Scenario 1:  Kinase Screen Using TR-FRET

TR-FRET (time-resolved fluorescence energy transfer) is a common assay platform utilizing a lanthanide cryptand donor and a fluorescent acceptor which generate a fluorescence signal under excitation when the donor and the acceptor are in close proximity. For a kinase assay, a common strategy is to utilize a donor labeled phospho-specific antibody or donor labeled peptide substrate.  

Common Issues

  • Cost – Labeled peptide substrates or antibodies for full screens can be a substantial expense, >$100,000.
  • Antibody issues – The selectivity and reactivity of antibodies can vary from batch to batch and also change over time making reproducibility and consistency problematic.

MS2 Array Solution

  • Lower peptide substrate costs – Fluorous modified peptides utilize low cost reagents and standard synthetic techniques resulting in substrate costs <20% TR-FRET labeled substrates
  • Elimination of antibodies – Direct readout of phosphorylation by MS eliminates the need for antibodies entirely.

Scenario 2:  Protease Assay Using FRET

Peptide substrates for the protease of interest are labeled with a fluorescence label and a quencher resulting in no signal upon excitation for the intact substrate. Cleavage of the peptide releases the quencher from the fluorescence label resulting in a fluorescence signal.

Common Issues

  • Compound interference – Auto-fluorescence or fluorescence interference resulting in a false signal
  • Non-specific cleavage – Hydrolytic cleavage of the peptide substrate not due to the protease of interest resulting in a false positive signal
  • Fluorescent label effects – Presence of the fluorescent tag can significantly alter the level or mechanism of activity of the substrate with the enzyme

MS2 Array Solution

  • Analyte enrichment and MS detection – Eliminates compound interference issues by removing potential interfering compounds through analyte enrichment before MS detection.
  • Structural information from MS detection – Products are measured directly by mass therefore non-specific cleavages not corresponding to the product of interest are not hits
  • Minimal label effects – The perfluorocarbon modification is significantly smaller than most fluorescent tags and contains no additional hydrogen donors or acceptors.

Scenario 3: Methyltransferase Assay Using LC/SPE-MS

Methylation or demethylation of a histone peptide substrate is monitored by mass spectrometry following sample preparation either by liquid chromatography or solid phase extraction.  MS detection allows for the simultaneous identification of various methylated states.

Common issues

  • Insufficient throughput for primary screening – Sampling rates of ~10sec/sample limit LC/SPE-MS use to secondary screening
  • Cost – For large campaigns SPE cartridge costs can become prohibitive
  • Lack of physiological relevance – Cell lysates or nuclear extracts difficult to use due to sample preparation issues.

MS2 Array Solution

  • High-throughput platform – MALDI-MS format provides sample rates >1 sec/sample
  • Reusable capture surface – MS2 Array's capture surface capable of conducting 384 or 1536 analyses in a single pass than being cleaned and reused
  • Complete bio-compatibility – MS2 Array's capture process is both hydrophobic and liphobic and has been demonstrated to enrich analytes of interest from cell lysates and nuclear extracts

Do these scenarios sound familiar? Contact us today to get started.