The ability to quantitatively probe diverse panels of proteins and their post-translational modifications (PTMs) across multiple samples would aid a broad spectrum of biological, biochemical, and pharmacological studies. A novel microarray analytical technology that can support highly multiplexed, targeted proteomic assays has been developed by Adeptrix.1–6 The technology, termed Affinity-Bead Assisted Mass Spectrometry (Affi-BAMS), combines immunoaffinity capture with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and it enables development of highly specific and customizable assay panels for simultaneous profiling of multiple proteins and PTMs.
The Affi-BAMS workflow uses a bead-based enrichment approach in which each bead contains one type of antibody. Each antibody has enough binding capacity to enable quantification within three orders of magnitude. Multiplexing capability is achieved by combining Affi-BAMS beads that have different specificities. With Affi-BAMS, both intact proteins and protein fragments can be monitored.
The Affi-BAMS assay workflow (Figure 1) combines three common bioanalytical technologies: magnetic beads, microarrays, and MALDI-TOF MS to uniquely provide scalable detection and quantification of multiple protein targets in a miniaturized, multiplexed, single-bead format.
Biological input material is lysed and digested with a protease; the resulting peptides are captured by antibody-conjugated magnetic beads in a bead suspension format. An array is formed from the reacted beads on a microwell array plate. The bead array is exposed to an aerosol containing a MALDI matrix solution, which causes the bound peptides to be eluted from the spatially separated beads into their respective microwells. The solvent is allowed to evaporate, which causes the eluted peptides to co-crystallize with the MALDI matrix solution.
The silicone gasket is removed to expose the array of spots, which is subsequently measured by MALDI-TOF MS and analyzed to identify peptides present in individual spots and to extract quantitative data that is used to calculate abundance changes of corresponding precursor proteins.
Affi-BAMS assay beads and slide arrays
Each Affi-BAMS bead includes a covalently coupled antibody that is validated to specifically capture a proteolytic fragment of a target protein (or PTM). The agarose beads contain a magnetic particle core to assist with bead manipulation during the sample preparation. Individual beads have a diameter of about 375 µm. The companion gasket contains microwells with dimensions that are slightly larger than a single bead (500-µm depth and diameter) to ensure one bead per well occupancy.
Peptide preparation and enrichment on target-specific immunoaffinity beads
For Affi-BAMS assays, peptides are prepared using the standard bottom-up proteomics methods. Under denaturing conditions, 100–200 µg of total protein extract from a cell culture, tissue, or liquid biopsy is subjected to reduction, alkylation, and proteolytic digestion. Immediately after digestion, proteolytic peptides can be assayed directly for liquid biopsy samples or they can be C18-purified for cell line or tissue samples prior to Affi-BAMS enrichment. Purified peptides are lyophilized and stored (−80 °C) until needed for Affi-BAMS assay.
A singleplex Affi-BAMS assay contains 2–3 replicate beads conjugated to an antibody that recognizes a specific protein target. A multiplexed Affi-BAMS assay contains a mixture of beads conjugated to different antibodies to carry out simultaneous enrichment of multiple protein targets. Affinity capture of target peptides is typically done for 12 h at 4 °C but can be performed within as little as 3 h. Nonspecifically bound peptides are removed by a series of brief washes with phosphate-buffered saline, ammonium bicarbonate buffer, and deionized water.
Bead arraying and conversion to an array of peptide microspots
The microwell array plate for assaying the reacted beads includes a silicone elastomer gasket containing an array of through-holes, which is attached to a gold-coated glass microscope slide. The number of wells exceeds the number of beads used in the affinity assays to ensure that every bead is singularly deposited. A multichamber frame serves to subdivide sets of microwells for the parallel processing of multiple sample sets. The number of chambers that can be accommodated on a single slide is between 1 and 64.
Washed Affi-BAMS beads containing bound target peptides are transferred into the sample chamber wells with a magnetic bead picker, dispersed within chamber wells by gentle agitation, and then settled into individual gasket microwells by gravity. The multichamber frame is subsequently detached, and bulk water is removed from the array surface using a slide spinner.
The bead array is then exposed to an aerosol containing cyano-4-hydroxycinnamic acid (CHCA) in 50% acetonitrile and 0.4% trifluoroacetic acid (TFA), generated by a matrix sprayer. The low pH of the solution dissociates bound peptides from each bead, while the sidewalls of the microwell gasket prevent the possible diffusion of eluted peptides into adjacent wells.
Following the elution cycle, the residual solvent evaporates, and the eluted peptides cocrystallize and are incorporated into a MALDI matrix in confined spots at the bottom of the microwells. Once the matrix is dry, the silicone gasket is lifted off the slide, and any remaining dry agarose beads are removed using a gentle burst of compressed air. Via this process, an array of microspots containing purified and concentrated target peptides is generated for subsequent MALDI-TOF MS measurement.
MS data acquisition
Arrays of peptide microspots can be measured on any MALDI-TOF MS instrument equipped to accept standard 25 × 75 mm microscope slides. The pattern of spots forms an array of 26 × 88 spots (with 0.8-mm spacing between each spot) that can be interrogated via MALDI-TOF MS. MS spectra are acquired sequentially from each spot in positive (or negative) ion mode. The laser beam is typically focused to approximately 10–20% of the diameter of the spot to ensure that the individual targets are sampled without contamination from adjacent spots. A MALDI-TOF MS instrument operating at a speed of 2 kHz can analyze a single 500-µm spot in less than 1 s and an entire 2288-spot array within approximately 1 h.
Exemplary Affi-BAMS assays: Monitoring brain and CSF biomarkers
Human brain samples were obtained from Maine Medical Center Research Institute BioBank (Scarborough, ME). Human cerebrospinal fluid (CSF) samples were obtained from Johns Hopkins School of Medicine, Alzheimer’s Disease Research Center (Baltimore, MD).
Figure 2 shows the results of an on-bead multiplexed Affi-BAMS assay used to monitor amyloid beta (Aβ) from undigested CSF derived from normal (red) and Alzheimer’s disease (black) patients (two normal and two diseased, triplicate beads). The assay retrieves 19 specific Aβ peptides between aa 672 and aa 713. After all MALDI-TOF MS spectra to the Aβ1–40 peptide (blue box) are normalized, clear separation of normal and diseased patient samples is seen from specific Aβ peptides.
Figure 3 shows the results of an on-bead multiplexed Affi-BAMS assay to monitor histone H3 Lys9 acetylation (H3K9ac) in ArgC-digested pooled brain tissue samples derived from normal (black) and Alzheimer’s disease (red) patients. The assay retrieves over 40 proteolytic H3 peptides between aa 1 and aa 21, all peptides containing acetylated Lys9. Many peptides additionally contain PTMs at Arg2 and/or Arg17 (citrullination) and Lys4, Lys14, and/or Lys18 (mono-, di-, and trimethylation and acetylation). A 10- to 25-fold increase in the MALDI MS signal indicates greater abundance of specific histone H3 PTM combinations (for example, H3K9ac + H3R2cit) in the diseased patient sample.
Affi-BAMS is highly effective at quantitating specific PTMs, such as phosphorylation, acetylation, and methylation, and can be configured to readily distinguish and quantitatively measure protein isoforms and orthologues. This platform can be readily adjusted to a multiplex assay using a defined panel of immunoaffinity beads (that is, focused target set) for large numbers of samples. The latter is advantageous for monitoring multiple biomarkers in high-throughput screening applications.
1. Hamza GM, Bergo VB, Mamaev S, et al. Affinity-Bead Assisted Mass Spectrometry (Affi-BAMS): A Multiplexed Microarray Platform for Targeted Proteomics. Int. J. Mol. Sci. 2020; 21(6): 1–36. DOI: doi:10.3390/ijms21062016.
2. Silva JC, Dikler S, Mamaev S, et al. A Targeted Multiplexed MALDI MS Assay Platform using Affinity-Bead Assisted Mass Spectrometry (Affi-BAMS) for Monitoring Brain and CSF Biomarkers. Poster presented at: The Association of Biomolecular Resource Facilities (ABRF) Annual Meeting; March 3, 2020; Palm Springs, CA.
3. Hamza GM, Kawatkar A, Browne CM, et al. From Chemical Proteomics to Translational Chemical Biology. In Chemical Biology. Poster presented at: THE HUB Symposium; September 19, 2019; Merck Research Laboratories, Boston, MA.
4. Mamaev S, Silva JC, Worsfold C, Bergo VB. A High-Throughput Multiplexed Assay Platform for Monitoring Protein Abundance in 96-Well Cell Cultures or Product Profiles from Enzyme-Substrate Reactions. Poster presented at: 67th ASMS Conference on Mass Spectrometry and Allied Topics; June 2–6, 2019; Atlanta, GA.
5. Mamaev S, Silva JC, Worsfold C, et al. Bead Assisted Mass Spectrometry (BAMS™): An Affinity Capture MALDI TOF MS Method for Multiplexed Biomarker Screening. Poster presented at: 66th ASMS Conference on Mass Spectrometry and Allied Topics; June 3–7, 2018; San Diego, CA.
6. Mamaev S, Silva JC, Worsfold C, et al. Bead Assisted Mass Spectrometry (BAMS™): A Robust Affinity Capture, MS Method for Multiplexed Biomarker Profiling. Poster presented at: 66th ASMS Conference on Mass Spectrometry and Allied Topics; June 3–7, 2018; San Diego, CA.