted in Table 1 and can be identified by their retention times.

stability of the IGF-I peptide retention time in the quantitative experiment was highly consistent with a run-to-run CV of 0.32% over the 112 injections. This demonstrates the excellent separation capability of the 2.7 µm fused-core particles, giving similar chromatographic characteristics to the smaller 1.7 µm particle columns, while maintaining a back pressure of 4000 psi as opposed to 10 000 psi with smaller particle columns. Comparison of Protein Concentrations in Extracted Plasma Digests. A method for the extraction of low molecular weight proteins from plasma using mixed-mode SPE was developed (for details see Experimental Procedures). To compare the effectiveness of the SPE techniques for plasma protein extraction to a previously characterized ACN depletion technique, a comparative experiment was performed. Aliquots of SHP were extracted using either the SPE technique with three sample loading conditions or the ACN depletion. For reference, aliquots of samples were also diluted into buffer as a control.

High-Throughput Chemical Extraction Techniques

research articles Likewise, apolipoprotein F levels are 20 times more concentrated in the basic conditions. This pH-dependency is apparent in the majority of the proteins analyzed, and suggests that when the Plexa SPE material is to be used for extracting specific proteins from plasma, loading conditions should be investigated thoroughly. The loading conditions allow the dissociation of smaller proteins (e.g., IGF-I) from complexes with larger binding proteins prior to the size-based enrichment. Interestingly, no correlation between protein pI and optimal preloading condition was found; indicating the prebinding conditions for a protein may depend on both its physicochemical properties and those of its binding partners.

Figure 2. Protein enrichment using ACN or SPE extraction. Proteins were extracted from plasma using ACN or SPE and analyzed by protein digestion/LC-MS/MS alongside unextracted plasma diluted into buffer. Eight extraction and analysis replicates were performed. Data on the ratio of signal in each extraction condition relative to that in unextracted plasma is shown for (a) high-abundance peptides and (b) low-abundance peptides in the LC-MS/MS analyses.

All samples were reduced, alkylated and digested prior to analysis by LC-MS/MS. Peptide peak areas were expressed as an enrichment factor with regard to the levels obtained in the unextracted plasma digest. This transformation was performed on all four extraction data sets and sorted by maximum enrichment; for clarity, see Figure 2. The graphs demonstrate that the ACN depletion technique is effective at removing high molecular weight proteins such as apolipoprotein B100, R-2-macroglobulin, caeruloplasmin, and albumin, which have molecular weights of 512, 160, 120, and 66 kDa, respectively. These proteins had an aggregate depletion factor in the extract of 40-fold relative to diluted plasma. The remaining extract (representing 0.4% of plasma protein by mass14) was enriched in lower molecular weight proteins, such as IGF-I and the small apolipoproteins. However, the ACN depletion method demonstrated poor recovery of R-1-antitrypsin, apolipoprotein E, and IGF-II which are 44, 34, and 7.8 kDa, respectively. This may indicate that enrichment is not based on size alone. Two proteins absent in the ACN extract were C-reactive protein and IGF-BPIII. The SPE-based extraction method demonstrated a lower efficiency for removing the high molecular weight proteins tested than the ACN method, with a depletion factor relative to dilute plasma of only 2.5- to 3-fold for the four large proteins described above. Bradford assay analysis demonstrated that the SPE extract retained approximately 2.1% of plasma protein by mass, compared to the 0.4% of protein retained by the ACN depletion method. However, the SPE extraction demonstrated similar, and in some cases superior, enrichment capabilities for the majority of the remaining proteins. The loading conditions dramatically affected the enrichment characteristics of the SPE material. For example, levels of albumin in the basic loading conditions are half that in the acidic conditions.

The optimum condition for extracting the target of interest IGF-I from plasma using the SPE cartridge was the basic loading conditions, which also gave the highest enrichment factor of all the extraction methods. The ACN method has been proven to be effective at extracting IGF-I from human serum,14 and this also appears to be the case for plasma. However, the ACN approach demonstrates a slightly lower enrichment factor than the basic SPE conditions (5.2 compared to 6.6, respectively). Absolute Quantitation of IGF-I in Human Plasma. For relevant clinical analysis of biomarker levels, assay accuracy, including both trueness and precision of measurement, is critical. Following development of two appropriate extraction techniques for IGF-I quantification (ACN depletion and SPE extraction), a comparison was made to assess the precision and trueness of measurement of the two approaches. Further, as protein LC-MS/MS is an emerging technique for protein quantification, an experiment was undertaken to compare these methods against a well-characterized, state-of-the-art immunological quantitative technique, the Immulite 2000 IGF-I assay.11 Standard addition curves, QC and SHP samples were extracted, processed and analyzed by LC-MS/MS. With the data generated, precision and trueness (accuracy) of the ACN and the basic SPE extraction methods were assessed using the same IGF-I calibration and QC plasma samples. To generate an IGF-I standard curve, the peak areas relating to the IGF-I T1 peptide were expressed as a ratio of the endogenous to the stable isotope labeled peptide. This was performed using standard addition for both the ACN and SPE extracted samples. Ratios were plotted and regression analysis provided a function of the expected concentration. The function of each line was used to identify the concentration of IGF-I in the pooled plasma. Concentrations of IGF-I were 125 and 100 ng/mL for the ACN and SPE extractions, respectively. These IGF-I concentrations were subsequently added to the expected concentrations of all calibration standards to generate standard addition curves (Figure 3a). The ACN and SPE derived standard addition calibration lines demonstrated good linearity of fit with R 2 values of 0.991 and 0.996, respectively. With the use of the equation of the two lines in Figure 3a, the IGF-I levels identified in the calibration standards were compared against the values identified by Immulite (Figure 3b). The R 2 values obtained from this comparison indicates a very high correlation between the LC-MS/MS and Immulite derived values. Furthermore, both the slope and intercept values of the two lines were very similar. The accuracy (measured based on inaccuracy or relative error (RE)) and precision (CV) for all calibration standards in both lines (Table 2) were adequate to pass the criteria required for validated methods for the quantitation of macromolecular compounds as specified in the guidelines for analysis in a regulated environment.20 Journal of Proteome Research • Vol. 9, No. 1, 2010 337

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Barton et al. derived from a pool of citrated plasma samples. Furthermore, K3 EDTA plasma was used to generate the IGF-I calibration samples, which could explain the differences between the analysis techniques. Assessment of the IGF-I Concentration in SHP. The developed assay was used to assess the concentration of IGF-I in SHP, for comparison to other techniques. The levels of IGF-I in SHP were calculated as 117 ( 10, 183 ( 25 and 119 ( 14 ng/mL for the SPE, ACN and Immulite assays, respectively. The IGF-I concentration assigned by the SPE and Immulite values differ by only 2 ng/mL, demonstrating extremely good comparability. However, the level assigned by the ACN depletion assay was significantly higher. This is potentially due to the SHP being a surrogate plasma matrix, which has been modified to increase protein stability for clinical analyses. This modification to the plasma may adversely affect the ACN depletion, as the ACN assay was successful when using K3 EDTA plasma. Therefore, the application of the ACN depletion technique to modified plasma could generate spurious results.

Conclusions

Figure 3. (a) Standard addition curves for IGF-I using ACN or SPE extraction. Plasma was spiked with plasma of a known IGF-I level, extracted and analyzed by LC-MS/MS. Peak area ratio is plotted against total plasma IGF-I concentration (including endogenous IGF-I) for SPE extraction (n ) 3; red) and ACN extraction (n ) 3; black) (b) Comparison of IGF-I concentrations identified in the calibration standards using both LC-MS/MS and Immulite approaches. The mean plasma IGF-I concentrations in the calibration standards for both SPE and ACN extraction were compared against the level identified by Immulite.

The concentration of IGF-I in the QC samples was assigned using the equation of the calibration line and the precision and trueness of the measurements calculated. The CVs of the concentrations for the two LC-MS/MS based approaches ranged from 1.5% to 6.4% and 6.8% to 11.1% for the SPE and ACN analyses, respectively. The trueness of measurements, reflected by RE achieved for the QC samples, ranged from 3.5% to 12.6% and 5% to 14.3% for the SPE and ACN approaches, respectively (Table 2). The Immulite derived concentrations for the calibration and QC plasma samples were similar to those achieved using the LC-MS/MS based approach. The analyzer assigned a blank plasma IGF-I concentration of 125.5 ng/mL, which is in close agreement with the ACN derived concentration of 125 ng/mL. One noticeable difference between the LC-MS/MS and Immulite values is that the HPLC-MS/MS derived IGF-I values are consistently higher. Respective differences are common when different assays are compared. This is particularly true for completely different technologies, even more so when calibration and traceability are different. For example, it was noted in a previous study that LC-MS/MS assigned Apolipoprotein A1 concentrations were consistently higher than the corresponding clinical analyzer values.3 The discrepancy may be due to differences in calibration and commutability. Of note, the appropriate specimens to be tested by the Immulite assays are serum and heparinized plasma. SHP is a processed specimen 338

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Two plasma protein extraction methods were used to demonstrate that HPLC-MS/MS and SRM detection can accurately quantify clinically relevant proteins in plasma. A direct comparison of the two extraction approaches demonstrated that both techniques reduced the levels of high abundant and high molecular weight plasma proteins, while enriching for the lower molecular weight protein fraction. The ACN depletion technique was highly effective at removing albumin, however, failed to enrich for specific low molecular weight proteins such as IGFBP-III and CRP. The mixed-mode SPE material demonstrated pH-dependent extraction characteristics, with the basic loading conditions giving the best enrichment factors for the majority of proteins. The pH-dependent recovery indicates that multiple loading conditions should be investigated when using the mixed-mode SPE material to identify optimal recovery of specific proteins. The different extraction efficiencies and characteristics of each of the depletion methods indicate that, as for small molecule bioanalysis, different proteins may require different techniques for reproducible extraction and quantification in plasma. For example, high molecular weight proteins, such as albumin, gave more reproducible precision in unextracted plasma, while the lower molecular weight IGF-I was more reproducibly measured by SPE extraction and Apolipoprotein F by ACN extraction (see Supplementary Table 1). The use of chemical-based extraction technologies, therefore, provides a complementary tool to immuno-affinity techniques prior to LC-MS/MS. Our data suggests that both ACN and SPE extraction techniques could be used for method development purposes when targeting specific midabundance proteins in plasma using an LC-MS/MS based detection strategy. For example, the SPE extraction method demonstrated enrichment for two GH-related biomarkers, IGF-I and IGFBP-III, with levels in plasma in the nanogram per milliliter (ng/mL) range. The method could, therefore, be used in combination with tryptic digestion and LC-MS/MS for detecting GH abuse. For lower abundance GH biomarkers, for example, PIIINP, another GH abuse marker, a combination of either immuno-enrichment of the target analyte21 or immuno-depletion of abundant proteins7 would be required to reach an appropriate lower limit of quantification.

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High-Throughput Chemical Extraction Techniques

Table 2. Precision and Accuracy Data for Standards and QCs Using ACN or SPE Extraction and HPLC Analysis concentration (ng/mL) spiked

Endogenous (0) 31.3 62.5 125 250 500 1000 2000 LLOQ (125) LOW QC (250) MED QC (500) HIGH QC (1000) SHP

basic SPE

ACN

endogenous + spiked (ng/mL)

calculated (ng/mL)

CV (%)

RE (%)

endogenous + spiked (ng/mL)

calculated (ng/mL)

CV (%)

RE (%)

100 132 162 225 350 600 1100 2100 225 350 600 1100 N/A

85.0 128 150 218 345 633 1120 2070 233 360 657 1237 117

8.7 11.5 7.5 10.0 11.9 9.5 7.6 9.6 6.4 2.5 1.5 6.4 6.9

-13.3 -0.8 -7.0 -4.7 -0.8 5.9 1.8 -1.4 4.3 3.5 9.9 12.6 N/A

125.0 155.0 187.0 250.0 375.0 625.0 1125.0 2125.0 250 375 625 1125 N/A

150 162 199 243 378 609 1182 2160 282 410 713 1180 183

14.5 4.5 7.3 0.8 1.4 5.7 10.0 0.8 6.8 11.1 7.4 6.9 8.6

21.2 4.7 6.4 -2.4 1.1 -2.4 5.0 1.6 13.2 9.5 14.3 5.0 N/A

The use of SHP allowed for the comparison of multiple extraction methodologies. The material is supplied in a lyophilized form, and is stable for a long period of time prior to rehydration, making it an attractive material for the development and optimization of methods as well as interlaboratory method comparison experiments and benchmarking exercises. As already suggested, SHP as well as other reference materials used in clinical chemistry for calibration and QC purposes could be circulated throughout the proteomics community for QC purposes, enabling precision and trueness of measurement calculations, for example, for quantitative LC-MS/MS assays. Reference materials with assigned values for specific proteins would be of particular interest.5,12 The protein extraction and sample analysis processes used in this study did not require specialist equipment such as antibody depletion columns or high pressure capable LC instrumentation. This negates the need for costly equipment and means that the assays described in this paper could be easily transferred to other laboratories for LC-MS/MS based protein quantitation experiments. This includes clinical laboratories where triple quadrupole MS instrumentation is already used routinely for newborn screening to detect inborn errors of metabolism,22,23 toxicology and forensic applications,24 immunosuppressive drug testing,25 drugs of abuse26 and dope testing of athletes.27 The method developed in this study may in principle be automated. To determine the impact of automation on precision was beyond the scope of this study, but it is justified to assume that precision will further increase upon automation. Automation would also increase throughput. A very short turnaround-time is not crucial for the biomarkers addressed in this study. Thus, the time period for sample extraction and digestion is not an obstacle and clinical laboratories with a sufficiently high number of requests for individual determinations could establish an automated, efficient high-throughput setting. Furthermore, the technology is capable of quantifying multiple proteins in a single sample aliquot, which would increase throughput even further. In addition, this could reduce the costs and required sample volume of using multiple immunoassay based approaches. The ACN and SPE extraction techniques were applied to quantify levels of IGF-I in plasma, and both demonstrated very high precision and trueness of measurement characteristics. Aliquots of the same plasma samples were also analyzed using the Immulite 2000 utilized in clinical laboratories worldwide

and a good comparability between the three approaches was found. This demonstrated that the application of tryptic digestion and LC-MS/MS analysis to the quantitation of clinically important plasma proteins is possible in reproducible and accurate manner. Abbreviations: ELISA, enzyme linked immuno sorbent assay; IGF-I, insulin-like growth factor I; GH, growth hormone; rhIGFI, recombinant human insulin-like growth factor I; SRM, single reaction monitoring; SHP, standard human plasma; SPE, solid phase extraction.

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