Antibody-Free Biomarker Determination: Exploring Molecularly

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Antibody-Free Biomarker Determination: Exploring Molecularly Imprinted Polymers for Pro-Gastrin Releasing Peptide Cecilia Rossetti,† Abed Abdel Qader,‡ Trine Grønhaug Halvorsen,† Börje Sellergren,‡,§ and Léon Reubsaet*,† †

Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo, NO-0316 Oslo, Norway Department of Environmental Chemistry and Analytical Chemistry, Institute for Environmental Research (INFU), Technical University of Dortmund, D-44221 Dortmund, Germany § Department of Biomedical Sciences, Faculty of Health and Society, University of Malmö, 205 06 Malmö, Sweden ‡

S Supporting Information *

ABSTRACT: Biomarker mass spectrometry assays are in high demand, and analysis of pro-gastrin releasing peptide (ProGRP) as a small cell lung cancer marker has been recently investigated by mass spectrometry after immunoextraction. In this article, we introduce an assay based on molecularly imprinted polymers (MIPs) targeting the proteotypic peptide of ProGRP as a possible alternative to current immuno-based assay. The MIPs were prepared by surface-initiated reversible addition−fragmentation chain transfer polymerization and were introduced as sorbents for the cleanup and enrichment of a ProGRP signature peptide from tryptically treated serum samples. The use of an appropriate solid-phase extraction protocol allowed specific extraction of the target peptide while depleting other peptides that arose from the sample digestion, hence resulting in reduced background. The selective extraction of a ProGRP signature peptide, after digestion of serum samples, translates into a time- and cost-effective method suited for bottom-up analysis wherever targeted peptide extraction from complex matrices is required.

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molecule analysis.13,14 MIPs have thus been employed as solidphase extraction (SPE) sorbents for a wide range of lowmolecular-weight food and environmental targets in complex matrices.15−19 This contrasts with the few reports describing assays or enrichment of biomacromolecular targets. This can be ascribed to the added complexity in generating the corresponding receptors in terms of template stability and scarcity, conformational matching, and mass transfer limitations. Nevertheless, recent progress in this field20−23 has shown that MIP-based sample pretreatment successfully addresses the main drawbacks of antibodies, suggesting the use of MIP extraction as a new proteomics sample preparation strategy.24 In order to demonstrate and compare these two affinitybased enrichment techniques in mass spectrometry-based assays, we have here focused on assays for pro-gastrin releasing peptide (ProGRP), a well-established biomarker for small cell lung cancer (SCLC),30−37 a highly metastatic form of lung cancer.38 Several SCLC marker studies have demonstrated the diagnostic and prognostic value of ProGRP.30−34,36,37 Applied solely, ProGRP has an SCLC diagnostic sensitivity of around 60−70% in early stage, limited disease cases and 75−90% in

ass spectrometry (MS) methods are in high demand in the field of clinical proteomics,1−4 providing sensitive, reproducible, and specific biomarker quantification. They represent a high-throughput option in clinical analysis, limiting the false positive rate and enhancing the specificity of diagnostic assays. Such precise quantification tools need to be selective to analyze complex clinical samples where the occurrence of highabundance proteins and low biomarker expression limits the dynamic range.5,6 Improvements have been achieved by adopting high-resolution MS and new strategies in sample cleanup, such as immunoextraction techniques. Immunoaffinity coupled with MS has been demonstrated to be highly effective in biomarker determination.7 In spite of the efficiency of immuno-based methods, the generation of high-quality antibodies is costly and timeconsuming, which commonly hampers the development of analytical methods for routine diagnosis in the clinic. Indeed, methods that entirely avoid the use of antibodies are of growing importance in the bioanalysis field.8−12 An alternative approach intended to mimic antibody specificity is represented by molecular imprinted polymers (MIP), commonly referred to as plastic antibodies. These artificial receptors, with affinity recognition sites able to recognize target molecules by a lock and key mechanism, have been used extensively as sample preparation tools in small © XXXX American Chemical Society

Received: September 23, 2014 Accepted: November 14, 2014

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concentrations in a protein mixture were prepared using 50 mM ABC. Internal standards (IS) NLLGLIEA[K_13C615N2] and ELPLY[R 13C615N4], both AQUA Peptide with purities above 95% (Sigma-Aldrich), were diluted according to the custom AQUA peptides storage and handling guidelines by SigmaAldrich and stored at −20 °C. TPCK-treated lyophilized trypsin from bovine pancreas was sequencing grade (Sigma-Aldrich). All other chemicals used were of analytical grade. ProGRP Digestion. Short ProGRP and ProGRP isoform 1 standard solutions were diluted using 50 mM freshly prepared ABC buffer to achieve a final concentration of 50 ng/mL (6.5 and 3.5 nM, respectively). Digestion was carried out by using an enzyme-to-substrate ratio of 1:20 (w/w) at 37 °C overnight (no reduction and alkylation needed). ProGRP and NSE Digestion. For the protein mixture standard, NSE (γ-enolase) was added to ProGRP isoform 1 to give a final concentration ratio of 3:1. A volume of 2.5 μL of 50 mM DTT (freshly prepared in ABC buffer) was added to the protein mixture in 50 mM freshly prepared ABC buffer and incubated at 800 rpm at 60 °C for 20 min. Afterward, the solution was cooled, and 2.5 μL of 200 mM IAA (freshly prepared in ABC buffer) was added. Incubation was carried out for 15 min at room temperature in the dark. Digestion was then accomplished by adding trypsin under the experimental conditions described above. Serum Samples. Serum samples were spiked with ProGRP isoform 1 and vortexed for 30 s, obtaining a final concentration of 4 nM. In order to reduce sample complexity and thus to avoid overloading the cartridges, the following protocol was used (as described by Winther et al.27). Protein precipitation was performed by adding an equal volume of cold MeCN (−32 °C) to serum and vortexing for 1 min. Samples were then centrifuged at 10 000 rpm, and the supernatant was evaporated under a nitrogen stream at 50 °C to dryness. ABC (50 mM) was used to reconstitute the samples, and digestion was subsequently performed as described above. Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Analysis. LC-MS/MS analysis was performed using a triple quadrupole (QqQ) mass spectrometer according to established methods for ProGRP and NSE analysis.25,43,44 The chromatographic system consisted of LPG-3400 M pumps with a degasser, a WPS-3000TRS autosampler, and a FLM3000 flow-manager (all Dionex, Sunnyvale, CA, USA). The LC system was controlled by Chromeleon v. 6.80 SR6 (Dionex). The chromatographic separation was carried out using an Aquasil C18 analytical column (Thermo Scientific) (100 Å, 3 μm, 50 mm × 1 mm) preceded by an Aquasil C18 guard cartridge column (Thermo Scientific, Rockford, IL, USA) (100 Å, 5 μm, 10 mm × 1 mm). The chromatographic separation was performed by loading 40 μL of sample with mobile phase A (20 mM formic acid (FA) and acetonitrile (MeCN) 99:1, v/v) and eluting with a 30 min linear gradient from 1 to 85% mobile phase B (20 mM FA and MeCN 1:99, v/v). After the gradient was run, the column was washed for 3 min with 90% mobile phase B and re-equilibrated with mobile phase A. Column temperature was set and kept constant at 30 °C. A mass spectrometry detector (Thermo Scientific) was used for quantification of signature peptides by selected reaction monitoring (SRM). The following transitions pairs were monitored (qualifier and quantifier, respectively): for total

advanced stage, extensive disease cases; therefore, its prognostic significance was correlated with clinical staging during chemotherapy.39 Hence, early detection is highly crucial for positive treatment outcomes, and new diagnostic modalities for SCLC biomarker determination assume absolute importance.40 Absolute quantification of ProGRP by MS was formerly investigated by means of its signature peptide, NLLGLIEAK, a unique prototypic nonapeptide detectable with high signal intensity.25−29 Among immuno-MS assays developed for ProGRP, we recently demonstrated that immunocapture using antibodies coupled to magnetic beads followed by tryptic digestion and quantification of the signature peptide on a triple quadrupole mass spectrometer (QqQ) provides a method for robust and sensitive quantification of this biomarker.25 The aim of this work was to develop an alternative enrichment strategy in targeted proteomics by exploring MIPbased peptide capture. In contrast to MIPs targeting proteins, the imprinting of peptides obviates the need for labile and expensive proteinaceous template and is compatible with a wide range of solvents, monomers, and elevated temperatures. Apart from its synthetic ease, this approach, moreover, allows for the enrichment of the only tryptic peptide of interest, yielding cleaner extracts to be used in bottom-up target experiments. In a preliminary report aimed at the optimization of compositional parameters (template, monomer, solvent), we identified polymers prepared using the hydrophobic cross-linker divinilbenzene (DVB) and the functional monomers N-(2aminoethyl) methacrylamide hydrochloride (EAMA) and imprinted with N- and C-protected forms of the signature peptide of ProGRP (NLLGLIEAK) as displaying particularly promising recognition properties.41 Polymers prepared using this combination exhibited significant imprinting, yielding higher peptide recovery in the MIP extract when compared with that of the respective nonimprinted polymer. With this as a starting point, we have here developed novel peptideimprinted thin-film composite beads and used them in conjunction with tandem mass spectrometry (MS/MS) in the bottom-up workflow for absolute quantification. A selectivity study was performed by evaluating both imprinted and nonimprinted polymers with respect to the retention behavior of the target peptide (NLLGLIEAK) and other, nontarget, peptides. Ultimately, the MIP’s potential in analysis of biological samples was evaluated with respect to affinity and selectivity by applying the developed protocol to a ProGRP-fortified serum sample.

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EXPERIMENTAL SECTION Protein and Peptide Standard. Recombinant ProGRP products, short ProGRP (AA 31−98), and ProGRP isoform 1 (AA 1−125 + 8) were cloned from human cDNA (OriGene Technologies), expressed in Escherichia coli (Promega) using pGEX-6P-3 constructs (GE Healthcare), and purified as described elsewhere.42 ProGRP concentrations were assessed by absorbance at 280 nm (A280), diluted to the desired concentration with 50 mM ammonium bicarbonate (ABC) buffer solution, and stored at −20 °C. The NSE standard (γ-enolase) was purchased from Scripps Laboratories (San Diego, CA, USA). Stock solution was prepared by diluting Scripps with 5% bovine serum albumin (BSA) in phosphate buffered saline (PBS), pH 7.4, to a concentration of 1 μg/mL. Successive dilutions to desired B

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Figure 1. Synthesis of MIP for NLLGLIEAK via RAFT-modified mesoporous silica. Template: Z-NLLGLIEA-Nle-OH.

ProGRP signature peptide NLLGLIEAK, 485.8 → 630.3 and 485.8 → 743.4; for the internal standard NLLGLIEA[K_13C615N2], 489.9 → 638.3 and 489.9 → 751.4; for ProGRP isoform 1 signature peptide LSAPGSQR, 408.2 → 272.6 and 408.2 → 544.4; for NSE(γ) signature peptide ELPLYR, 395.72 → 274.72 and 395.72 → 548.3 were acquired together with transition 401.02 → 279.52 and 401.02 → 558.3 for its internal standard ELPLY[R13C615N4]. TSQ data were processed by Xcalibur’s QualBrowser (Thermo Scientific), and MS responses based on the peak areas, automatically processed by genesis peak detection algorithm, were used. Among them, only peaks a with signalto-noise (S/N)-ratio above 10 and with retention time and ion ratios corresponding to those of reference samples at high concentration were considered. Preparation, Packing, and Use of MIP and NIP Materials. Off-line SPE was performed by using MIPs complementary to the ProGRP signature peptide NLLGLIEAK. Nonimprinted polymers (NIP) were also tested in order to assess MIP imprinting efficiency. The MIPs were prepared by reversible addition−fragmentation chain transfer (RAFT) polymerization from porous silica supports, resulting in thin, uniform coatings of imprinted polymer on the silica support beads (see Supporting Information). To prepare MIP and NIP cartridges, 20 ± 0.5 mg of both MIP and NIP was, respectively, slurry-packed under vacuum sedimentation into empty SPE cartridges (1 mL, Agilent, Gemany) using 2 mL of methanol and capped between two fritted polyethylene disks (20 μm pore size). Optimized SPE Protocol for NLLGLIEAK. After activation and conditioning with 1 mL of MeOH and 1 mL of ABC 50 mM, the MIP and NIP SPE cartridges were loaded with sample (500 μL). Each cartridge was subsequently washed with 5 mL of 7.5% MeCN and eluted with 500 μL of 80% MeCN acidified with 3% FA. Prior to chromatographic injection, the collected fractions were diluted (1:10) by a solution of PEG-20000 (0.001%) acidified with 0.1% FA. Both cartridges were then reconditioned with MeOH (500 μL) to remove the acidic elution solvent completely and, finally, were stored with 500 μL of MeOH until the next analysis. MIP and NIP cartridges were reused in a series of extractions (n > 25) after verifying the absence of a carry-over effect. The analysis of ProGRP using the MIP SPE method was completed in 40 min starting from the digested sample and including the entire chromatographic run. No incubation time was needed, and both extraction and cartridge regeneration are conducted by percolation of solutions at a 1 mL/min flow rate. Recovery. In order to assess differences between MIP and NIP retention mechanisms, the percolated fractions were analyzed for their peptide content. The recovery percentage in each extracted fraction (R%EF) was then calculated, based on the chromatographic peak area generated by the extracted

fraction (AEF) and the peak area of the unextracted sample (AUS), according to eq 1 R %EF =

AEF × 100 AUS

(1)

Experimental replicates were compared by normalization of all fraction recoveries by setting as 100% the sum of all recoveries achieved measuring areas of the sample flow through, the entire wash step, and elution, according equation eq 2 R% =

R %EF × 100 (R % sample flow through + R % wash + R % elution)

(2)

At least two replicates were performed for each experiment.

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RESULTS AND DISCUSSION Polymer Preparation. The preparation of the receptor for the proteotypic peptide was based on an extensive screening of combinatorial libraries comprising different cross-linking monomers, functional monomers, templates, and solvents, with the aim of finding polymers displaying strong reversible affinity for the target peptide in aqueous or hydro-organic solvents. 41 In this study, we have used hydrophobic divinylbenzene copolymerized with 2-aminoethylmethacrylamide and an N-terminally Z-protected peptide as template for generating the surface-imprinted beads (Figure 1). The surface-initiated polymerization was performed by suspending RAFT-modified wide-pore (50 nm average pore diameter) silica beads in a dilute monomer solution followed by thermal initiation. On completion of the grafting process, the beads were washed from unreacted monomers, the template was removed by solvent extraction, and the beads were subsequently characterized by elemental analysis, thermal gravimetry, and nitrogen sorption (Supporting Information Figure S3 and Tables S1−S3). The mass loss (%), carbon content (% C), and average pore diameter changed in accordance with the formation of homogeneous thin films on the silica pore walls. Hence, very similar film thickness values were obtained (ca. 3 nm) regardless of whether the calculation was based on mass loss or compositional results. The scanning electron microscope images (shown in Supporting Information Figure S4) revealed the anticipated spherical composite beads but also some irregular nongrafted polymer particles, with the latter being more frequent in the imprinted polymer. Prior to development of the SPE procedure, binding curves for the target template were plotted from data of the adsorbed amount of target peptide versus the concentration of free peptide with increasing total concentrations in HEPES buffer (shown in Supporting Information Figure S1). The curves are non-Langmuirian, indicating a heterogeneous distribution of binding sites. Nevertheless, the values corresponding to the binding capacity as well as the affinity constant are markedly higher for the imprinted polymer compared to those for the C

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dryness after SPE and reconstituted in mobile phase A. The poor reproducibility and recovery in this step led us to replace the evaporation and reconstitution step by a dilution step, 1:10 with PEG-20000 (0.001%) acidified with 0.1% FA, which resulted in less adsorptive losses of peptides (e.g., on autosampler vials) and hence higher reproducibility (see Stejskal et al.45) (Supporting Information, Table S5). The results of these initial experiment showed that the standardized MIP protocol applied was not selective enough to differentiate between specific MIP interactions and nonselective NIP interactions. Efforts were therefore focused on optimizing the protocol, specifically to find a wash step that exclusively disrupts nonselective interactions while retaining the selective interactions with the recognitive sites. Optimization of SPE Protocol. As the initial protocol revealed similar recoveries for MIP and NIP, we embarked on a careful optimization of the washing and elution conditions of the SPE protocol. We decided to first investigate the influence of solvent composition on the elution profiles of both MIP and NIP. The screening was performed by conditioning the cartridges and loading the sample as previously described and then subsequently collecting all of the sequential flow through fractions obtained by increasing the MeCN percentage. The volume of the applied solutions was 500 μL, and all collected fractions were diluted as described in the Experimental Section. A first experiment was performed by increasing MeCN in 10% intervals (between 5 and 90%); the elution profiles obtained by plotting the sum of the NLLGLIEA[K_13C615N2] recoveries against the MeCN content are shown in Figure 2A. Similar performances of MIP and NIP were observed, and complete peptide elution was obtained at around 30% MeCN.

nonimprinted polymer. Hence, the weighted average affinity (KPT2 = 140 000 M −1) was more than 20-fold higher than that of the NIP (KNIP = 6000 M −1) in the measured concentration range. From the steep initial slope, it is clear, however, that a significant number of sites with K > 106 M−1 are present (Figure S1). Evaluation Framework. Performance of the imprinted polymers was investigated in two different ways, by comparing MIP and NIP recoveries and by evaluation of MIP’s ability to differentiate between NLLGLIEAK and other peptides in the sample. By starting out with samples consisting of a single peptide standard solution, an optimized SPE protocol was developed. The sample complexity was then increased to determine the MIPs’ discriminative potential. Choice of Standards and Reusability of the SPE Cartridges. In order to understand if effective interaction occurs between the signature peptide and the polymer, the presence of other interfering peptides at this stage was avoided. For this purpose, the labeled peptide NLLGLIEA[K_13C615N2] was employed to develop the SPE protocol. Hence, interference due to carry-over effects and disturbance originating from other proteins and peptides in the sample were avoided, and, although the standard differs from NLLGLIEAK in its m/z value (Δm = +8 Da), it exhibits identical chromatographic properties and appears as an easily detectable peak in the LC-MS/MS method used. In addition, sample application was always performed using 50 nM ABC buffer to ensure direct downstream compatibility with digest samples to avoid additional sample preparation steps. The reusability of the cartridges was tested by assessing carry-over effects. Hence, the conditioning fractions obtained before sample application were collected and checked for analyte signal in both NIP and MIP cartridges. The absence of carry over (≤1% of applied sample) allowed us to reuse the same cartridges for several extractions (>25), ensuring a sufficient number of experimental replicates (n ≥ 2) (Supporting Information, Figure S5). Given the extensive reuse, the total peptide recoveries from the cartridges and the standard deviation between experimental replicates (75 ± 5% on average) was considered to be satisfactory. Initial Tests on MIP and NIP. First, a standardized SPE protocol41 was used to evaluate the materials as follows: activation and conditioning of the cartridges by 1 mL of MeOH and 1 mL of ABC buffer 50 mM (pH 7.8), sample loading of 500 μL of NLLGLIEA[K_13C615N2] (1 nM), two washes with 500 μL of 5% MeCN, and elution with 500 μL of MeOH/TFA 98:2 (v/v). However, this protocol showed comparable recoveries of MIP and NIP, both below 50% (38.3 and 45.2%, respectively) (Supporting Information, Figure S6). Analysis of the sample flow through and wash solution showed no presence of analyte, indicating that ABC buffer was a suitable sample loading media perfectly compatible with the tryptic digestion procedure and that the low recovery was presumably caused mainly by noneluted analyte remaining bound to the cartridges. Subsequent blank extractions were performed on the MIP and NIP cartridges and showed a combined additional recovery of 10 and 16%, respectively (no signal was observed in the eluent after the third blank extraction). The low recovery proved that NLLGLIEA[K_13C615N2] was still not completely eluted from the cartridges. Another important source of peptide loss and variation was the evaporation step prior chromatographic injection. During the initial tests, samples were evaporated to

Figure 2. Cumulative total recovery of NLLGLIEA[K_13C615N2] after loading 500 μL of a 1 nM solution in 50 mM ABC buffer on MIP (black) and NIP (gray) cartridges followed by sequential wash steps using aqueous solution (500 μL each) containing an increasing percentage of acetonitrile at (A) 10% intervals (5−100% MeCN) and (B) 2.5% intervals (5−30% MeCN).

A second screening was thus performed in the range between 5 and 30% MeCN, but MeCN was increased in steps of 2.5%. The small variation in organic composition revealed differences in the elution profile between MIP and NIP (Figure 2B). The higher NIP recoveries suggested the destabilization, in this solvent composition range, of mainly nonspecific and labile interactions occurring in the NIP cartridge. D

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On the basis of this information, the introduction of an extensive wash step, which could disrupt all of the weak, nonspecific interactions prior to elution, was evaluated. Strength of the Wash Solution. A focused investigation of different wash solvent compositions (5, 7.5, 10, 12.5, 20, and 30% in MeCN) was performed using a volume of wash solvent of 5 mL and 500 μL of MeOH/TFA 98:2 as the elution solvent. The wash efficiency was assessed by comparing the amount of peptide recovered in the elution step after SPE using MIP and NIP cartridges. Figure 3 illustrates the different elution

Figure 4. Elution efficiency in the MIP-SPE expressed as the recovery of target peptide NLLGLIEA[K_13C615N2] (1 nM solution in 50 mM ABC buffer) in the elution fractions (500 μL) divided by the overall recovery in the SPE protocol (N = 3).

was investigated by performing SPE on digested short ProGRP (AA 31−98) and digested ProGRP isoform 1 (AA 1−125 + 8) samples. This implies an increase in sample complexity from that of one standard peptide to a peptide mixture that contains, in the case of complete digestion, equimolar concentrations of 11 and 23 tryptic peptides with more than 4 amino acids for short ProGRP (AA 31−98) and ProGRP isoform 1 (AA 1−125 + 8), respectively, in addition to the target peptide. Washing and elution recoveries agreed closely with those obtained when only NLLGLIEA[K_13C615N2] was loaded onto the cartridges during sample optimization (Figure 5). These

Figure 3. Elution recoveries of NLLGLIEA[K_13C615N2] after loading 500 μL of a 1 nM solution in 50 mM ABC buffer on MIP (black) and NIP (gray) cartridges followed by washes (5 mL) using solutions containing different percentages of MeCN (N = 3).

recoveries plotted against the MeCN concentration in the wash solvents. Weak washes with low MeCN concentrations (5% MeCN) failed to produce differences in the elution recovery of MIP and NIP. However, using compositions of 7.5 and 10% MeCN, differences were clearly observed, implying the disruption of nonspecific binding under these conditions. Concentrations above 12.5% MeCN led to inadequate differentiation between polymers and also resulted in poor overall recoveries. Thus, using 5 mL of a wash solution containing 7.5% MeCN led to effective discrimination between MIP and NIP without compromising the recovery of the peptide from the MIP cartridge (>50%), unlike the use of 10% MeCN. Strength of the Eluent. Next, the eluent composition was optimized. The eluent efficiency, determined as the recovery, was investigated for different compositions (MeCN or MeOH containing different amounts of water and acidic modifier) after washing with 7.5% MeCN (Figure 4). Using MeCN instead of MeOH as basic eluent resulted in a slightly higher recovery, probably due to the increased peptide solubility in MeCN. The highest efficiency was obtained using MeCN/H2O/FA (80:20:3), confirming the importance of high concentrations of acid modifier to unlock the MIP binding sites. In addition, FA is preferred over TFA because it avoids any possible ion suppression in the mass spectrometer. The final protocol thus consisted of conditioning the cartridges with 1 mL of MeOH and 1 mL of 50 mM ABC buffer, sample loading in 50 mM ABC buffer, washing with 5 mL of 7.5% MeCN, and eluting with 500 μL of MeCN/H2O/FA (80:20:3). Application of Optimized SPE Protocol to Digested ProGRP. Once the optimized protocol was established, the affinity of the MIP for NLLGLIEAK in more complex samples

Figure 5. Comparison of the recoveries of the NLLGLIEA[K_13C615N2] standard solution (darker color) and the target peptide (NLLGLIEAK) resulting from the digestion of short ProGRP (AA 31−98) (brighter color) using the optimized extraction protocol.

results confirmed the MIP and NIP retention differences, indicating that the MIPs’ affinity for NLLGLIEAK was not compromised by the presence of multiple peptides in the sample. In Figure 5, it can further seen that the signature peptide is retained, although to a lesser extent, by the NIP cartridge. This indicates that the retention observed on the MIP under the current conditions also has a less selective contribution that is not related to imprinting. The extraction of digested ProGRP isoform 1 (AA 1−125 + 8) aimed to verify the MIP retention mechanism. This protein is characterized by two distinguishable regions: a constant region, common to all ProGRP isoforms harboring the amino acid sequence NLLGLIEAK, and a variable region specific for isoform 1 only, harboring the sequence LSAPGSQR. The E

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former is thus a signature peptide for total ProGRP that is included in all isoforms, whereas the latter represents an isoform 1 signature peptide.25 The number of tryptic peptides resulting from isoform 1 digestion is significantly higher than that from short ProGRP, implying the presence of a higher number of interfering peptides that could compete with the target peptide in the binding sites, thereby reducing the polymer’s selectivity for NLLGLIEAK. Results shown in Figure 6 confirmed the discriminatory power of the MIP to specifically enrich the target peptide.

Figure 7. Recoveries of NLLGLIEAK, LSAPGSQR, and ELPLYR in the SPE of a mixture of digested ProGRP isoform 1 (AA 1−125 + 8) and NSE (γ-enolase) (1:3) on MIP (black) and NIP (gray) cartridges.

Application of Final SPE Protocol to Serum Samples. To further challenge the MIP cartridge, we next loaded tryptically treated ProGRP spiked serum samples. Samples were first spiked with ProGRP isoform 1 followed by protein precipitation with cold acetonitrile. This preliminary depletion was considered to be essential to reduce abundant proteins such as albumin and to avoid overloading the cartridges. ProGRP concentration in the supernatant is not affected by protein precipitation, as previously described by Winther et al.27 MIP and NIP extraction differences are displayed in their SRM chromatograms (Figure 8A). The MIP eluate displayed a large and quantifiable peak that was absent in the NIP eluate. This implies that the MIP can successfully capture the signature peptide from real-life samples. Full-scan chromatograms (m/z =

Figure 6. Recoveries of NLLGLIEAK and LSAPGSQR in the SPE of a sample (500 μL of 3.5 nM in 50 mM ABC buffer) of digested ProGRP isoform 1 (AA 1−125 + 8) on MIP (black) and NIP (gray) cartridges.

Meanwhile, the isoform 1 peptide (LSAPGSQR) was effectively removed by 5 mL of 7.5% MeCN, resulting in the exclusive elution of NLLGLIEAK from the MIP cartridge. Selectivity Tests. In order to gain a more detailed understanding of the MIP recognition properties, we decided to further increase the sample complexity. Hence, an excess of neuron specific enolase (NSE) was added to the ProGRP isoform 1 standard solution, resulting in a protein concentration ratio of 3:1. NSE is a current SCLC marker that shows enhanced concentration in patience samples;34 thus, it is reasonable to investigate potential interference originating from this protein. Peptides LSAPGSQR, ELPLYR, and NLLGLIEAK, corresponding to the signature peptides of ProGRP isoform1, NSE, and total ProGRP, respectively, were monitored in the fractions resulting from sample application, wash, and elution, and the resulting peptide recoveries are plotted in Figure 7. Again, it was shown that a significant amount of NLLGLIEAK was recovered in the elution from the MIP well, exceeding that obtained in the NIP. Meanwhile, the signature peptides of NSE and ProGRP isoform 1 were similarly depleted in the wash fractions of both MIP and NIP. Hence, mainly NLLGLIEAK was found in the elution step, whereas less than 4% of LSAPGSQR and ELPLYR were recovered. The MIPs were thus demonstrated to be highly specific for NLLGLIEAK with a robust retention mechanism, leading to reproducible peptide extraction from complex samples.

Figure 8. Spiked serum (1 mL) sample extraction. (A) SRM chromatograms of NLLGLIEAK transitions (acquisition window 19−25 min) in MIP (red) and NIP (black) elution. (B) Full-scan chromatograms of MIP elution (red) and unextracted sample (black). F

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resulted in a lower NLLGLIEAK elution recovery than that with smaller serum volumes. This also agrees with the different amounts of NLLGLIEAK recovered in the sample flow through (14, 0, and 0% respectively). A third cause could possibly be ascribed to a partial coprecipitation during the serum pretreatment or to protein interference, which could interact and ultimately prevent the signature peptide from accessing the binding sites. In spite of this shortcoming, the specificity of the MIP is evident, as judged from the clean elution of the target peptide with a complete absence of the other signature peptides. The combined use of smaller serum volumes and an increased amount of MIP could thus overcome issues related to material capacity and yield improved recovery. Low recovery levels also occur in affinity-based enrichment procedures; using an immuno-extraction protocol for hCG in serum (similar to the one for ProGRP), for instance, recoveries of 32% were reported.46 The limit of detection (LOD) using the optimized MIP-SPE procedure on spiked serum samples was estimated to be 625 pM ProGRP isoform 1 using a signal-to-noise ratio (S/N) of 3. This detection limit is ca. 80 times higher than the reference level for the clinical screening of ProGRP in serum samples (7.6 pM), and we believe that this is mainly caused by the incomplete recovery combined with the 1:10 dilution step before the injection. These are the first results on MIP extraction of ProGRP from serum samples; therefore, overcoming these issues could yield improved performance.

250−1100) of the unextracted sample and MIP elution were acquired and overlapped (Figure 8B), showing a reduction in peak numbers and overall peak intensity in the MIP extract (red chromatogram) compared to that of the unextracted sample (black chromatogram). The substantial decrease in sample complexity has to be attributed to the cleanup effect of the MIP SPE because both solutions were otherwise identically treated. Neither unextracted nor MIP extracted full-scan chromatograms reveal a distinct peak ascribable to NLLGLIEAK, although we know from the SRM experiments that 86% of the peptide initially present in the sample was absorbed onto the MIP cartridge (Figure 9A). This circumstance is probably due to the small amount of ProGRP added in the spiking process in combination with poor sensitivity of fullscan acquisition using a QqQ system.

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CONCLUSIONS A combination of extensive material and method optimization was shown to be crucial for addressing the analytical challenge of specific and sensitive peptide quantification using MIPs. Surface imprinting of mesoporous silica relying on RAFTmediated grafting-from resulted in MIP composite beads featuring high affinity and selectivity for a proteotypic peptide in aqueous buffered media. Nonspecific binding could be suppressed by an extensive wash after sample loading and an elution, using a good solvent for the target peptide and a relatively high acid concentration. Using the above settings the MIPs showed specific retention of NLLGLIEAK when compared with that of other peptides (LSAPGSQR and ELPYR) occurring in the sample. Hence, increasing sample complexity did not affect the MIPs’ ability to selectively retain NLLGLIEAK, and serum samples fortified with ProGRP were successfully analyzed after MIP cleanup. Although the incomplete recovery yielded a higher LOD than that of the immunoaffinity extraction method,25 we expect further improvements to be relatively straightforward and to comprise a further optimization of the MIP per se as well as the SPE procedure. Comparing the extraction time of the two different techniques, the MIP protocol was clearly faster due to the lack of the incubation time required for the immunoextraction (1 h). The new strategy proposed is intended to enrich ProGRP peptide after the digestion of serum samples, producing cleaner extracts to analyze in the mass spectrometer. To the best of our knowledge, this is the first time that MIPs have been demonstrated to be selective for tryptic peptides in serum samples. This fundamental property gives relevance to the employment of MIPs in biological sample cleanup, with special focus on targeted proteomics, and represents a novel approach for time- and cost-effective biomarker analysis.

Figure 9. Serum sample extraction. Recovery of NLLGLIEAK and LSAPGSQR after SPE on MIP (black) and NIP (gray) of digested serum samples spiked with ProGRP isoform 1 (AA 1−125 + 8). Serum volumes: 1 mL (A), 500 μL (B), and 250 μL (C).

Different serum volumes were tested (1 mL, 500 μL, and 250 μL) while keeping the ProGRP isoform 1 concentration constant at 4 nM. Fractions from sample load, from the first three 500 μL of the wash step, and from the elution were collected for both MIP and NIP. Recoveries were calculated and plotted in Figure 9. For all sample volumes, NLLGLIEAK elution is significantly higher in all MIP fractions, highlighting the efficiency of the imprinting process to generate selective binding sites within the polymer. The recovery of NLLGLIEAK from serum was lower (