BAMS™: Bead Assisted Mass Spectrometry

A new tool for targeted, multiplexed proteomics that combines bead-based affinity enrichment with the power of mass spectrometry

BAMS™ assay workflow

Sample Type

tissue, cell line cultures, liquid biopsies

Generate Lysate

proteins are extracted from original source to make accessible for BAMS™ assay

Digest Protein Lysate

proteins are digested with protease (e.g. trypsin or chymotrypsin) for immuno-affinity capture

Affinity Capture

BAMS™ affinity capture beads are used to enrich for corresponding peptides to each target protein or post-translational modification (PTM)

Prepare BAMS™ Array

BAMS™ affinity capture beads are washed and then assembled into an ordered array using Adeptrix’ pico-well chamber gasket

Scan BAMS™ Array

captured peptides are eluted from beads and deposited onto microarray slide for MALDI MS scanning

Identify Protein Targets

acquired MALDI MS signal (mass & intensity) is compared to spectral library for identification of target protein or PTM

Quantify Protein Targets

MALDI MS intensity signal is used to determine relative quantitation based on internal standard reference

Monitor Biological Pathways

BAMS™ assay can be assembled with a specific panel of proteins and PTMs to monitor key signaling networks or specific areas of biology

BAMS Workflow

1. What is BAMS™?

BAMS™ stands for Bead-Assisted Mass Spectrometry. It is a multiplexed analytical platform technology that is based on single bead immunoaffinity capture and single bead analysis by mass spectrometry (MS).

2. What types of analytes can be measured by BAMS™?

The majority of currently available BAMS™ assays are configured for detecting proteins and protein modifications (PTMs).

3. What types of samples are analyzable by BAMS™?

In short, any biological or environmental sample can be analyzed. We have successfully measured proteins from serum/plasma, CSF, cell lysates and tissue lysates.

4. How does BAMS™ work?

Antibody-conjugated magnetic beads are incubated with a biological sample to capture the target analytes of interest. Multiple samples may be processed in parallel in a 96-well plate. The beads are washed to remove non-specifically bound biomolecules and arrayed on a BAMS™ microwell array slide that consists of an elastomer gasket attached to a microscope slide. The bead array is exposed to an aerosol containing matrix for MALDI mass spectrometry. The captured compounds are eluted from beads into individual microwells. The gasket and the beads are subsequently removed to make an array of spots containing concentrated and purified analytes incorporated into the matrix (BAMS™ array). Individual spots in the array are then measured by MALDI MS.

5. What is the advantage of BAMS™ for protein analysis?

Unlike other bead array platforms, BAMS™ uses direct detection by MS, meaning there is no ambiguity when measuring closely related protein targets, such as sequence variants, mutants, and fragments produced by an endogenous proteolytic activity. Whatever your application is, BAMS™ can achieve a greater level of precision and confidence in target identification.

6. How does BAMS™ detect protein modifications?

Protein modifications such as phosphorylation, acetylation, methylation, ubiquitination and glycosylation are detected and identified based on their characteristic mass shifts. The exact location of the PTM site may be confirmed by MS-MS sequencing. BAMS™ is particularly suitable for measuring combinatorial PTM signatures in histones and heavily phosphorylated proteins.

7. How is BAMS™ different from flow cytometry-based assays?

BAMS™ is a bead-based immunoassay platform that uses MS instead of fluorescence to detect protein targets captured on individual beads. BAMS™ does not require detection antibodies and may be used in a wider range of proteomic applications including bottom-up, middle-down and top-down protein analysis. The microarray format employed by BAMS™ provides convenient means for sample measurement and archiving.

8. How does BAMS™ compare against Western blot, ELISA and antibody microarrays?

How does BAMS™ compare against Western blot, ELISA and antibody microarrays? BAMS™ can measure a greater number of proteins in a single assay. Valuable samples such as primary cells, tissue biopsies and liquid biopsies may be screened for tens or even hundreds of protein targets using the same quantity of material that one would load in a single lane of a Western blot. Importantly, BAMS™ is able to detect the molecular heterogeneity in a sample, which is missed by the other methods.

9. How does BAMS™ compare against LC-MS?

BAMS™ replaces the laborious and time-consuming chromatographic separation with a robust, reproducible antibody capture using a simple magnetic bead-based workflow. As a result, tens to hundreds of samples per day may be measured making BAMS™ attractive for screening applications. The direct enrichment employed by BAMS™ is particularly suitable for analyzing complex biological samples, such as non-depleted serum and plasma, where an amount of input material exceeds the capacity of the chromatographic column.

10. Can BAMS™ measure multiple proteins in a single assay?

Yes. Multiplexing is achieved by combining different BAMS™ assay beads, each containing different antibodies. Furthermore, on-bead multiplexing is achieved using an antibody that recognizes several targets, such as wild-type protein and a mutant (or phosphorylated and non-phosphorylated protein site). Certain BAMS™ reagents also recognize an epitope that occurs in more than one protein. The MS readout resolves multiple targets captured on a single bead based on their unique molecular weights.

11. Can BAMS™ measure multiple sites within a single protein?

Yes. Once the protein is digested with a suitable protease, different proteolytic fragments may be captured individually on separate beads and analyzed independently.

12. Is BAMS™ quantitative?

Yes. BAMS™ is compatible with various quantification methods including metabolic labeling, N-terminal labeling and internal standards. Unlike LC-MS, BAMS™ does not require the use of stable isotope labeled standards (SIS). The quantitative reproducibility of BAMS™ is typically under 10% CV (coefficient of variation) as determined by replicate BAMS™ assay beads.

13. What is the analytical sensitivity of BAMS™?

The sensitivity is ultimately determined by the MS instrument. Linear MALDI TOF MS have attomole sensitivity. A total of 10-20 μg of input protein is often sufficient for profiling multiple proteins. Such amount is available from a tissue biopsy or a cell culture grown in a single well of a 96-well plate.

14. Is antibody cross-reactivity a problem?

No. Unlike sandwich immunoassays, BAMS™ does not use detection antibodies.

15. Does the sample need to be protease digested in order to be analyzed by BAMS™?

No, however it will depend up the specific BAMS™ assay. BAMS™ can analyze proteins and peptides via direct enrichment from non-digested lysates, biofluids and cell culture supernatants. For example, amyloid beta protein fragments Aβ38, Aβ40, Aβ42 may be simultaneously detected in a single BAMS™ assay. However, the assay sensitivity is often significantly improved when the protein is digested and protein fragments are measured.

16. Which proteases are compatible with BAMS™?

The catalog of BAMS™ reagents includes the list of proteases that are compatible with a specific protein target.

17. Can BAMS™ samples be archived?

Yes. The pass-through portion of a sample may be lyophilized and stored at -80°C for future enrichments. Also, analyte-containing spots in the BAMS™ arrays are typically only partially depleted during the measurement and may be re-analyzed using reflector MS or MS-MS sequencing. When properly stored, BAMS™ arrays are stable for several months.

18. Can BAMS™ be used in epigenetic studies?

Yes. BAMS™ has been successfully applied to identify signature PTM combinations in histone tails.

19. Can BAMS™ be used to profile signaling pathways?

Yes. BAMS™ has been successfully applied to analyze cellular pathways such as mTOR.

20. Can BAMS™ be used to measure enzyme activity?

Yes. BAMS™ has been successfully applied to measure inhibition of a kinase activity.

21. What equipment do I need to run BAMS™?

The magnetic bead enrichment workflow requires only the standard lab equipment. In addition, a matrix sprayer is required to perform elution from bead arrays.

22. What type of MS instrument do I need to measure BAMS™ assays?

Any commercial MALDI TOF instrument that is equipped to accept microscope slides. The majority of BAMS™ assays are configured for linear MALDI TOF MS, which is the most basic type of MALDI mass spectrometry.

23. What if I do not have access to a MALDI instrument?

BAMS™ arrays may be measured as a service by many university core facilities or contract research organizations (CROs). Contact us for more information.

24. Can I measure BAMS™ arrays using an electrospray MS instrument?

Several companies manufacture MALDI sources for ESI MS that may be used to read BAMS™ arrays. Contact us for more information.

25. What software do I need to run BAMS™?

BAMS™ arrays are measured using standard data acquisition software provided with the MS instrument. Custom analytical software is available from Adeptrix. Contact us for more information.

26. How many beads can be measured on a BAMS™ microwell slide?

A medium-capacity BAMS™ microwell slide can accommodate over 2,000 beads. A large-capacity BAMS™ microwell slide can accommodate over 5,000 beads. In practice, tens to hundreds of beads are typically measured per assay.

27. How many samples can be measured on a BAMS™ microwell slide?

The slide may be subdivided into 2, 4, 8, 16, 24 or 64 chambers to accommodate the corresponding number of samples.

28. Are BAMS™ components reusable?

Multi-chamber frames, spring clips and slide trays may be used multiple times. Individual parts of the BAMS™ microwell array slide, i.e. the gasket and the microscope slide may be washed and reused several times.

29. How long does a BAMS™ experiment take?

The immunoaffinity enrichment requires as little as 1 hour but may be extended overnight for capturing low abundance targets. Arraying the beads and eluting compounds from a bead array take less than 1 hour. The above steps may be performed in parallel for multiple samples. A BAMS™ array may be measured in as little as 10 minutes or longer, depending on the number of spots.

30. Can BAMS™ assay results be integrated into follow-up bio-analytical studies?

Yes. Many antibodies in BAMS™ assays are validated for applications such as western blot, ELISA, immunohistochemistry (IHC) and flow cytometry.

31. Can BAMS™ be used in discovery proteomics?

Yes. BAMS™ can screen tens to hundreds to thousands of samples in a cost-efficient manner. It is particularly suitable for discovering novel protein modification sites and novel PTM combinations.

32. Can I use fluorescent probes in BAMS™ assays?

Yes. Optically transparent ITO-coated BAMS™ microwell slides can support dual MS and fluorescence readout.

33. What are BAMS™ reagents?

Adeptrix offers a catalog of bead-conjugated affinity probes that have been pre-validated to recognize selected protein targets and protein sites, as well as a number of pre-configured panels for performing multiplex immunoaffinity enrichment.

34. How do I choose the right quantification method for my BAMS™ assay?

BAMS™ reagents are compatible with various quantification strategies. Internal standard-based methods are commonly used but require pre-configured mixtures of synthetic peptides. In some cases, an internal standard may already be present in the sample, for example when measuring protein isoforms, modification sites or wild-type/mutant ratios. Semi-quantitative methods based on analyte depletion produce results to label-free LC-MS. Alternatively, samples may be analyzed using metabolic or chemical labeling strategies.

32. Can I use fluorescent probes in BAMS™ assays?

We are continuously adding more protein targets and can perform custom assay development. Contact us for more information.