Noncovalent Antibody Immobilization on Porous Silicon Combined

May 6, 2011 - Proteomics has spread widely into every field of life science and medicine as an important part of the postgenomic research era. The ana...
2 downloads 0 Views 3MB Size
Download PDF
ARTICLE pubs.acs.org/ac

Noncovalent Antibody Immobilization on Porous Silicon Combined with Miniaturized Solid-Phase Extraction (SPE) for Array Based ImmunoMALDI Assays Hong Yan,†,^ Asilah Ahmad-Tajudin,†,‡ Martin Bengtsson,† Shoujun Xiao,^ Thomas Laurell,†,‡,§ and Simon Ekstr€om*,†,‡ †

Department of Measurement Technology and Industrial Electrical Engineering, Division of Nanotechnology, Lund University, Lund, Sweden ‡ CREATE Health, Lund University, Lund, Sweden § Department of Biomedical Engineering, Dongguk University, Seoul, Korea ^ State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing National Laboratory of Microstructures, Nanjing University, Nanjing 210093, PR China ABSTRACT: This paper presents a new strategy to combine the power of antibody based capturing of target species in complex samples with the benefits of microfluidic reverse phase sample preparation on an integrated sample enrichment target (RP-ISET) and the analysis speed of MALDI MS. The immunoaffinity step is performed on an in-house developed 3D-structured high surface area porous silicon (PSi) matrix, which allows efficient antibody immobilization by surface adsorption without any coupling agents in 30 60 min. The hydrophilic nature of the porous silicon surface at the molecular level displays a low adsorption of background peptides when exposed to complex digests or plasma samples, improving the conditions for the antigen specific extraction and subsequent readout. At the same time, the hydrophobic behavior, due to the nanostructured surface, of the PSi material facilitates liquid confinement during the assay. Using a footprint conforming to the standard for 384 well microplates, direct adaption of the protocol into standard sample handling robots is possible. The performance of the proposed immunoaffinity PSi-ISET immunoMALDI (iMALDI) assay was evaluated by specific detection of angiotensin I at a 10 femtomol level in diluted plasma samples (10 μL, 1 nM).

roteomics has spread widely into every field of life science and medicine as an important part of the postgenomic research era. The analysis of the proteome requires high-resolution separation technologies interfaced to high sensitivity detection. With the progress of proteomics and the ability to measure markers in complex biofluids, we now see a major effort to transfer this knowledge into clinical proteomics for detailed mapping of signaling pathways and protein expression profiles related to disease.1 4 Along this route, quantitative and targeted proteomics using liquid chromatography (LC)-electrospray ionization (ESI) MS and multiple reaction monitoring (MRM) has the potential to uncover a multitude of new biomarkers,5 7 although it is improbable that MRM based assays will completely replace standard immunoassays in the immediate future. There is, however, room for new methodologies that can provide more in depth structural information of the target protein than a standard immunoassay while still being cost-effective and perhaps better suited for high throughput biomarker monitoring in larger clinical studies. In clinical proteomics, the protein targets are often known and, thus, it is possible to bypass the need for a high-resolution separation utilizing more specific capturing techniques. In this perspective, the combination of immunocapture and mass spectrometry is a highly desirable combination since the immunocapture reduces the complexity of the sample to be analyzed at the same time as the target species is enriched, thereby increasing

P

r 2011 American Chemical Society

the possibility of detecting the analyte of interest at lower abundant levels.8 This need is also reflected in the fact that a direct MS analysis of biofluids will often only detect changes in high abundant molecules, e.g., surface-enhanced laser desorption ionization spectrometry (SELDI).9 While a mass spectrometry readout of an immunoassay currently cannot match the detection levels of the most sensitive optical techniques, the mass spectrometric approach can serve to provide unequivocal identification of antigens and be used to pinpoint different structural variants of an antigen coupled to a disease state.10,11 Many different immuno-MS approaches have been presented in the literature. A noteworthy example is the use of immunocapture combined with LC-ESI MS in the stable isotope standards and capture by anti-peptide antibodies (SISCAPA) method pioneered by Anderson et al.12 SISCAPA is based on target specific enrichment using an antipeptide antibody on a small precolumn (100 nL), which is coupled online to LC-MS. While LC-MS based approaches have the advantage of inherent sample decomplexing and enrichment, it is less amenable to high-throughput sample processing. An attractive alternative is to use MALDI MS to analyze immunocaptured analytes, providing a simpler system Received: March 17, 2011 Accepted: May 6, 2011 Published: May 06, 2011 4942

dx.doi.org/10.1021/ac200679t | Anal. Chem. 2011, 83, 4942–4948

Analytical Chemistry with less risk of sample memory effects and higher throughput capability. A prerequisite is, however, that sufficient sample decomplexing is accomplished in the immunoaffinity step. As the MRM based assays are developed for peptides, it is foreseeable that peptide standards will be applied to the development of antipeptide antibodies for immuno-MS assays of important pathways.13 The very first immunoMALDI (iMALDI) methodologies were based on immunoprecipitation,14,15 and surface plasmon resonance biomolecular interaction analysis (SPR-BIA) combined with MALDI MS.16 18 A number of different approaches using various antibody covered MALDI target surfaces,19 23 columns, tips,24,25 and beads26,27 have been reported in the literature. In fact, many recently presented iMALDI methods have included quantification and, thus, are at the verge of being truly ready for clinical studies. Each approach to immunoMALDI has its strengths and weaknesses. Regardless of approach the ideal method should provide high capacity, low background, and simple coupling/immobilization of the antibody as well as be amendable for automation. One rapidly emerging approach for multiplex immunoaffinity assaying is the chip based microarray assay using rapid optical readout of a large number of analytes in a single array experiment.28 34 These do, however, suffer from less quantitative aspects and most importantly do not indicate the sequence of the captured structure, which ideally an MS based identification would provide. In order to make microarray based immunocapture strategies amenable for MALDI MS readout, the capturing capacity has to be addressed. In view of this, porous silicon has in the past years emerged as an interesting support for high capacity immobilization of proteins.35 41 Recently, combined immmunocapture laser desorption/ionization MS on porous silicon of benzodiazepine drugs was demonstrated.42 Extraordinary material properties, such as a high surface area-to-volume ratio, hundreds of square meters per cubic centimeter,43,44 a tunable pore geometry, morphology, and density, make porous silicon (PSi) efficient for antibody immobilization. Previously, we have used the porous silicon antibody array approach to perform biomarker assays with fluorescent detection.35 37 Also, early MALDI MS work using antipeptide antibodies against angiotensins have also been presented by our group.22 A big advantage of the porous silicon platform is that the antibody is immobilized on the chip by physical adsorption, circumventing the tedious steps required for covalent coupling, yet displaying high loading capacities per chip area. A key aspect in immunoaffinity MALDI assays is the sample preparation step after the immunocapture, removing buffer adducts, yet enabling reconcentration of the captured and displaced analytes onto well-defined MALDI spots. In earlier work, we have reported a chip integrated microarray based solid phase extraction platform for MALDI MS sample preparation, the integrated selective enrichment target (ISET).45 47 ISET provides improved MS sensitivity by the integrated and area confined sample processing protocol, minimizing both the number of sample transfers and the total surface area to which the analyte is exposed. Using reverse phase ISET, sample preparation immunoaffinity captured analytes can be reconcentrated after elution. If needed, additional sample preparation steps such as digestion of immunocaptured intact proteins can also be performed on-bead in the ISET microarray.48 In this paper, we present technical and methodological improvements of the macroporous silicon protein array technology previously developed in our group. Using macroporous silicon as a

ARTICLE

substrate for the immunocapture step, vast time and cost saving can be made compared to chemical immobilization protocols. Furthermore, the high surface area 3-D macroporous silicon has low wetting properties, due to the nanostructured surface; i. e., it behaves like a hydrophobic material which assists in confining the sample solutions in a small area, although Psi actually has an entirely hydrophilic surface at the molecular level (SiO2). Most importantly, the hydrophilic backbone of the porous silicon provides low unspecific binding and a nondenaturing surface for antibody adsorption. We evaluated the proposed methodology in a model system with an angiotensin 1 (Ang I) antibody in the immunocapture step on the porous silicon followed by a Solid-Phase Extraction (SPE) step on the ISET platform with MALDI MS readout to achieve a rapid approach readily adaptable for automation of immunoMALDI assays. The porous silicon ISET enrichment immunoMALDI assay (PSi-ISET iMALDI) approach is demonstrated by successful analysis of 10 μL plasma samples spiked with angiotensin I (Ang I) at a level of 1 nM.

’ EXPERIMENTAL SECTION Chemicals. Unless otherwise specified, all chemicals were purchased from Sigma-Aldrich Co. (St. Louis, MO) and used without any further purification. Rabbit polyclonal antibody angiotensin I (Ab-Ang I) was from abcam (UK) and rabbit antiHuman. Magnetic beads, Dynabeads M-270 Carboxylic Acid, were purchased from Invitrogen Dynal Biotech AS (Oslo, Norway), and Poros R2 50 μm beads were from Applied Biosystems (Carlsbad, CA, US). HPLC grade water was used for all aqueous preparations. Samples for Immunocapture. Alcohol dehydrogenase (ADH) was digested with trypsin from Promega (Madison, WI, USA) in a 1:100 ratio (enzyme:protein) for 90 min in 37 C. The digested peptide mixture was diluted 100 times with 10 μM PBS, pH 7.4, and frozen to stop the digestion process. A stock solution of 1 μM ADH was used to prepare standard samples by dilution and spiking with antigen. The Universal Proteomics Standard (UPS 1, Sigma-Aldrich. Co., St. Louis, MO, US) containing 48 different proteins was reconstituted and digested with trypsin according to the manufactures specification. Human Plasma was diluted 1:10 with 10 mM PBS containing protease inhibitor cocktail Sigma-Aldrich Co. (St. Louis, MO) and spiked with Ang I to specified concentrations. Porous Silicon Fabrication. The silicon wafers, 5 10 Ω 3 cm, Æ100æ, P type and Boron doped, were from Addison Engineering Inc. (San Jose, CA). The chips were patterned using the standard UV-lithography protocol with silicon dioxide as a mask. The circular area defining the antibody capture zone on the chip was a 3 mm diameter with a surrounding silicon dioxide ring of 200 μm width; the pitch between positions was 4.5 mm, chosen to conform to standard Society for Biomolecular Sciences (SBS) measurements of a 384 microplate and aligned with robotic liquid handling. The detailed porosification protocol has been reported previously.36,37 The porosified silicon wafer was diced into chips holding 3  4 array positions, Figure 1. Magnetic Beads. The angiotensin antibody was immobilized onto Dynabeads M-270 Carboxylic Acid beads from Invitrogen, Life Technologies Corporation (Carlsbad, CA, USA), according to the manufacturers protocol (http://www.invitrogen.com). The protocol used for affinity capture and elution was as follows: (1) 10 μL of antibody coated magnetic beads in PBS was added to 10 μL of sample in a 0.6 mL Axygen (Union City, CA, US) 4943

dx.doi.org/10.1021/ac200679t |Anal. Chem. 2011, 83, 4942–4948

Analytical Chemistry maximum recovery tube and allowed to bind for 1 h at RT, with shaking. (2) The tube was placed on a magnet, and magnetic beads were pelleted to the side of the tube. The supernatant was removed, and the beads were washed with 20 μL of PBS. This wash step was repeated 3 times. In order to minimize background, the magnetic beads were transferred to a new tube after the first wash cycle. (3) After the final wash, the magnetic beads were resuspended in 10 μL of 5% acetic acid and incubated for 5 min in order to elute the captured target. (4) The eluate (10 μL) was directly transferred to the ISET and treated in the same way as the poros silicon assay samples. Assay for the Ang I on Porous Silicon. First, 10 μL of angiotensin I antibody 0.11 mg/mL in 10 mM PBS was deposited onto the capture zones on the porous silicon chip and the antibody was allowed to bind for 30 min. After this step, the spots were washed with 3  10 μL 10 mM PBS in order to remove antibody not bound to the surface by physical adsorption. While maintaining the chip wet, each spot was incubated with 10 μL of sample solution for 30 min and then washed with 3  10 μL of 10 mM PB buffer. Elution was made using 2  10 μL of 5% acetic acid for each spot; after 15 min of incubation, the elution liquid was transferred to the ISET plate for reversed phase solid phase extraction. Figure 2 shows the workflow of the Psi-ISET iMALD sample preparation. ISET Protocol. In this paper, the previously described46,47 integrated selective enrichment target (ISET) was used to purify and concentrate the captured antigen after elution from the porous silicon surface prior to MALDI MS analysis. Poros R2 50 μm beads (Carlsbad, CA, US) were used to purify and

ARTICLE

concentrate the peptides. The ISET target was placed in an vacuum fixture, and the vacuum was supplied by a vacuum pump (Vacuubrand GMBH, Wertheim, Germany), controlled and gauged with a valve (Qiagen vacuum regulator, product NO. 19530), inserted between the vacuum pump and the fixture. The ISET reverse phase (RP)-SPE protocol was as follows: (1) Prior to application of the eluted sample, each perforated nanovial was filled with approximately 100 nL of R2 beads in 60% ACN/0.1% TFA and washed with 2  2.5 μL of 60% ACN/0.1%TFA followed by 0.1% TFA twice, at maximum vacuum. (2) Ten μL of eluted sample in acetic acid was applied to each position and after 5 min drawn through the beads by applying a lower vacuum ( 5 mmHg). (3) The beads where then washed with 2  3 μL 0.1% TFA twice, under high vacuum ( 10 mmHg). (4) Prior to elution with matrix, the ISET was removed from the vacuum fixture and the backside of the target was dried with a tissue. (5) Elution of the analytes onto the backside of the ISET was performed with 2  0.3 μL, 60% ACN/0.1%TFA containing 1 mg/mL of cyano-4-hydroxy-cinnamic acid (CHCA). The elution was done at a low vacuum ( 2 mmHg). For the final MALDI MS analysis, the ISET chip was turned upside down, i.e., with the matrix spots facing upward into a MALDI target having a milled recession accepting the chip. MALDI MS Analysis. MALDI-TOF MS was performed on a M@LDI (Waters, Milford, MA, US) running Masslynx 4.0. All the spectra were acquired manually, and approximately, 100 shots were averaged. The peptide analysis (800 3000 Da) was made in reflection mode and positive ion mode. The instrument was calibrated by ProteoMass Normal Mass Calibration Mix (Sigma). Matrix solution was prepared by dissolving 10 mg/mL of R-CHCA in 60% ACN/0.1% TFA.

’ RESULTS AND DISCUSSION

Figure 1. Photo shows a patterned porous silicon microchip and the deposition of 10 μL performed with a pipet. Note confinement of the sample solution within the SiO2 ring.

Sample Handling during the PSi-ISET iMALDI Protocol. The silicon dioxide ring around each porous silicon spot effectively confined the samples to each antibody covered PSi array position, avoiding carryover and allowing loading of up to of 10 μL of solution. Although most of the presented data was run in small manually prepared series, the SBS compliant array formats of both the PSi-array and the ISET platform allows for easy automation of the PSi-ISET iMALDI protocol, Figure 3. It is important to note that the sample handling process includes the antibody immobilization by physical adsorption. This allows the immunoaffinity capture chips to be activated immediately prior

Figure 2. Schematic workflow of immunocapture; top (I): antibody directly adsorbed on the high-surface area porous silicon is used to capture the antigen Ang I; bottom (II): the eluted antigen is transferred and subjected to RP-SPE sample preparation protocol on the ISET chip followed by MALDI MS detection. 4944

dx.doi.org/10.1021/ac200679t |Anal. Chem. 2011, 83, 4942–4948

Analytical Chemistry

ARTICLE

Figure 3. Photo showing a porous silicon chip where the immunocapture takes place (top left inset: 10 μL aliquots/position) and the ISET platform in a robotic setup (lower right inset) where the eluted antigen (10 μL sample elution) is subject to SPE for purification and concentration prior to MALDI MS. The use of SBS compatible pitches on both chip platforms allows for automation of the assay protocol using standard liquid handlers.

to use, avoiding covalent antibody immobilization protocols. Also, concerns about the stability of the chip immobilized antibody during storage in between assays are eliminated. The hydrophobic behavior of the PSi surface is a prerequisite for successful sample handling using robotics, yet the hydrophilic nature of PSi at the molecular level, (SiO2), allows for a nondenaturing immobilization of the antibody by adsorption, which stands in contrast to adsorption on hydrophobic surfaces.49 In a recent paper by Reid et al.,27 a quantitative iMALDI assay for Ang I was reported using magnetic Protein G Dynabeads for the immunocapture followed by transfer of the beads directly onto the MALDI target. The authors commented particularly on the high background generated by Tween 20. Indeed, the use of detergents will generate an intense background during MALDI MS that hampers ionization efficiency and, worse, competes with the peptide antigen for the binding to the RP beads during purification. In the outlined PSi-ISET iMALDI approach, the Tween could be eliminated from the protocol. In our magnetic bead reference experiments, we used Dynabeads M270, which analogous to the PSi has a hydrophilic backbone, as these have considerably lower background than beads with a hydrophobic backbone (data not shown). The Psi-ISET iMALDI protocol reported here uses no detergents in any of the steps, as the avoidance of detergents is of utmost importance to benefit from the RP-SPE step in the subsequent ISET platform. Compared to a direct immobilization of the antibody on a MALDI target or placing antigen antibody beads directly onto the MALDI target, the use of a reverse phase sample preparation on an integrated sample enrichment target (RP-ISET) purification and concentration step prior to MS solves several problems commonly encountered during immunoMALDI: (1) The sample is desalted. (2) The eluted sample is efficiently concentrated into a confined MALDI spot. (3) The MALDI readout is performed on a sample without interference from the antibody. The ease of the PSi-ISET iMALDI sample handling and time and cost savings is definitely one of the biggest benefits of the approach. Specific Capture of Angiotensin 1 on PSi-ISET Platform. A series of experiments were performed to verify the performance

of the PSi-ISET iMALDI approach. In a first step, a sample, containing 5 nM Ang I spiked in 50 nM ADH digest before and after assay, was assayed with or without antibody on the PSi. When analyzing a 10 μL sample directly after RP-ISET sample preparation (no immunocapture), the Ang I could clearly be observed surrounded by a very high background of ADH peptides, Figure 4A. After the PSi-ISET iMALDI assay, the Ang I peak is observed at a somewhat lower intensity but without the background peaks from the ADH digest, Figure 4B, demonstrating successful immunocapture and enrichment of the Ang I. Figure 4 C shows the MS readout after processing the sample on a blank porous silicon (no antibody immobilized), demonstrating the low unspecific binding of the porous surface. Binding Capacity. The capturing capacity of the porous silicon can be derived from previous data where an antibody deposition/density of more than 140 amol/60 μm spot diameter saturated the porous silicon surface,37 yielding a corresponding loading capacity of approximately 350 fmol/position for the 3 mm diameter spot. In experiments using 10 nM of spiked Ang I in the sample, the resulting spectra from the magnetic beads (Figure 5A) and the PSi array (Figure 5B) generates comparable intensities, indicating that at this level there is no significant capacity difference between the two capturing matrixes. When depositing antibody on a nonporosified chip surface (Figure 5C), the Ang I could not be detected, due to the lack of capacity. Hence, for work with antipeptide antibodies, the observed capacity of the 3 mm porous silicon spot should be adequate. Handling Samples of Increasing Complexity. One of the most important aspects of any immunoMALDI protocol is that the background from nonspecific binders is minimized. In order to take advantage of the high-throughput and avoid expensive and time-consuming chromatographic separation steps after the affinity capture, the ion suppression phenomena50 in MALDI MS must be avoided. In a first experiment, the complexity of the sample was increased by spiking 10 nM Ang 1 into a 100 nM tryptic digest mixture of 48 proteins (Sigma-Aldrich, UPS1). This type of mixture contains thousands of peptides, and Figure 6A shows the mass spectra resulting from a direct 4945

dx.doi.org/10.1021/ac200679t |Anal. Chem. 2011, 83, 4942–4948

Analytical Chemistry

ARTICLE

Figure 4. Specific capture of angiotensin 1. The top spectra (A) results from a direct ISET RP-SPE of 10 μL of a sample solution containing 5 nM Ang I and 50 nM ADH digest, without any immunocapture. Middle spectra (B) shows specific capture of Ang I (red arrow) from 10 μL of the sample used above, after immunocapture on porous silicon and ISET RP-SPE. Bottom spectra (C) shows the results of an assay without Ang I-Ab on the porous spot. Note the reduction of complexity where very little unspecific binding can be observed on the porous silicon. Inset mass range zoom indicates in (B) the isotope resolution of the immunocaptured Ang I and in (C) that no Ang I can be seen in the unspecific signal.

Figure 5. Capacity of porous silicon. The mass spectra shows the readout after immunocapture and ISET RP-SPE from 10 μL samples of 5 nM Ang I spiked in 50 nM ADH, using 10 μL of magnetic beads (A), porous silicon (B), and a planar surface nonporous silicon chip (C). As can be noted, the porous silicon and the magnetic beads both provide very similar capacity at this level, whereas a planar surface does not provide enough antibody binding/capacity to enable the affinity capture.

RP-ISET sample preparation (no preceding immunocapture step) of a 10 μL mixture. Only a fraction of the peptides present in this complex sample can be observed by direct MALDI MS, and the 10 nM Ang I spiking could not be observed. Only after performing the PSi-ISET immunoaffinity protocol could the Ang I be clearly detected, Figure 6B. Note that due to the complexity of the sample the signal intensities (TIC) are actually lower for the direct sample preparation (Figure 6A) than for the immunocaptured sample (Figure 6B). Even with the low intrinsic background of the PSi, there is still some background peaks visible (Figure 6B). It can, however, be expected that some background will always be present from complex samples and that the use of isotope spiking with internal standard will be necessary for clinical work using an iMALDI approach.

The complex digest represents one type of sample that can be assayed with the PSi-ISET iMALDI approach. It is foreseeable that crude biofluids, e.g., plasma, will constitute a complex matrix that must be addressable. Plasma represents the most widely available clinical material for large-scale studies. In experiments using undigested plasma diluted 1:10 with PBS, Ang I could not be detected after a direct ISET SPE sample preparation step at low nM Ang I levels. Most notably, after processing the same samples on the PSi-ISET iMALDI platform, Ang I could be clearly observed both at 5 and at 1 nM levels, Figure 7. It is noteworthy that most of the data presented here were generated on an old MALDI MS instrument with a low sensitivity. Thanks to the generic format of the ISET, the processed samples can also be run on a more modern MALDI Orbitrap XL (Thermo Scientific, Waltham, MA, USA), with MS/MS capability. Figure 8 4946

dx.doi.org/10.1021/ac200679t |Anal. Chem. 2011, 83, 4942–4948

Analytical Chemistry

ARTICLE

Figure 6. Handling samples of increasing complexity. Top spectra shows a direct ISET RP-SPE (no immunocapture) of a 10 μL sample consisting of 10 nM Ang I spiked at a 1:10 ratio in a 50 proteins digest; the bottom spectra show the analysis result after immunocapture of the same sample amount using the PSi-ISET iMALDI approach. It can be seen that before the assay the sample complexity prevents the detection of Ang 1 and after the assay the Ang 1 can be clearly detected (red arrow).

Figure 7. Immunocapture from plasma samples. 10 μL of plasma 1:10 diluted in PBS was spiked with low concentrations of Ang 1. Top spectra (A) plasma at a level of 5 nM, and bottom spectra (B) spiked with 1 nM Ang 1. No Ang 1 signal could be observed by a direct ISET RP-SPE.

spectrum shows the subsequent MS/MS data that provides structural information of the bound species. The performance of modern MALDI instruments also offers improved means of quantitation by use of single reaction monitoring (SIM) and selected reaction monitoring (SRM), which opens the route to high-speed MALDI MS biomarker assays in complex biofluids.

Figure 8. FT MS spectra acquired on a MALDI Orbitrap XL of a sample comprising 1 nM Ang I spiked in 1:10 plasma after affinity capture; inset shows the MS/MS spectra that enables identification of the antigen.

shows Ang I identified after PSi-ISET iMALDI processing of a 1 nM Ang I sample in 1:10 diluted plasma. The inset mass

’ CONCLUSIONS A new platform, PSi-ISET iMALDI, for immunocapture and MS readout has been developed. Antibody immobilization by adsorption on PSi is made just prior to use, avoiding tedious covalent immobilization protocols. The hydrophobic behavior of the hydrophilic PSi surface enables successful sample handling using robotics and low unspecific binding. Improved MS sensitivity is accomplished by concentration of the eluted antigen by ISET sample preparation that minimizes both the number of sample transfers and the total surface area to which the analyte is exposed. Concentrations down to 1 nM Ang I in diluted plasma were successfully assayed. The presented PSi-ISET iMALDI platform opens the possibility to combine the benefits of immunoaffinity capturing with the structural and high speed readout of MALDI MS. Oncoming work will target multiplex arrays on the 4947

dx.doi.org/10.1021/ac200679t |Anal. Chem. 2011, 83, 4942–4948

Analytical Chemistry PSi capturing platform linked to automated sample handling and RP-ISET and MALDI MS readout in clinical samples.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

’ ACKNOWLEDGMENT Financial support is acknowledged from Swedish Research Council (VR 2009-5361 and VR/Vinnova/SSF MTBH 20067600), the Royal Physiographic Society, the Crafoord Foundation, the Carl Trygger Foundation, the SSF Strategic Research Centre (Create Health), and Vinnova (Vinn Verifiera 200702614). The authors are also very grateful to the China Scholarship Council and National Construction of high-quality University projects of graduates for providing a scholarship for H.Y. ’ REFERENCES (1) Anderson, N. L. Clin. Chem. 2010, 56 (2), 177 185. (2) Kato, H.; Nishimura, T.; Hirano, T.; Nomura, M.; Tojo, H.; Fujii, K.; Kawamura, T.; Mikami, S.; Kihara, M.; Bando, Y.; Tsuboi, M.; Ikeda, N.; Marko-Varga, G. J. Proteome Res. 2011, 10 (1), 51 57. (3) Surinova, S.; Schiess, R.; H€uttenhain, R.; Cerciello, F.; Wollscheid, B.; Aebersold, R. J. Proteome Res. 2011, 10 (1), 5 16. (4) Zhang, Q.; Faca, V.; Hanash, S. J. Proteome Res. 2011, 10 (1), 46 50. (5) Beck, M.; Claassen, M.; Aebersold, R. Curr. Opin. Biotechnol. 2011, 22 (1), 3 8. (6) Lam, H.; Aebersold, R. Methods Mol. Biol. 2010, 604, 95 103. (7) Nilsson, T.; Mann, M.; Aebersold, R.; Yates, J. R., 3rd; Bairoch, A.; Bergeron, J. J. Nat. Methods 2010, 7 (9), 681 685. (8) Sparbier, K.; Wenzel, T.; Dihazi, H.; Blaschke, S.; Muller, G. A.; Deelder, A.; Flad, T.; Kostrzewa, M. Proteomics 2009, 9 (6), 1442–1450. (9) Diamandis, E. P. Mol. Cell Proteomics 2004, 3 (4), 367–378. (10) Nedelkov, D. Proteomics 2008, 8 (4), 779–786. (11) Nedelkov, D.; Phillips, D. A.; Tubbs, K. A.; Nelson, R. W. Mol. Cell Proteomics 2007, 6 (7), 1183–1187. (12) Anderson, N. L.; Jackson, A.; Smith, D.; Hardie, D.; Borchers, C.; Pearson, T. W. Mol. Cell Proteomics 2009, 8 (5), 995–1005. (13) Anderson, N. L.; Anderson, N. G.; Pearson, T. W.; Borchers, C. H.; Paulovich, A. G.; Patterson, S. D.; Gillette, M.; Aebersold, R.; Carr, S. A. Mol. Cell Proteomics 2009, 8 (5), 883–886. (14) Papac, D. I.; Hoyes, J.; Tomer, K. B. Anal. Chem. 1994, 66 (17), 2609–2613. (15) Macht, M.; Fiedler, W.; Kurzinger, K.; Przybylski, M. Biochemistry-Us 1996, 35 (49), 15633–15639. (16) Nelson, R. W.; Krone, J. R.; Jansson, O. Anal. Chem. 1997, 69 (21), 4363–4368. (17) Nedelkov, D.; Nelson, R. W. J. Mol. Recognit. 2000, 13 (3), 140–145. (18) Nedelkov, D. Anal. Chem. 2007, 79 (15), 5987–5990. (19) Sun, S. Q.; Mo, W. J.; Ji, Y. P.; Liu, S. Y. Rapid Commun. Mass Spectrom. 2001, 15 (18), 1743–1746. (20) Borrebaeck, C. A.; Ekstrom, S.; Hager, A. C.; Nilsson, J.; Laurell, T.; Marko-Varga, G. Biotechniques 2001, 30 (5), 1126–1130. 1132. (21) Liang, X. L.; Lubman, D. M.; Rossi, D. T.; Nordblom, G. D.; Barksdale, C. M. Anal. Chem. 1998, 70 (3), 498–503. (22) Finnskog, D.; Ressine, A.; Laurell, T.; Marko-Varga, G. J. Proteome Res. 2004, 3 (5), 988–994. (23) Brauer, H. A.; Lampe, P. D.; Yasui, Y. Y.; Hamajima, N.; Stolowitz, M. L. Proteomics 2010, 10 (21), 3922–3927. (24) Niederkofler, E. E.; Tubbs, K. A.; Gruber, K.; Nedelkov, D.; Kiernan, U. A.; Williams, P.; Nelson, R. W. Anal. Chem. 2001, 73 (14), 3294–3299.

ARTICLE

(25) Trenchevska, O.; Kamcheva, E.; Nedelkov, D. J. Proteome Res. 2010, 9 (11), 5969 5973. (26) Hurst, G. B.; Buchanan, M. V.; Foote, L. J.; Kennel, S. J. Anal. Chem. 1999, 71 (20), 4727–4733. (27) Reid, J. D.; Holmes, D. T.; Mason, D. R.; Shah, B.; Borchers, C. H. J. Am. Soc. Mass Spectrom. 2010, 21 (10), 1680–1686. (28) Phizicky, E.; Bastiaens, P. I.; Zhu, H.; Snyder, M.; Fields, S. Nature 2003, 422 (6928), 208–215. (29) Ptacek, J.; Devgan, G.; Michaud, G.; Zhu, H.; Zhu, X.; Fasolo, J.; Guo, H.; Jona, G.; Breitkreutz, A.; Sopko, R.; McCartney, R. R.; Schmidt, M. C.; Rachidi, N.; Lee, S. J.; Mah, A. S.; Meng, L.; Stark, M. J.; Stern, D. F.; De Virgilio, C.; Tyers, M.; Andrews, B.; Gerstein, M.; Schweitzer, B.; Predki, P. F.; Snyder, M. Nature 2005, 438 (7068), 679–684. (30) Zhu, H.; Bilgin, M.; Bangham, R.; Hall, D.; Casamayor, A.; Bertone, P.; Lan, N.; Jansen, R.; Bidlingmaier, S.; Houfek, T.; Mitchell, T.; Miller, P.; Dean, R. A.; Gerstein, M.; Snyder, M. Science 2001, 293 (5537), 2101–2105. (31) Emili, A. Q.; Cagney, G. Nat. Biotechnol. 2000, 18 (4), 393–397. (32) Reid, J. D.; Parker, C. E.; Borchers, C. H. Curr. Opin. Mol. Ther. 2007, 9 (3), 216–221. (33) Voshol, H.; Ehrat, M.; Traenkle, J.; Bertrand, E.; van Oostrum, J. FEBS J. 2009, 276 (23), 6871–6879. (34) Anderson, K. S.; Sibani, S.; Wallstrom, G.; Qiu, J.; Mendoza, E. A.; Raphael, J.; Hainsworth, E.; Montor, W. R.; Wong, J.; Park, J. G.; Lokko, N.; Logvinenko, T.; Ramachandran, N.; Godwin, A. K.; Marks, J.; Engstrom, P.; LaBaer, J. J. Proteome Res. 2011, 10 (1), 85 96. (35) Jaras, K.; Tajudin, A. A.; Ressine, A.; Soukka, T.; Marko-Varga, G.; Bjartell, A.; Malm, J.; Laurell, T.; Lilja, H. J. Proteome Res. 2008, 7 (3), 1308–1314. (36) Ressine, A.; Corin, I.; Jaras, K.; Guanti, G.; Simone, C.; Marko-Varga, G.; Laurell, T. Electrophoresis 2007, 28 (23), 4407–4415. (37) Ressine, A.; Ekstrom, S.; Marko-Varga, G.; Laurell, T. Anal. Chem. 2003, 75 (24), 6968–6974. (38) Yakovleva, J.; Davidsson, R.; Bengtsson, M.; Laurell, T.; Emneus, J. Biosens. Bioelectron. 2003, 19 (1), 21–34. (39) Yakovleva, J.; Davidsson, R.; Lobanova, A.; Bengtsson, M.; Eremin, S.; Laurell, T.; Emneus, J. Anal. Chem. 2002, 74 (13), 2994–3004. (40) Chen, L.; Chen, Z. T.; Wang, J.; Xiao, S. J.; Lu, Z. H.; Gu, Z. Z.; Kang, L.; Chen, J.; Wu, P. H.; Tang, Y. C.; Liu, J. N. Lab Chip 2009, 9 (6), 756–760. (41) Chen, Y. Q.; Bi, F.; Wang, S. Q.; Xiao, S. J.; Liu, J. N. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2008, 875 (2), 502–508. (42) Lowe, R. D.; Szili, E. J.; Kirkbride, P.; Thissen, H.; Siuzdak, G.; Voelcker, N. H. Anal. Chem. 2010, 82 (10), 4201–4208. (43) Drott, J.; Lindstr€ om, L.; Rosengren, L.; Laurell, T. J. Micromech. Microeng. 1997, 7, 14–23. (44) Drott, J.; Rosengren, L.; Lindstr€om, K.; Laurell, T. Thin Solid Films 1998, 330, 161–166. (45) Ekstrom, S.; Wallman, L.; Helldin, G.; Nilsson, J.; Marko-Varga, G.; Laurell, T. J. Mass Spectrom. 2007, 42 (11), 1445–1452. (46) Ekstrom, S.; Wallman, L.; Hok, D.; Marko-Varga, G.; Laurell, T. J. Proteome Res. 2006, 5 (5), 1071–1081. (47) Ekstrom, S.; Wallman, L.; Malm, J.; Becker, C.; Lilja, H.; Laurell, T.; Marko-Varga, G. Electrophoresis 2004, 25 (21 22), 3769–3777. (48) Tajudin, A. A.; Ekstrom, S.; Jaras, K.; Marko-Varga, G.; Malm, J.; Lilja, H.; Laurell, T. In MALDI-target integrated microfluidic PSA assay, Proceedings of Micro Total Analysis Systems Conference, The Chemical and Biological Microsystems Society: San Diego, CA, USA, 2009; pp 1064 1066. (49) Vermeer, A. W.; Giacomelli, C. E.; Norde, W. Biochim. Biophys. Acta 2001, 1526 (1), 61–69. (50) Knochenmuss, R.; Dubois, F.; Dale, M. J.; Zenobi, R. Rapid Commun. Mass Spectrom. 1996, 10 (8), 871–877.

4948

dx.doi.org/10.1021/ac200679t |Anal. Chem. 2011, 83, 4942–4948