Original Paper Immuno-capture as ultimate sample cleanup in LC-
Transcription
Original Paper Immuno-capture as ultimate sample cleanup in LC-
J. Sep. Sci. 2009, 32, 2937 – 2943 Bjørn Winther1 Marianne Nordlund2 Elisabeth Paus2 Lon Reubsaet1 Trine Grønhaug Halvorsen1 1 Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo, Oslo, Norway 2 Central Laboratory, Radiumhospitalet, Rikshospitalet, Oslo University Hospital, Oslo, Norway B. Winther et al. 2937 Original Paper Immuno-capture as ultimate sample cleanup in LCMS/MS determination of the early stage biomarker ProGRP This paper presents a selective and efficient sample preparation procedure for NLLGLIEAK, signature peptide for the small cell lung cancer (SCLC) biomarker ProGRP, in human serum. The procedure is based on immuno-capture of ProGRP in 96-wells microtiter plates coated with the mAb E146. After immuno-capture and thorough rinse, trypsin was added for in-well digestion. Subsequently the signature peptide was enriched by SPE and determined by LC-MS/MS. Various steps in the procedure were optimized to achieve a low LOD such as dilution of sample, tryptic digestion, and SPE cleanup and peptide enrichment conditions. A single quadropole MS was used during optimization of the method. A triple quadropole MS was used in the method evaluation in order to improve sensitivity. The evaluation showed good repeatability (RSD, 11.9 – 17.5%), accuracy (3.0 – 6.6%), and linearity (r2 = 0.995) in the tested range (0.5 – 50 ng/mL). LOD and LOQ were in the pg/mL area (0.20 and 0.33 ng/mL, respectively), enabling the determination of clinically relevant concentrations. The method was applied to two patient samples and showed good agreement with an established immunological reference method. The final method was compared to a previous published LC-MS method for the determination of ProGRP in serum based on protein precipitation and online sample cleanup. Both showed acceptable method performance, however, the immuno-capture LC-MS method was superior with respect to sensitivity. This illustrates the large potential of immunocapture sample preparation prior to LC-MS in protein biomarker quantification. Keywords: Immuno-capture / LC-MS/MS / ProGRP / Signature peptide / Tryptic digestion / Received: April 2, 2009; revised: May 25, 2009; accepted: May 25, 2009 DOI 10.1002/jssc.200900233 1 Introduction Progastrin releasing peptide (ProGRP) is a protein biomarker, which among other biomarkers, in the case of increased concentrations indicates small cell lung cancer (SCLC) [1 – 4]. This aggressive disease, with rapidly growing neoplasm and early metastasis, is highly sensitive to early initiated systemic chemotherapy and radiation. This makes early diagnosis and treatment monitoring crucial. The reference value for ProGRP in human serum is l60 pg/mL [5], but the ProGRP concentration can, in the Correspondence: Dr. Lon Reubsaet, School of Pharmacy, Department of Pharmaceutical Chemistry, University of Oslo, P.O. Box 1068 Blindern, NO-0316 Oslo, Norway E-mail: [email protected] Fax: +47-22854402 Abbreviations: PPT, protein precipitation; ProGRP, progastrin releasing peptide; RAM, restricted access media; SCLC, small cell lung cancer i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim case of SCLC, increase to low and medium ng/mL levels [2]. Concentration determination is being currently carried out using immunoassay with enzymatic or fluorescence detection [5, 6]. In general, this methodology with reason accounts for a large amount of protein determinations at low concentrations. With the rapid growing interest in protein analysis using proteomic approaches, chromatographic separations coupled to mass spectrometric detection (LC-MS) is an emerging technique in absolute protein quantification. Although more complex and less robust compared to the common immunochemical methods, it is less prone to cross-reactivity and has a competitive sensitivity. It has been demonstrated that several proteins like prions in brain tissue [7], the kidney dysfunction marker cystatin C [8], hemoglobin A2 in whole blood and dried blood spots [9], as well as the potential serum biomarker for prostate cancer Zn-a2 glycoprotein [10] can be quantitatively determined by this approach. Determination was achieved by first subjecting the sample to tryptic digestion and then subse- www.jss-journal.com 2938 B. Winther et al. quently monitoring tryptic peptide(s) specific for the proteins to be determined. A single specific tryptic peptide will then be sufficient to determine the equimolar amount of protein it originates from assuming reproducible digestion. Such a specific tryptic peptide is defined as a signature peptide. ProGRP has also been determined using this approach. In this case, the tryptic peptide NLLGLIEAK was identified as signature peptide. Concentrations down to 1.5 ng/mL (l200 fmol/mL) ProGRP in serum could be detected using acetonitrile (MeCN) induced protein precipitation and online sample cleanup prior to chromatography. Although clinically relevant, this LOD is still above the reference value [11, 12]. Attempts to improve the LOD by using a triple quadropole mass spectrometer instead of a single quadropole mass spectrometer only contributed to a less complex chromatogram but not to a lower LOD. This was discussed in ref. [12] and explained by possible ion-suppression, which might be circumvented by better sample preparation. The combination of advantages from both immunoaffinity and LC-MS is fronted as the next phase of clinical applications to determine very low abundant proteins in complex biological matrices. Two different strategies have been described using either antibodies for protein or antibody for proteolytic peptides, both reporting promising results [13 – 17]. The antibody for protein strategy has one great advantage as these antibodies are often already available, making the strategy more easily employable. In the current paper, the potential of an immuno-capture step in the LC-MS based ProGRP determination was evaluated. The low reference value of ProGRP has proven to be a challenge. Even optimized conventional sample preparation techniques, such as protein precipitation combined with online restricted access media (PPT-RAM), have fallen short with respect to sample cleanup and sensitivity [12]. The convenience of available antibodies for ProGRP [5] made it feasible to investigate immuno-capture as a sample cleanup step prior to tryptic digestion and LC-MS. A comparison of immuno-capture sample preparation and the PPT-RAM technique, applied to ProGRP, is performed with focus on sample cleanup, and effect on the LOQ. In addition applicability of the immuno-capture LC-MS method on realistic samples will be discussed. 2 Experimental 2.1 Chemicals ProGRP (31 – 98) was produced and patient samples were provided by the Central Laboratory, Radiumhospitalet, Rikshospitalet, Oslo University hospital (Oslo, Norway). All participants had given written informed consent to i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim J. Sep. Sci. 2009, 32, 2937 – 2943 participate in the study. TPCK treated and lyophilized sequencing grade trypsin from bovine pancreas was purchased from Sigma – Aldrich (St. Louis, MO, USA) and human serum samples from healthy subjects were obtained from Oslo University Hospital, Ullevaal (Oslo, Norway). All other chemicals used were of analytical grade. 2.2 Preparation of standards Stock solutions of ProGRP (31 – 98) were prepared in 100 mM aqueous triethanoleamine (TEA) at pH = 7.3. These solutions were stored at – 328C and care was taken to avoid excessive freeze – thaw cycles. The stock solutions were used to spike serum samples for the immunocapture experiments and to prepare the ProGRP in 50 mM freshly prepared ammonium bicarbonate buffer used in the in-solution digestion experiments. Serum samples and ammonium bicarbonate buffer were spiked on the day of the experiments. The volume of spiking was kept negligible compared to the total volume of sample. 2.3 LC-MS 2.3.1 Single quadropole The system consisted of a Shimadzu SIL-10ADvp auto injector, two Shimadzu LC-10ADvp gradient pumps, a Shimadzu DGU-14A degasser, a Shimadzu SCL-10Avp system controller, and a Shimadzu LCMS-2010A singlequadropole MS detector. Data acquisition and processing were carried out using Shimadzu LCMS Solution software Version 2.04-H3 (all Bergman, Lillestrøm, Norway). The ESI source was operated in the positive ionization mode. The MS was set to monitor the ProGRP-specific digest peptide NLLGLIEAK [M + 2H]2+ m/z 485.8. The MS operating conditions were as follows: drying gas between 10 and 20 L/min, nebulizer gas 1.5 L/min, CDL temperature 2008C, block temperature 2008C and probe voltage +4.5 kV. 2.3.2 Triple quadropole This system consisted of a Waters 2795 liquid chromatograph, Waters 600 LCD controller and a Waters Quattro micro-MS/MS detector. System control and data acquisition was performed with MassLynxm version 4.0 SP4 (all Waters Corporation, Milford, MA, USA). The interface was ESI operated in the positive ionization mode. The MS was set to monitor the precursor-fragment ion transition of the ProGRP-specific digest peptide NLLGLIEAK [M + 2H]2+ (m/z 486.01) to the y7 ion [M + H]+ (m/z 743.74). The optimized fragmentation conditions [12] for the SRM of NLLGLIEAK were with a cone voltage of 25 V and www.jss-journal.com J. Sep. Sci. 2009, 32, 2937 – 2943 collision energy at 14 eV. The dwell time was 600 ms with an inter-scan delay of 100 ms. 2.4 Chromatographic conditions Chromatographic separation was carried out on a Biobasic-C8 (Teknolab AS, Kolbotn, Norway) column with average pore size 300 , particle diameter 5 lm and column dimensions 50 mm61 mm id. The mobile phases consisted of A: 20 mM aqueous formic acid and MeCN (95:5 v/v) and B: 20 mM aqueous formic acid and MeCN (5:95 v/v). A two-step linear gradient was used. The system was first kept isocratic at 5% mobile phase B for 1 min after injection of the sample. The first gradient was then run from 5% mobile phase B to 20% mobile phase B in 4 min, and was immediately followed by the second gradient running from 20 to 30% mobile phase B in 11 min. Mobile phase B was then increased to 60% in 0.1 min and kept constant for 2 min before it was returned to starting conditions in 0.1 min. The column was regenerated with 10 column volumes. Flow rate was set to 50 lL/min and the injection volume was 20 lL. Liquid Chromatography 2939 After each step, the flow-through fraction was collected. All fractions were evaporated to dryness under a stream of N2 gas at 608C, reconstituted in 25 lL of 20 mM aqueous formic acid and analyzed on the content of NLLGLIEAK. 2.5.3 Final SPE procedure Up to 125 lL of sample was applied. After a washing step (50 lL of 15% MeCN in 20 mM aqueous formic acid) the peptide was eluted using 50 lL of 45% MeCN in 20 mM aqueous formic acid. The eluate was evaporated to dryness under a stream of N2 gas at 608C, reconstituted in 25 lL of 20 mM aqueous formic acid and analyzed on the content of NLLGLIEAK. 2.5.4 Calculation of extraction recoveries The extraction recoveries were defined as the percentage of the total analyte amount (originally applied to the SPE tip), which was recovered after SPE elution, evaporation to dryness and reconstitution. 2.6 Preparation and use of 96-well immuno plates 2.6.1 Coating of mAb E146 to microtiter plates 2.5 SPE SPE was carried out by using in-house made SPE tips. All tips were activated using 100 lL of 95% MeCN in 20 mM aqueous formic acid and then washed with 100 lL of 20 mM aqueous formic acid prior to use. Each step in the SPE procedure was carried out by loading the solution on the top of the tip. In order to press the liquids through the SPE tip, it was placed on a 10 mL syringe. Pressure was applied manually. 2.5.1 Preparation of in-house SPE tips A Pasteur pipette was used to punch six small cushions (diameter l1 mm) from a 3M Emporem C18 disk (Teknolab AS, Kolbotn, Norway). The C18 material was transferred from the Pasteur pipette to the bottom of a 300 lL pipette tip (TIP 300 lL Bevel bulk, VWR International, Oslo, Norway) using a thin metal wire. After transfer, the six cushions were carefully pressed together in the narrow, lower part of the tip. 2.5.2 Determination of optimal SPE conditions Optimization of the SPE procedure was performed in samples digested in-solution (sample volume 300 lL containing 50 ng/mL ProGRP in 50 mM freshly prepared ABC and 100 ng trypsin, digested o/n at 378C). The optimization was carried out as follows: after application of 50 lL of digested sample the flow-through of the sample solution from the SPE was collected. Then six 50 lL aliquots of solutions with increasing amounts of MeCN (0 – 90% in 20 mM aqueous formic acid) were applied to the tip. i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim The mAb E146 was produced by immunizing mice with recombinant ProGRP (31 – 98) and establishing a hybridoma cell line as described by Nordlund et al. [6]. Immobilization of purified E146 (1 lg/well) was then performed on Maxisorp microtiter plates (Nunc, Copenhagen, Denmark) as described earlier [18]. In brief, the antibody solution (1 mg/mL) was first acid treated for 10 min at pH 2.8 by adding six volumes of 100 mM glycine buffer pH 2.5, and then neutralized with 200 mM sodium dihydrogen phosphate buffer pH 4.3 to an antibody concentration of 5 lg/mL. After dispensing 200 lL of solution in each well, the microtiter plates were incubated under humidified conditions at 378C for 20 h. Then the plates were washed twice with Delfia wash solution (50 mM Tris, 150 mM NaCl, 0.1% Germall, and 0.05% Tween 20 pH 7.8, PerkinElmer Life and Analytical Sciences, Waltham, MA, USA) and incubated with 300 lL/well of blocking buffer (50 mM Tris, 6% sorbitol, 0.05% azide pH 7.0) for another 20 h under humidified conditions at room temperature. The plates were aspirated and kept dry until use. 2.6.2 Immuno-capture The immuno-capture method was based on the immunocapture step in the immunofluorometric assay described in ref. [6]. The samples (fortified serum from healthy subjects and patient samples) were applied to the 96-well immuno microtiter plates in 200 lL aliquots. When the effect of sample dilution prior to immuno-capture was tested, the serum samples were diluted with an aqueous protein-containing buffer consisting of 50 mM Tris-HCl www.jss-journal.com 2940 B. Winther et al. pH 7.8, 150 mM NaCl, 20 lM diethylene triamine pentaacetic acid, 100 mg/L Tween 20, 3 mg/L tartrazine, 1 g/L Germall, 5 g/L BSA, 1 g/L bovine IgG, and 60 mg/L MAK 33-IgG pPoly [5]. One hundred microliter of this buffer was added to wells containing 100 lL of serum, thus diluting the sample twice. To capture the proteins the plates were subsequently shaken for 1 h at 500 rpm on a Heidolphm Vibramax vibrating platform shaker (Kelheim, Germany) to facilitate antigen – antibody interaction. Afterward, the plates were washed six times with Delfia wash solution using a Delfia Platewash (both PerkinElmer). Subsequently, the plates were washed manually twice with 10 mM Tris-HCl pH 7.4 to ensure removal of Tween and Germall prior to digestion since these components might interfere with SPE and LC-MS analysis. J. Sep. Sci. 2009, 32, 2937 – 2943 Figure 1. Plot of the accumulating amount of NLLGLIEAK normalized to 100% (lstdev) against the eluting strength in SPE (n = 3). Extraction recovery, 62%. After washing, 200 lL of 50 mM freshly prepared ammonium bicarbonate buffer was added to the wells. Tryptic digestion was carried out by adding 1 lL of freshly prepared trypsin in 50 mM ammonium bicarbonate buffer. During optimization the trypsin concentration varied from 0.001 to 10 mg/mL. The wells were sealed using parafilm and placed on the Heidolph Vibramax vibrating platform shaker and shaken for 5 min at 800 rpm. Following this, the wells were placed in a 378C stove allowing overnight digestion. Up to 125 lL of the sample was subjected to SPE. retention. With immuno-capture and in-well digestion, it is in addition important that the signature peptide is not a part of the amino acid sequence of the antibodies and proteins present on the wall of the wells. A BLAST experiment (http://blast.ncbi.nlm.nih.gov/) for the sequence NLLGLIEAK in databases for Homo sapiens (tax. ID 9606), murine (tax. ID 10090), and bovine (tax. ID 9913) showed that in similarity with proteins originating from humans, none of the proteins used in the immunocapture procedure contained this amino acid sequence. We could therefore conclude that NLLGLIEAK is a valid signature peptide for this study. 2.7 Evaluation of the final method 3.2 SPE To evaluate the potential of immuno-capture, SPE, and LC-MS/MS analysis of ProGRP, repeatability (n = 5) and accuracy (n = 3) were tested at 2, 15, and 50 ng/mL ProGRP in human serum. In addition, the linearity of the response was evaluated at seven concentrations (n = 2): 0.5, 2, 7, 15, 25, 50, and 100 ng/mL. LOD and LOQ were estimated from the peak heights of samples fortified with 0.5 and 2 ng/mL ProGRP in relation to the noise level. S/N of 3:1 was used to estimate LOD and S/N of 5:1 was used to estimate LOQ. The method evaluation was performed using the triple quadrupole. 3.2.1 Application, wash, and elution 2.6.3 In-well digestion 3 Results and discussion 3.1 Selection of signature peptide As reported in earlier studies [11, 19], NLLGLIEAK can be used as signature peptide for ProGRP. Selection of the signature peptide and the specificity of this peptide is thoroughly described earlier [11]. In short, this selection was based on peptide signal intensity, absence of missed cleavage sites, peptide specificity, and adequate column i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim To optimize the SPE wash- and elution-steps, three inhouse made SPE tips were used. After sample application flow-through fractions from each elution-step with increasing amounts of MeCN in 20 mM aqueous formic acid were determined on their content NLLGLIEAK. Figure 1 shows the cumulative relative amount of NLLGLIEAK plotted against the MeCN content of the eluent. The cumulative amount was determined by summing the signal intensities of each elution step with the signal intensity of all prior elution steps for the individual SPE tip. From Fig. 1 it is clear that washing can be performed using 20 mM aqueous formic acid containing 15% MeCN without loosing NLLGLIEAK, while 30% MeCN results in virtually complete elution. To ensure a robust system with complete elution of NLLGLIEAK from the SPE column, eluent containing 45% MeCN in 20 mM aqueous formic acid was used for the remainder of the studies. 3.2.2 Recovery of SPE after in-well digestion After optimization of the SPE wash and elution conditions the final conditions were applied to enrich an inwww.jss-journal.com J. Sep. Sci. 2009, 32, 2937 – 2943 Liquid Chromatography 2941 Figure 2. Plot of NLLGLIEAK intensity against the amount of trypsin added (n = 2). well digest of 25 ng/mL ProGRP in serum after immunocapture. Extraction recovery was determined to 65% (n = 2; average peak height prior to SPE, 5242; average peak height after SPE, 16 963; preconcentration factor (125 lL fi 25 lL), five times). Linearity up to 50 ng/mL, as shown later, indicates that this value for recovery is achieved at all tested concentration levels. Figure 3. Single MS chromatogram (SIM at m/z = 485.8) of the signature peptide for (a) undiluted serum and (b) diluted serum after immuno-capture, in-well digestion and SPE. Both samples were fortified with 1 ng/mL ProGRP. 3.3 Evaluation of the in-well digestion 3.3.1 Effect of trypsin concentration on the in-well digestion The amount of trypsin to be used is related to the amount of protein present. In an in-solution digest this ratio varies from l1:25 (w/w) to lower ratios. In the case of inwell digestion, it is difficult to estimate the total protein amount since in addition to the protein of interest both antibodies and other proteins utilized during preparation and immuno-capture will be present in the well during digestion. To circumvent this challenge the effect of the amount of trypsin added was investigated. Figure 2 shows the intensity of the signature peptide plotted against the amount of trypsin added. The lowest trypsin amount added to the wells, which gave good digest with high NLLGLIEAK formation, was 100 ng. This amount was achieved by adding 1 lL of 100 lg/mL trypsin to each well, and was used throughout the study. 3.3.2 Dilution of the samples In immunoaffinity assays, serum samples are often subjected to dilution prior to application on the microtiter plates. In order to investigate the possible positive effects of serum dilution during immuno-capture, a comparison was made between undiluted serum and serum diluted 1:1 with a protein-containing buffer, as described in Section 2. Figure 3 shows the typical SIM-traces (m/z = 485.8) of LC-MS (single-quadropole) analyses of both undiluted and diluted serum. It is clear that the undiluted serum (Fig. 3a) yields higher peak intensities for both the signa- i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ture peptide and impurities compared to that of the diluted serum (Fig. 3b). The noise level was similar in both analyses, hence, highest S/N was obtained for undiluted serum. It was, therefore, decided to use undiluted serum for the subsequent experiments. 3.4 Evaluation of the method 3.4.1 Method performance The final method was validated using an LC-MS/MS (triple quadrupole) for increased sensitivity. Optimal SRM conditions had been determined previously [12]. Repeatability, accuracy, linearity and LOD, LOQ were determined for the ProGRP signature peptide NLLGLIEAK in human serum after immuno-capture, in-well digestion and SPE. Table 1 shows the results for repetitive extractions at three different ProGRP concentrations. RSDs were similar at low, medium, and high ProGRP concentrations. Table 2 shows the values for accuracy determined at three concentrations of ProGRP. These values were also independent of the concentration of ProGRP analyzed. A calibration curve was constructed from 0.5 to 100 ng/mL ProGRP in human serum. A quadratic polynomial curvature (y = – 0.6709x2 + 213.25x + 83.095) fitted concentrations up to 100 ng/mL well (r2 = 0.997). However, as apparent linearity was observed between 0.5 and 50 ng/mL ProGRP (y = 182.1x + 226, r2 = 0.995), it was decided to evaluate the typical validation parameters for www.jss-journal.com 2942 B. Winther et al. J. Sep. Sci. 2009, 32, 2937 – 2943 Figure 4. Triple quadrupole ionextract of the SRM transition 486.01 fi 743.74 of (a) blank human serum, (b) human serum from subject 1, and (c) human serum from subject 2. The signal intensity of the NLLGLIEAK fragment (m/z = 743.74) at tr = 17.07 min in (c) equals 100% signal intensity. Table 1. Within-day repeatability of ProGRP from human serum samples (n = 5) Spiked ProGRP conc. (ng/mL) Signal intensity RSD (%) NLLGLIEAKa) (average l SD) 2 15 50 564 l 70 3295 l 576 9647 l 1144 12.4 17.5 11.9 Table 3. Analysis of patient samples Subject 1 Subject 2 ProGRP immuno- ProGRP immunoMS (ng/mL) fluorometric assaya) (ng/mL) Bias (%) 0.72 26.62 – 29 15 1.01 23.24 a) a) SRM transition: 486.01 fi 743.74. Table 2. Accuracy of ProGRP from human serum samples (n = 3) Spiked ProGRP conc. (ng/mL) Determined ProGRP conc. (ng/mL) Accuracy (%) 2 15 50 2.1 15.4 52.5 6.6 3.0 5.1 concentrations up to 50 ng/mL ProGRP in human serum using linear calibration. The LOD and LOQ were 0.2 and 0.33 ng/mL ProGRP in human serum, respectively. The evaluation shows acceptable repeatability, accuracy, and linearity. The LOD and LOQ were in the pg/mL area, which is of clinical relevance. 3.4.2 Analysis of patient samples The method was applied to the analysis of serum from two subjects with elevated serum-ProGRP level. Table 3 i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Described by Nordlund et al. [5], and regarded as reference method as it is used on routine basis for analyzing patient samples. Comparison with established immunofluorometric assay. shows the average ProGRP concentration (n = 2) of the two subjects determined both by the established immunofluorometric assay (described by Nordlund et al. [5]) and with the immuno-capture LC-MS/MS method. Serum levels of ProGRP assayed by the two procedures were comparable, indicating that immuno-capture LC-MS/MS has a potential in ProGRP quantification. However, the LOQ (16 pg/mL) of the immunofluorometric assay is still superior to that of the present immuno-capture method. Figure 4 shows the ion-extracts of the SRM transition 486.01 fi 743.74 for a blank human serum sample as well as for subjects 1 and 2. 3.4.3 Method comparison The analytical performance of the immuno-capture method was compared with the performance of the previously described LC-MS method utilizing protein precipitation and online sample cleanup (PPT-RAM method [12]). The LOD improved with a factor of 7.5 utilizing the www.jss-journal.com J. Sep. Sci. 2009, 32, 2937 – 2943 immuno-capture method. This improvement was achieved despite a five-fold reduction in the sample volume (from 1 to 0.2 mL), illustrating the large potential of combining immuno-capture and LC-MS/MS. Both the immuno-capture method and the PPT-RAM method showed acceptable and comparable repeatability and calibration performance. While the accuracy of the two methods was comparable at higher ProGRP concentrations the accuracy of the immuno-capture method was superior to the PPT-RAM method in the lower range (accuracy of 22% at 5 ng/mL vs. 6.6% at 2 ng/mL, respectively). However, contrary to the PPT-RAM method the values for the immuno-capture method was obtained without the use of internal standard, indicating the potential of even better method performance by applying internal standard for quantification. Liquid Chromatography 2943 any of these strategies is chosen a more thorough validation including the use of internal standard should be employed in order to ensure reliable quantification of the biomarker(s). The authors wish to thank Niclas Lunder from the Department of Psychopharmacology, Diakonhjemmet Hospital, Oslo, Norway for his valuable contribution to the analysis on the triple quadrupole MS/MS. The authors declared no conflict of interest. 5 References [1] Molina, R., Filella, X., Aug, J. M., Clin. Biochem. 2004, 37, 505 – 511. [2] Yamaguchi, K., Stieber, P., J. Lab. Med. 2003, 27, 26 – 30. 4 Concluding remarks The present work is the first report on an LC-MS based ProGRP analysis using a selective immuno-capture step. Selective immuno-capture of ProGRP in 96-wells microtiter plates followed by in-well digestion and subsequent SPE enrichment of the signature peptide prior to LC-MS/ MS analysis resulted in efficient sample cleanup and enrichment from human serum. Good repeatability, accuracy, and linearity were seen in the tested range. In addition, LOD and LOQ were in the pg/mL area, enabling reliable determination of clinical relevant concentrations. Two patient samples were analyzed and the method showed good agreement with an established immunological assay. A comparison with an LC-MS method for ProGRP utilizing conventional sample preparation methods demonstrated the potential of immuno-capture sample pretreatment especially with respect to sensitivity. 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D., Otnes, B., Skovlund, E., J. Urol. 1998, 159, 1599 – 1605. [19] Winther, B., Reubsaet, J. L. E., J. Sep. Sci. 2007, 30, 234 – 240. www.jss-journal.com