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High throughput SISCAPA quantitation of peptides from human plasma digests by ultrafast, liquid chromatography-free mass spectrometry Morteza Razavi, Lauren E Frick, William A LaMarr, Matthew E Pope, Christine A Miller, Leigh Anderson, and Terry William Pearson J. Proteome Res., Just Accepted Manuscript • Publication Date (Web): 05 Nov 2012 Downloaded from http://pubs.acs.org on November 6, 2012

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Journal of Proteome Research

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High throughput SISCAPA quantitation of peptides from human plasma digests by ultrafast, liquid chromatography-free mass spectrometry

Morteza Razavi,1 Lauren E. Frick,2 William A. LaMarr,2 Matthew E. Pope,3 Christine A. Miller,4 N. Leigh Anderson,3,5 and Terry W. Pearson*1,3

1Department

2Agilent

of Biochemistry and Microbiology, University of Victoria, Victoria, BC Canada.

Technologies, Inc., 11 Audubon Road, Wakefield, MA 01880, USA.

3SISCAPA

Assay Technologies, Inc., P.O. Box 53309, Washington, DC 20009, USA.

4Agilent

Technologies, Inc., 5301 Stevens Creek Boulevard, Santa Clara, CA 95051, USA.

5Plasma

Proteome Institute, Red Cross Holland Lab, Rockville, MD 20855, USA.

ACS Paragon Plus Environment


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ABSTRACT: We investigated the utility of an SPE-MS/MS platform in combination with a modified SISCAPA workflow for chromatography-free MRM analysis of proteotypic peptides in digested human plasma. This combination of SISCAPA and SPE-MS/MS technology allows sensitive, MRM-based quantification of peptides from plasma digests with a sample cycle time of ~7 seconds, a 300-fold improvement over typical MRM analyses with analysis times of 30-40 minutes that use liquid chromatography upstream of MS. The optimized system includes capture and enrichment to near purity of target proteotypic peptides using rigorously selected, high affinity anti-peptide monoclonal antibodies and reduction of background peptides using a novel treatment of magnetic bead immunoadsorbents. Using this method, we have successfully quantitated LPS-binding protein and mesothelin (concentrations of ~ 5000 ng/mL and ~10 ng/mL, respectively) in human plasma. The method eliminates the need for upstream liquid-chromatography and can be multiplexed, thus facilitating quantitative analysis of proteins, including biomarkers, in large sample sets. The method is ideal for high-throughput biomarker validation after affinity enrichment and has the potential for applications in clinical laboratories. KEYWORDS: SISCAPA, anti-peptide antibodies, multiple reaction monitoring, mass spectrometry, SPE-MS/MS, RapidFire, biomarkers, validation


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INTRODUCTION

Mass spectrometry (MS) is increasingly used to measure proteins in biological fluids, where it provides an effective method for reproducible quantitation of clinical biomarkers1. The most widely applied quantitative MS method, multiple reaction monitoring (MRM)-MS analysis of proteotypic peptides from trypsinized biological samples, provides good precision for measuring relatively abundant peptide (protein) analytes2 but has typically required lengthy chromatographic separations upstream of the mass spectrometer. The limited sensitivity of such assays (µg/mL – high ng/mL levels in unfractionated digests, even with nanospray ionization, although this sensitivity is rapidly improving; see Picotti and Aebersold 3) and the low throughput (typically 30 - 40 min/sample) associated with peptide chromatography represent major limitations in the validation of candidate clinical biomarkers. Effective validation of biomarker proteins in plasma, of which thousands have been reported, requires analysis of 1,000 - 2,000 samples2, often with sensitivities typically in the sub-ng/mL range4,5. Improvements in MRM throughput and sensitivity necessary to generate such data are therefore critical for biomarker translation, and might also allow MS to replace immunoassays for routine measurement of protein markers in clinical laboratories6.



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In order to boost MRM performance to the required level, the method known as stable isotope standards and capture by anti-peptide antibodies (SISCAPA ) has been developed to enrich low abundance peptides from complex mixtures such as trypsin digested human plasma or serum7. While SISCAPA significantly improves the sensitivity of MRM assays, the workflow has usually employed peptide separation chromatography upstream of MS analysis to reduce MRM interferences from remaining non-specific peptide background. The cycle times for SISCAPA assays have decreased from 30 - 40 minutes using nanoflow (~300 nL/min) high performance liquid chromatography/MS8 to approximately 3 minutes using standard flow (~ 1.2 mL/min) HPLC, while retaining equivalent sensitivity through use of an Agilent 6490 mass spectrometer equipped with iFunnel technology9. Recently, we reported a modification of the SISCAPA workflow that provides a further significant reduction of non-specific background peptide binding by the immunoaffinity matrices required for peptide enrichment10. This improvement allows enrichment of highly pure peptide analytes, thus eliminating the requirement for chromatographic separation prior to MS analysis and enabling direct analysis of target peptides by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) MS10. The ability to prepare highly pure analyte peptides led us to examine the use of a solid-


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phase extraction (SPE) -MS/MS system11,12 as a means to achieve very high throughput peptide quantitation while retaining the specificity and sensitivity advantages of MRM detection. Here we describe a SISCAPA-SPE-MS/MS workflow that allows quantitation of multiple peptide analytes with a sample cycle time of ~7 seconds. More specifically, the time that is required to transfer the sample (after trypsin digestion and peptide enrichment) to the mass spectrometer and acquire the necessary transitions is ~7 seconds. The optimized methods include an “addition only” protocol to enable facile, efficient and reproducible tryptic digestion of plasma samples, the use of “crude” tryptic digests without prior solid-phase peptide extraction, selective capture of target proteotypic peptides using very high affinity anti-peptide rabbit monoclonal antibodies, reduction of background peptides to extremely low levels using a novel treatment of magnetic bead immunoadsorbents and ultra-fast injection of samples into an Agilent 6490 triple quadrupole mass spectrometer using SPE-MS/MS technology (RapidFire system-see Materials and methods). The SPE-MS/MS system that we used is an ultra-fast online SPE platform coupled to a mass spectrometer11,12 that has been extensively employed for the MS analysis of high



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throughput screening campaigns against large compound libraries and for absorption, distribution, metabolism and excretion (ADME) studies of drug candidates. The instrument aspirates a portion of each sample (directly from 96-or 384-well assay plates) onto a small SPE trapping column chosen to retain the analyte(s) of interest. After a washing step, the sample is reverse eluted from the SPE trapping column and sprayed directly into a mass spectrometer for detection. The entire cycle time between aspiration of one sample and the next is ~7 seconds.

MATERIALS AND METHODS

Target Protein Analytes and Surrogate Proteotypic Peptides. Protein analytes of varying abundance in human plasma were selected to test the general utility of the methods developed in this work (Table 1). Synthetic tryptic proteotypic peptides chosen as surrogates of protein analytes were used. Peptides that occur in a single protein encoded within the human genome and that yield several, strong MRM transitions in a triple quadrupole mass spectrometer were selected according to criteria previously described7. Peptides were synthesized by solid-phase methods by the Chinese


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Peptide Company (Hangzhou, China) or by the UVic-Genome BC Proteomics Centre (Victoria, BC, Canada). Stable-isotope-labeled versions of selected peptides were made by chemical synthesis at the UVic-Genome BC Proteomics Centre or by JPT Peptide Technologies GmbH (Berlin, Germany). A mass increment was added in each case through the use of labeled C-terminal arginine (+ 10 amu) or lysine (+ 8 amu), providing mass shifts of m/z = + 5 or + 4 for typical doubly-charged peptide ions. All peptides were of greater than 90 % purity as determined by HPLC. Upon receipt from the vendors, peptide stocks were adjusted to approximately 10 nmol/µL (based on dry weight) in 30 % acetonitrile/0.1 % formic acid. However, stock concentrations determined by peptide weight are inaccurate due to differing hygroscopic properties, hydrophobicity and solubilities of peptides; thus aliquots of these initial stocks were sent for quantitation by amino acid analysis (AAA; Advanced Protein Technology Centre, The Hospital for Sick Children, Toronto, Ontario). Using AAA data, the peptide stocks were then readjusted to 10 nmol/µL. Peptides were diluted immediately prior to use and were stored for short periods (2 weeks or less) at 4 °C in solution phase. After thawing and/or just before use, all peptides were analyzed by MALDI-TOF-MS to determine their integrity and to assess the presence of altered forms.



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Peptide Combination Curves. Peptide curves were generated by using a ‘combination strategy’ to generate samples containing both light and heavy peptides (L+H). To generate a combination curve for each target, 10 pmol/μL stocks of both light and heavy peptides were prepared based on the AAA results. Two mixtures were prepared from these stocks representing the ends of an 11-point curve: Mixture A with L:H ratio of 50:1 and Mixture B with L:H ratio of 1:50 at a final peptide concentration of 510 fmol/μL covering a dynamic range of 2500 fold. Equal volumes of Mixture A and Mixture B were combined to make a 1:1 dilution representing the center of the curve. Other points on the curve were similarly generated by mixing samples with varying ratios of L:H peptides. Using this strategy, the overall peptide concentration (L+H) remains the same in all samples while varying L:H ratios are created. Five different tryptic peptides were used in a 5-plex format and the curves were generated in triplicate.

Anti-Peptide Monoclonal Antibodies.


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Rabbit anti-peptide monoclonal antibodies (RabMAbs) specific for surrogate tryptic peptides of mesothelin and LPS Binding Protein (LBP) were made by Epitomics Inc. (Burlingame, CA) after immunizing rabbits with peptide coupled via thiol linkage through N- or C-terminal cysteines to keyhole limpet hemocyanin (KLH). Anti-peptide antibodies in hybridoma supernatants were first screened by “peptide ELISA”13. To select high affinity anti-peptide RabMAbs, screening of ELISA positive hybridoma supernatants was performed using a combination of surface plasmon resonance13 and MALDI immunoscreening (MiSCREEN)14 . It is important to emphasize that the anti-peptide antibodies must be of at least nanomolar affinity, which roughly translates to a half off-time of ~10 minutes13, 14, to bind peptides from solution and to hold on to them through extensive washing steps to obtain optimal enrichment with minimum non-specific background binding of peptides.

Tryptic Digestion of Human Plasma Proteins. We developed an “addition only” method to enable facile, efficient and reproducible tryptic digestion of plasma samples. We also developed an optimized washing procedure to remove both lipid impurities and peptides that bind non-specifically to the magnetic



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immunoadsorbent beads after incubation with trypsin-digested plasma (see below). This washing procedure allows the trypsin-digested plasma to be used directly, without the need for expensive and time-consuming off-line SPE of each digest. The method has been tested using plasma volumes of 10-1000 µL. To do this, the appropriate amount of urea, the reducing agent tris(2-carboxyethyl)phosphine (TCEP) and Tris buffer were first mixed and lyophilized. TCEP was added to 2.0-fold molar excess over the estimated average cysteine concentration of 0.25 M in plasma and urea was added to 9 M final concentration to achieve protein reduction and denaturation. In the experiments reported here, 1000 µL of plasma were digested by adding the plasma to the denaturation mix followed by 30 min incubation at room temperature. Iodoacetamide was added to the solution at 1.5 molar excess followed by an additional incubation for 30 min at room temperature in the dark. Lastly, the reaction mixture was diluted 1:9 (final urea concentration of 1 M), trypsin (Worthington Cat. No. LS003740) was added at a 1:20 ratio of enzyme to substrate and the sample was incubated 16 hours at 37 °C before adding a 2-fold excess of tosyl-L-lysine chloromethyl ketone (TLCK) to stop the tryptic cleavage. The digested samples were used without further processing.


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SISCAPA Assay Protocol. The SISCAPA assay described here used trypsin-digest from 30 µL of plasma for antibody capture of peptides. All procedures were performed using a KingFisher 96 bead-handling robot (Thermo Electron Corporation, Vantaa, Finland). A schematic diagram of the generic SISCAPA peptide enrichment and assay procedure is shown in Figure 1. For the work reported here, KingFisher 96-deepwell microplates were used for antibody and peptide capture whereas KingFisher 96-well standard microplates were used for peptide elution. A series of KingFisher plates was first prepared as follows: Plate 1: A Bead Wash Plate containing 1.43 µL/well of MyOne Protein G Dynabeads (Invitrogen-Dynal; custom made 1.0 micron diameter; low peptide binding) brought up to 200 µL/well in PBS/0.03% of the zwitterionic detergent 3-[(3cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPS). The detergent was used to prevent peptide loss throughout sample handling. CHAPS is compatible with mass spectrometry and elutes late in reversed-phase peptide separation15. It is important to note that with the LC-free RapidFire system used here, the CHAPS is removed from our sample (by extensive bead washing prior to peptide elution from the magnetic bead immunosorbent) before the peptides are loaded onto the RapidFire SPE



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column. If other magnetic protein G beads are to be used, such as the commercially available Protein G Dynabeads, then the bead concentration must be adjusted so that each well contains enough beads to allow binding of 1.0 µg of antibody. Plate 2: An Antibody Capture Plate containing 1.0 µg/well of desired antibody brought up to a final volume of 100 µL/well in PBS/0.03% CHAPS. Plate 3: A Peptide Capture Plate containing the trypsin digest of 30 µL plasma. The corresponding Stable Isotope Standard (SIS) peptides were added at this stage at 500 fmol/well. Here the samples can be prepared as a “master mix” before addition to the plate as long as the final concentration of the digest and the amount of SIS peptide per well remain unchanged. Plates 4 and 5: Two Wash Plates (#1 and #2) containing 250 µL/well of PBS/0.03% CHAPS. Plate 6: A Wash Plate (#3) containing 400 µL/well of 75% acetonitrile in PBS/0.03% CHAPS. This step removes non-specifically bound background peptides. Plate 7: An Elution Plate containing 75 µL/well of 0.1% formic acid. Briefly, the washed beads were transferred to the antibody plate where they are allowed to capture 1 μg of the relevant antibodies. The bead-antibody complex was then transferred to the plasma digest


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plate to capture the target peptides. The non-specifically bound peptides were washed away by the optimized wash buffer in the Wash Plates and finally the captured peptides were eluted in the elution buffer, which in the current work was split equally for analysis using both RapidFire/6490 QQQ and LC/6490 QQQ platforms.

Determination and Selection of Optimum MRM Transitions. MRM transitions for each surrogate peptide were first determined by direct infusion into an Agilent 6490 QQQ mass spectrometer. The transition list was used to examine each peptide after RapidFire 300 direct injection into the MS and transitions that gave robust signals with no interferences were selected for our subsequent SISCAPA-SPE-MS/MS experiments. The optimum collision energies for these MRM transitions were also determined on an Agilent 6490 triple quadrupole mass spectrometer using ultra highpressure chromatographic (UHPLC) separation. The transitions and conditions used are shown in Table 2. Mass Spectrometric Peptide Detection (SPE-MS/MS) The predetermined optimum MRM transitions for antibody-enriched peptide were analyzed on an Agilent RapidFire 300 High-throughput Mass Spectrometry System using an



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Agilent 6490 QQQ mass spectrometer fitted with an electrospray ionization source and operating in the positive MRM mode. The RapidFire/MS platform is a fully automated, online, microfluidic sample preparation system. Samples are aspirated directly from 96well (or 384 well) assay plates and loaded onto a micro-scale solid-phase extraction (SPE) cartridge. The salts, buffers, and other polar “contaminants” are washed through the cartridge while the analytes of interest are retained. The purified analytes are then eluted from the cartridge directly into the mass spectrometer for analysis. More specifically, the instrument aspirated aliquots of each sample sequentially, removing sample until the sip sensor determined that the 10 µL loop was full (usually about 200 ms). The contents of the loop were then applied to an SPE cartridge A (C4 packing material; Agilent) and washed with ultrapure H20 supplemented with 0.1% formic acid for 3000 ms. The purified sample was reverse-eluted using 90% ultrapure acetonitrile supplemented with 0.1% formic acid in a 3000 ms step and sent to the mass spectrometer, which was already monitoring the mass transitions of interest for that well. A re-equilibration of 500 ms brought the total cycle time to approximately 7 s. The system is hereafter referred to as SPE-MS/MS. Data analysis was performed using RapidFire Integrator v3.4 software, which generated an output file of integrated peak areas for each MRM transition for each well in the sequence.


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Mass Spectrometric Peptide Detection (LC/6490 QQQ). Peptide samples were analyzed with a system consisting of a 6490 triple quadrupole mass spectrometer coupled to a 1290 Infinity UHPLC using a JetStream interface (Agilent). A 20 μL aliquot of each sample was separated on a 2.1 x 50 mm Zorbax 300 SB-C18 column with a flow rate of 1.2 mL/min. The target peptides were separated using a 3-minute gradient with 0.1% formic acid in water as solvent A and 90 % acetonitrile in 0.1% formic acid in water as solvent B. From initial conditions of 10 % B, a gradient was developed to 16 % B at 1 min, 22 % B at 1.5 min, 40 % B at 1.85 min, 70% B at 1.9 min, then back to 10 % B from 1.95 min to 3 min for column re-equilibration. Source conditions included drying gas at 200 °C, sheath gas at 250 °C and 11 L/min flow for both drying and sheath gases. Ions were isolated in Q1 using 1.2 FWHM resolution and in Q3 using 0.7 FWHM resolution. Peptide fragmentation was performed using collision energies optimized for each transition.

RESULTS AND DISCUSSION

Peptide Analysis by SPE-MS/MS.



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The utility of SPE-MS/MS for ultra-fast throughput MRM analysis of proteotypic peptides has not been investigated before, primarily because the SPE-MS/MS system, unlike standard LC/MS platforms used for MRM analysis, does not separate peptides chromatographically prior to injection into the mass spectrometer – all molecules in each sample are injected as a single peak approximately 2 seconds wide (Figure 2). We first tested the linearity and reproducibility of peptide responses in the SPE-MS/MS system using a range of concentrations of five pairs of pure synthetic tryptic peptides (unlabeled = light, L; stable isotope labeled = heavy, H) dissolved in 0.1% formic acid (for peptide sequences and masses see Table 1). The light and heavy peptides were combined in varying L:H ratios as previously described for SISCAPA-MALDI assays10. The results of these peptide analyses are shown in Figure 3. By pairing unlabeled peptides with stable isotope labeled chemically identical versions and measuring varying L:H ratios (as is the normal procedure in SISCAPA assays) the R2 values were > 0.99 for four of the peptides and > 0.97 for the surrogate peptide from LPS Binding Protein (LBP) (Figure 3A). These results suggest that the platform can be used for peptide quantitation in simple mixtures over at least a 2,500-fold range in abundance. Ten peptides were combined for this analysis, clearly demonstrating that multiplexed measurement is possible.


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Instrument reproducibility was tested using triplicate samples that formed combination curves (L:H ratio curves) for the five peptide analytes. As an example, the spectrum for the mesothelin surrogate peptide is shown in Figure 3B. The average percent coefficient of variation (% CV; within-run) over the 2500-fold dynamic range for the 5 peptides ranged from 6.0 % - 8.3 % (Figure 3C). It is likely that not every tryptic peptide will be captured on the C4 SPE matrix and not every peptide will be soluble in 90% acetonitrile. Fortunately, with the five peptides studied in this manuscript (and with several others not reported here that are being used in other projects) we did not observe any lack of peptide binding or insolubility on the SPE trapping column used. If we had, the analytical protocols could easily be adjusted as seven standard packing materials are available in the SPE cartridges (cyano, C4, C18, phenyl, graphitic carbon and HILIC) and any of several standard solvents (acetonitrile, methanol, ethanol, methylene chloride, etc) can be used at any concentration. This bodes well for designing assays for specific enriched peptides, for example in specific clinical applications.

Peptide Enrichment By High Affinity Monoclonal Antibodies.



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Effective peptide enrichment requires antibodies that specifically bind the peptide analyte with high affinity. Since the SISCAPA workflow involves stringent washing procedures, the capture antibodies must have slow off-rates to allow retention of the target peptides. Much effort was expended to select the appropriate antibodies using a combination of peptide ELISA13, surface plasmon resonance13 and MiSCREEN14 analysis. As shown by surface plasmon resonance kinetic analysis (Figure 4), the rabbit monoclonal antibodies that we selected for the work reported here had slow off-times (224 minutes for the anti-LBP peptide RabMAb- Figure 4A; and 33 minutes for the anti-mesothelin peptide RabMAbFigure 4B), both much greater than the minimum of 10 minutes required for the modified SISCAPA workflow.

Reducing Non-Specific Peptide Binding in SISCAPA Assays. We have previously found (unpublished observations) that a variety of magnetic bead immunoadsorbents tested in peptide immunoenrichment assays show non-specific (NS) binding of a number of peptides and that these are generally derived from major plasma proteins (such as albumin). To minimize NS peptide binding and thus maximize the purity of the analyte peptides, we tested a variety of different types of magnetic beads made by


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several different manufacturers and optimized washing steps to maximize removal of NS peptides. After extensive testing for antibody and peptide capture capacity, ease and efficiency of manipulation using the KingFisher® 96 bead-handling robot (Thermo Electron Corporation, Vantaa, Finland) and our ability to achieve low levels of background peptide contamination as assessed by MS, we found custom-made 1.0 micron diameter Dynabeads® MyOneTM Protein G (Invitrogen Dynal AS, Oslo, Norway, Cat No. 109.03D) to offer a good combination of properties. These beads exhibit low background binding and their small diameter (increased surface area/volume ratio) increases antibody binding capacity, making them more suitable for multiplex analyses where a number of antibodies (at 0.1-1 µg each) are used in a single SISCAPA capture reaction. If multiplexing of more than 10 analytes is not intended or required, we found that commercially available 2.8 micron diameter Protein G Dynabeads (Invitrogen Dynal AS, Oslo, Norway, Cat. No. 30015D) perform well. An important modification from previous published SISCAPA protocols is the inclusion of a final wash with 3:1 acetonitrile/PBS just prior to acid elution of the enriched peptides. MALDI-TOF analyses of processed samples with and without optimized binding and washing are shown in Figure 5.



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SPE-MS/MS Quantitation of Mesothelin. To demonstrate that the SISCAPA-SPE-MS/MS system can be used for measuring analytes in trypsin-digested human plasma, we analysed replicate SISCAPA peptide response curves for a low-abundance protein, mesothelin (~ 10 ng/mL, a level typically observed by SISCAPA-MRM analysis using LC-MS/MS). We compared the RapidFire/Agilent 6490 QQQ MS platform with a standard-flow LC/Agilent 6490 QQQ MS platform. Unfractionated, trypsin-digested human plasma was used in all experiments. The quantitated amount for mesothelin was identical between the two MS platforms (Figure 6A-D) and the average % CV of peak area ratios for endogenous mesothelin peptide (0 fmol spike level of the light peptide on the forward curve) was 7.4 % on the SPE-MS/MS platform, which is similar to the 8.1 % average precision observed when pure mesothelin peptide was analysed over a 2500-fold dynamic range. It is important to note that three different transitions for the L and H mesothelin peptides were measured to control for interferences and that triplicate samples were analysed for determination of the average % CV of peak area ratios. We hypothesized that the high purity of peptides obtained by immunocapture in our optimized SISCAPA workflow would allow a wide variety of peptides to be quantitated by the platform. This was the case, as was demonstrated using five proteotypic peptides


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derived from protein targets that differ widely in concentration in human plasma, even when multiplexed. It is important to note that these five peptides were not specifically selected to perform well using this SPEMS/MS system and were only chosen to yield good MRM transitions and to represent protein analytes of a range of concentrations in human plasma. Despite the lack of LC separation of peptides, we were able to monitor 10 MRM transitions from the 10 analyte peptides (5 L + 5 H) that eluted together in a single 2second-wide peak (Figure 2). This allows the quantitation of 5 protein analytes if only 1 transition per surrogate peptide is monitored (both L and H peptides must be measured for each analyte). Thus the method described here currently allows 5-plex detection of peptide analytes with a 7-second cycle time, although with careful selection of peptide transitions a higher level of multiplexing should be attainable. The ultra-fast analysis with the SPE-MS/MS system is made possible by the high purity of the enriched peptides achieved by enrichment with highly selected RabMAbs and by optimized immunoaffinity peptide capture. It is important to note that he overall time per sample includes the sample preparation time, trypsin digestion and peptide enrichment. Sample preparation time and digestion is required by all peptide-based protein analysis methods using serum/plasma samples and this is clearly a rate-limiting step, with one proviso: parallel



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processing of samples, which is performed offline, is being pursued by us and others using robotics and will become less of a bottleneck, especially since in a high-throughput, high capacity environment, further sample processing can occur on-line as part of the sample analysis process as demonstrated here. The SPE-MS/MS technique that we describe is designed to process already prepared samples with a 7-second sample cycle time, compared to the 30-40 minute sample cycle time typically required for LC-MS/MS methods. Some peptides (often with a C-terminal arginine) do ionize well by MALDI and are thus amenable to analysis by SISCAPA-MALDI10. However, many peptide analytes will require analysis by the SPE-MS/MS method described here. The method and platform was also used to successfully measure the plasma levels of LPS Binding Protein (LBP), a moderately high abundance protein (~ 5 μg/mL) in human plasma (data not shown). Thus, as proof of principle, we were able to enrich, from trypsindigested human plasma, surrogate proteotypic peptides from a moderate abundance protein (LBP) and a low abundance analyte (mesothelin) and to elute them with sufficient purity to be analysed by the SPE-MS/MS platform with no HPLC separation prior to MS, analysis, thereby decreasing the sample cycle time to ~ 7 seconds. Our data show that quantitation of both LBP and mesothelin was identical with both LC/MS and LC-free MS.


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Also, the presence or absence of liquid chromatography with the system we used here did not affect the sensitivity achieved. A coefficient of variation of less than 8% for the SISCAPA-SPE-MS/MS quantitation of both LBP and mesothelin was within the range of instrument variation observed when pure peptides were injected. By enriching target peptides to near purity with optimized SISCAPA immunoenrichment methods and by taking advantage of the speed of the RapidFire/6490 QQQ MS platform (SPE-MS/MS), we have developed a high throughput, multiplexible, biomarker validation platform that we believe is ready for wide-scale application. Using this method, a plate of 96 samples can be analyzed in less than 15 minutes and a 1,500 sample validation study can be performed in approximately 3 hours. By coupling the SISCAPA-SPE platform with the 6490 mass spectrometer, multiplexed analysis of 5 peptides was achieved (a total of 10 transitions; 5 L and 5 H), which bodes well for future multiplexed determination of protein concentrations.

ASSOCIATED CONTENT Supporting Information AUTHOR INFORMATION



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Corresponding Author *Phone: (250) 721-7080. E-mail: [email protected] Notes N.L.A and T.W.P. are founders and M.E.P. is an employee of SISCAPA Assay Technologies, Inc. a company formed to commercialize SISCAPA technology. L.E.F., W.A.L. and C.A.M. are employees of Agilent Technologies, Inc., the company that sells both the RapidFire platform and the 6490 ion-funnel mass spectrometer highlighted in the work reported here.

ACKNOWLEDGMENTS The authors thank Genome Canada and Genome BC for platform funding and support of the UVic-Genome BC Proteomics Centre where some of the peptide synthesis and determination of MRM transitions were performed. Specifically we are grateful to Angela Jackson and Alex Camenzind for their expert help with peptide MRM determinations and peptide quality control by MALDI-TOF MS, respectively. We also thank Peter Rye at Agilent for performing some of the RapidFire runs. We acknowledge the Government of British Columbia through the Pacific Century Graduate Scholarship Program and the University of


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Victoria for a Graduate Award in support of M.R. during his PhD research. This work was supported in part by the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant 8405-2006 to T.W.P.

ABBREVIATIONS MS, mass spectrometry; MRM, multiple reaction monitoring; SISCAPA, stable isotope standards and capture by anti-peptide antibodies; LBP, lipopolysaccharide binding protein; MALDI-TOF, matrix-assisted laser desorption ionization time of flight; QQQ, triple quadrupole; SPE, solid-phase extraction; AAA, amino acid analysis; RabMAbs, rabbit monoclonal antibodies; ELISA, enzyme-linked immmunoadsorbent assay, CHAPS, 3-[(3cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate; SIS, stable isotope standards; MiSCREEN, MALDI-immunoscreening.



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REFERENCES (1)

Hortin, G.L; Carr, S.A; & Anderson, N.L. Introduction: Advances in protein analysis for the clinical laboratory. Clin. Chem. 2010, 56, 149-151.

(2)

Anderson, N.L. The roles of multiple proteomic platforms in a pipeline for new diagnostics. Mol. Cell. Proteom. 2005, 4, 1441-1444.

(3)

Picotti, P; Aebersold, R. Selected reaction monitoring-based proteomics: workflows, potential, pitfalls and future directions. Nat Methods. 2012, 9, 555-9566.

(4)

Addona, T.A; et al. Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma. Nature Biotechnology. 2009, 27, 633-641.

(5)

Keshishian, H; et al. Quantitative, multiplexed assays for low abundance proteins in plasma by targeted mass spectrometry and stable isotope dilution. Mol. Cell. Proteom. 2007, 6, 2212–2229.

(6)

Kiernan, U.A; et al. High-throughput protein characterization using mass spectrometric immunoassay. Anal Biochem. 2002, 301, 49-56.

(7)

Anderson, N.L; et al. Mass spectrometric quantitation of peptides and proteins using Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA). J.


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Proteome Res. 2004, 3, 235-244. (8)

Kuhn, E; et al. Inter-laboratory evaluation of automated, multiplexed peptide immunoaffinity enrichment coupled to multiple reaction monitoring mass spectrometry for quantifying proteins in plasma. Mol. Cell. Proteom. 2011, E-pub PMID: 22199228.

(9)

Miller, C; Pope, M; Razavi, M; Pearson, T.W.; Anderson N.L. Optimization of a rapid chromatographic method for a multiplexed SISCAPA assay. J. Amer. Soc Mass. Spec. 2012, 23, 147.

(10) Anderson, N.L; et al. Precision of heavy-light peptide ratios measured by MALDITOF mass spectrometry. J. Proteome Res. 2012, 11, 1868-1878. (11) Hutchinson, S.E; et al. Enabling lead discovery for histone lysine demethylases by high-throughput RapidFire mass spectrometry. J. Biomol. Screen. 2012, 17, 39-48. (12) Highkin, M.K; et al. High-throughput screening assay for sphingosine kinase inhibitors in whole blood using RapidFire® mass spectrometry. J. Biomol. Screen. 2011, 16, 272-277.



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(13)

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Pope, M.E; et al. Anti-peptide antibody screening: selection of high affinity monoclonal reagents by a refined surface plasmon resonance technique. J. Immunol. Meth. 2009, 341, 86-96.

(14)

Razavi M; et al. MALDI immunoscreening (MiSCREEN): a method for selection of anti-peptide monoclonal antibodies for use in immunoproteomics. J. Immunol. Meth. 2011, 364, 50-64.

(15)

Anderson, N. L. et al. SISCAPA peptide enrichment on magnetic beads using an inline bead trap device. Molecular & Cellular Proteomics. 2009, 8, 995–1005.


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Table 1. Protein Targets and Proteotypic, Surrogate Peptides

Stable Surrogate Protein Target

Peptide

Endogenous

Isotope

Mass (Da)

Mass Shift

Peptide Sequence

Abbreviation (Da) Protein C PCI

EDQ YHY LLD R

1351.44

+10

Thyroglobulin

TgFSP

FSP DDS AGA SAL LR

1406.52

+10

Thyroglobulin

TgVIF

VIF DAN APV AVR

1271.48

+10

LBP

LAE GFP LPL LK

1197.48

+8

Meso

LLG PHV EGL K

1062.28

+8

Inhibitor

LPS Binding Protein Mesothelin



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Table 2. MRM transitions and collision energies for the 10 proteotypic peptides

Surrogate

Q1 (m/z)

Q3 (m/z)

Collision Energy

PCI light

451.2

403.2

11

PCI heavy

454.5

413.2

11

TgFSP light

703.8

586.8

19

TgFSP heavy

708.8

591.8

19

TgVIF light

636.4

541.3

16

TgVIF heavy

641.4

551.4

16

LBP light

599.4

680.5

17

LBP heavy

603.4

688.5

17

Meso light

354.9

418.7

4

Meso heavy

357.5

422.7

4

Peptide


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Figure Legends Figure 1. Schematic representation of the SISCAPA method. High affinity anti-peptide rabbit monoclonal antibodies (RabMAbs) coupled with optimized immunoadsorbent wash steps allow significant reduction of non-specifically adsorbed “background” peptides.

Figure 2. MRM transitions for a multiplex peptide assay. Light and heavy transitions from a 5-plex peptide analysis are shown eluting in a single 2-second duration peak. For clarity, only results from 3 peptide analytes (both H + L) are plotted (LPS binding protein, protein C inhibitor and mesothelin).

Figure 3. Peptide quantitation using the SPE-MS/MS platform. (A) An 11-point peptide titration curve was created by the combination of light and heavy peptides at different ratios (L:H of 50:1 to 1:50) such that a dynamic range of 2500 fold was covered. All 10 peptides (5 L + 5 H) were run as a multiplex. Using a power fit law, R2 values of greater than 0.99 were observed for four of the analytes while the R2 for LBP was > 0.97. (B) Combined spectra (overlaid) showing one light and one heavy transition for the


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mesothelin surrogate peptide through the 11-point curve. (C) % CV from triplicate runs of the 11-point curve for every point on the curve.

Figure 4. Surface plasmon resonance analysis of RabMAbs. (A) Surface plasmon resonance kinetic analysis of LBP-specific anti-peptide rabbit monoclonal antibody demonstrating its slow off-rate (Kd = 5.16 E -5; half off-time = 224 min). (B) SPRdetermined kinetic analysis for mesothelin-specific anti-peptide rabbit monoclonal antibody (Kd = 3.53 E -4; half off-time = 33 min). These highly selected antibodies allow specific peptide binding during extensive washing to achieve peptides of high purity, necessary for elimination of LC upstream of MS.

Figure 5. MALDI-TOF mass spectra showing low non-specific peptide background. (A) A MALDI-TOF spectrum showing poor specific peptide enrichment from trypsindigested human plasma due to the presence of non-specific background peptides, mainly derived from serum albumin. (B) A MALDI-TOF spectrum showing excellent peptide enrichment from the same sample described above using the optimized washing procedure.



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Figure. 6. SISCAPA quantitation of mesothelin. (A) Forward (red) and reverse (black) curves using trypsin-digested human plasma (no SPE cleanup) to measure the levels of mesothelin by SPE-MS/MS. The Forward Curve was generated by titrating the light peptide from 1000 fmol to 1 fmol with the last point containing 0 fmol spike (i.e. endogenous peptide level) while the heavy peptide remained constant at 500 fmol. The Reverse Curve was generated by titrating the heavy peptide from 1000 fmol to 0.5 fmol while the light spike was kept constant at 500 fmol. (B) Analysis of an aliquot of the same eluate using standard flow LC/6490 QQQ/MS. (C & D) MS spectra showing one light (red) and one heavy (black) transitions of the mesothelin peptide for the Forward Curve on the SPEMS/MS and LC/MS platforms, respectively.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Pl at e1:BeadWash 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36


MassSpec Anal ysi s Pl at e2:AbCapt ur e

Pl at e3:Tar getCapt ur e

Pl at e4:Wash#1

Pl at e5:Wash#2


Pl at e6:Wash#3

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4. 0E+04

MesoLi ght

3. 5E+04

Count s( cps)

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MesoHeavy

3. 0E+04

PCILi ght

2. 5E+04

PCIHeavy

2. 0E+04

LBPLi ght

1. 5E+04

LBPHeavy

1. 0E+04 5. 0E+03 0 5. 9

6. 0

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