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Technical Note
Bead-Extractor Assisted Ready-to-use reagent System (BEARS) for immunoprecipitation coupled to MALDI-MS Huiyan Li, Robert Popp, Michael Chen, Elizabeth M MacNamara, David Juncker, and Christoph H. Borchers Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04169 • Publication Date (Web): 03 Mar 2017 Downloaded from http://pubs.acs.org on March 4, 2017
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Analytical Chemistry
Technical Note
Bead-Extractor Assisted Ready-to-use reagent System (BEARS) for immunoprecipitation coupled to MALDI-MS
Huiyan Li1,2,3, Robert Popp1, Michael Chen4, Elizabeth M. MacNamara4, David Juncker2,3, and Christoph H. Borchers1,5
1
University of Victoria - Genome BC Proteomics Centre, #3101 - 4464 Markham Street,
Vancouver Island Technology Park, Victoria, BC V8Z 7X8, Canada 2
Biomedical Engineering Department, McGill University, Duff Medical Building, McGill
University, 3775, rue University, Room 316, Montreal, QC H3A 2B4, Canada 3
McGill University and Genome Quebec Innovation Centre, 740 Dr. Penfield Avenue, Room
6206, Montreal, QC H3A 1A4, Canada 4
Jewish General Hospital, 3755 Côte-Ste-Catherine Road, Montreal, QC H3T 1E2, Canada
5
Dept of Biochemistry and Microbiology, University of Victoria, Petch Building Room 207, 3800
Finnerty Rd., Victoria, BC V8P 5C2, Canada
Corresponding author: Christoph H. Borchers Email:
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Abstract Quantitative protein assays play an important role in the study of biological functions. Immunoassays and mass spectrometry are two main technologies for quantifying proteins in biological samples.
The combination of immunoprecipitation (IP) with MALDI technology
delivers high assay sensitivity and specificity, but the sample preparation procedure involves multiple washing and transfer steps. These steps can either be performed manually (requiring significant time and labor), or automatically (requiring the purchase of a complex liquidhandling workstation). This bottleneck has limited the widespread adoption of this technology. We present here the Bead-Extractor Assisted Ready-to-use reagent System (BEARS) technology for simplified, low cost protein and peptide immunoprecipitation combined with MALDI-MS detection. All of the reagents are stable during long-term storage and can be prepared in advance. In the BEARS technology, a magnetic-bead extractor is used to handle beads from 96 wells simultaneously. A BEARS-based method was developed for Plasma Renin Activity (PRA), and was evaluated on fifty-three clinical samples. These experiments showed that the BEARS assay had an LOD and linear range comparable to the manual method and an automated iMALDI PRA assay, but was 4-times faster than the manual approach. The BEARS iMALDI results also correlated well with a conventional ELISA PRA assay, with a coefficient of determination of 0.98.
The BEARS technology provides
convenience and affordability, and extends the use of IP-based mass spectrometry technology to most research and clinical laboratories, including those in developing countries.
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Introduction Since the first immuno-mass spectrometry assay presented in 1993,1 the combination of immunoprecipitation with mass spectrometry has shown great potential to address the challenge of low target-protein concentration in biological matrices.2-4 The target proteins or peptides are enriched by immuno-capture with specific antibodies, and then analyzed by mass spectrometry. In this way, the target molecules are pre-concentrated from the samples, and the potential problem of non-specificity of antibodies can be mitigated by a double-selection process consisting of both antibody capture and mass spectrometry. The immuno-enrichment step can be performed on beads,5 in 96-well plates,6 on chips,7-8 in columns,9 or in tips.10 For the quantitative analysis of peptides, a labeled internal standard is co-captured on anti-peptide antibodies, along with the endogenous target peptide, followed either by elution and LC/ESIMS (SISCAPA,11-12) or direct detection by MALDI (iMALDI,13-14)
Because of the immuno-
enrichment step, the assay sensitivity for a peptide can be in the low ng/mL range or better for blood plasma proteins, and a recent study has shown the multiplexing of as many as 172 peptides quantitated in a single assay.15 In IP experiments, antibodies are usually immobilized on beads. In the iMALDI technology,13-14, 16-17
these beads are placed directly onto the MALDI target, where the captured analytes are
released by the matrix solvent. For ease of handling during the capture, washing, and elution steps, magnetic beads are now commonly used.18 However, sample preparation has been either performed manually, which is time-consuming and tedious, or has required an expensive and complex liquid handling robot, limiting the implementation of the iMALDI technique by most laboratories.
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The BEARS system described here simplified this procedure by allowing the use of a magnetic bead-handling system. To evaluate this new BEARS technology, we have applied the BEARS system to a Plasma Renin Activity (PRA) assay for the screening of primary aldosteronism, a form of secondary hypertension, whose automation using magnetic beads and a liquid handling system has been recently published.19
Experimental The step-by-step comparison of the BEARS-iMALDI procedure with a conventional iMALDI experiment is shown in Figure 1. In conventional iMALDI (Figure 1a), anti-peptide antibodies are bound to beads, and are then aliquoted into a 96-well plate immediately before the assay. In BEARS-iMALDI (Figure 1b), the beads are pre-coated with antibodies, aliquoted in a 96-well plate, but instead of being used immediately, the plate is sealed and stored in a refrigerator for later use (up to 3 months). During the assay, in both methods, the beads are then incubated with samples to capture the target analytes and the internal standard peptides. After sample incubation, the beads are washed. To do this, in a conventional iMALDI experiment, the magnetic beads are washed by pelleting them in each well with magnets, pipetting out the sample solutions, and re-suspending the beads in wash buffers. The whole procedure requires several rounds of reagent exchange in 96 wells by either manual pipetting or by using an expensive robotic liquid handling device, which is time-consuming. In contrast, in BEARS-iMALDI, a magnetic bead extractor consisting of 96 magnetic pins is used to handle the beads from 96 wells simultaneously. Instead of exchanging the buffers in each well, beads are captured from the sample solutions and released into another 96-well plate containing the wash buffer. A PCR-plate is then clipped onto the set of 96 magnetic pins and acts as a
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"disposable glove" between the magnets and beads, thereby avoiding contamination of the magnets. Beads are captured on the outer surface of the PCR plate’s wells, and dropped into the wash buffer by releasing the PCR plate from the magnets. Compared to the conventional bead washing method, in this way beads from 96 samples can be simultaneously transferred from plate-to-plate quickly and efficiently, simplifying the bead-washing procedure. After bead washing, in conventional iMALDI, the next two steps are bead transfer and matrix delivery. To transfer the beads to the target plate, the beads are re-suspended in buffer (typically an ammonium bicarbonate (AmBic) solution), and 7-10 µL of beads and AmBic per spot are transferred to a target plate by either manual pipetting or by using a liquid-handling robot.19 Once the beads are dried on the plate, 1 µL of matrix solutions is pipetted or spotted onto each bead spot. Unlike in conventional iMALDI assays, after bead washing, in BEARS-iMALDI, the beads are transferred to the target with the aid of the magnetic bead extractor, and the bead transfer and matrix delivery are combined into a single step. Specifically, in this step, first the pre-coated MALDI target is retrieved from storage, and then 10 µL of matrix solvent, containing ammonium citrate and 0.1% trifluoroacetic acid (TFA) in 75% acetonitrile (ACN), is pipetted onto each spot to dissolve the α-cyano-4-hydroxycinnamic acid (HCCA). Next, the beads are captured on the outside of a PCR plate with the magnetic bead extractor, and, finally, the beads are released into the matrix solvent droplets by placing the PCR plate on the MALDI target. In this process, the matrix solvent solution performs three functions simultaneously: it dissolves the HCCA, it transfers the beads to the target, and it elutes the bound peptides or proteins from the beads. Thus, the conventional protocol for performing these two steps (first deliver the beads and then spot the matrix onto the target) has been reversed in the BEARS
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technique, and combined into a single convenient step. Only one round of pipetting is needed to deliver the matrix solvent, instead of the previous requirement of 3 rounds of pipetting for resuspending the beads in wells, transferring the beads onto the target, and delivering matrix solutions. This makes the immunoprecipitation technology easier and more convenient to use. In addition, different from SISCAPA-MALDI assays where the captured peptides are eluted and transferred onto a MALDI plate20-21, the beads carrying captured peptides are transferred onto a plate without an elution step, simplifying the sample preparation procedure. Importantly, the design of the BEARS-iMALDI technology is based on a standard 96-well plate format, compatible with most MALDI mass spectrometers, including the Bruker BioTyper instrument which is FDA approved for bacterial identification in clinics.
In a BEARS immunoprecipitation experiment, most reagents can be prepared and stored until needed for the assay. The magnetic beads are pre-coated with the antibodies, and aliquoted into a 96-well plate. The plate is then sealed to prevent evaporation, stored at 4°C and stable for at least 3 months (Figure S2a). The HCCA matrix is pre-pipetted onto a MALDI target plate, and after drying, the plate is stored in dark under argon at room temperature until use (Figure S2b). In addition to the beads and matrix solution, all of the other reagents were prepared beforehand. All of these reagents can be stored for 1-3 months before use, with the exception of the AmBic solution for washing the beads, which is stored at 4°C and which needs to be prepared fresh every week.
Results and Discussion
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The proof-of-concept of BEARS-iMALDI for measuring PRA and assay optimization are shown in Figure S1. PRA is defined as the Angiotensin I (AngI) generation rate in plasma. The limit of detection (LOD) and the linear range of the BEARS-iMALDI assay for measuring Ang I in human plasma were determined. Because of the endogenous Ang I in the samples, the LOD was determined based on stable isotope-labeled standard (SIS) Ang I (Figure 2). The assay LOD was defined as the lowest Ang I SIS concentration at which the S/N ratio was ≥ 3. The LOD was 6.72 fmol (0.02 ng/L/s), comparable to that obtained from the automated iMALDI method (4.9 fmol, 0.015 ng/L/s),19 and four times better than the sensitivity range for PRA assays recommended by the Endocrine Society’s Clinical Practice Guidelines ( 0.08-0.6 ng/L/s).22
The lower limit of detection (LLOD) was defined as the lowest SIS Ang I concentration with
