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RapidFire® FAQ
What is RapidFire®?
What are the Applications of RapidFire®?
How does RapidFire® work?
What do I do if I don't want to send you my proprietary compound library?
Can RapidFire® quantitate trace compounds in plasma/urine?
What kind of mass spectrometer is compatible with RapidFire®?
How many compounds can RapidFire® measure at once? If there are 2 or 3 substrates and products, can they all be accurately detected?
What kind of sample preparation does RapidFire® provide?
How do we ensure that substrate and product are not removed during RapidFire® sample preparation (where salts, detergents, etc. are removed)?
What is needed for RapidFire® quantitation of substrate and product peaks?
What are the mass ranges of substrates/products than can be detected with RapidFire®?
Are only singly charged species detected by RapidFire, or also doubly, etc.?
Does RapidFire® analyze samples in positive- or negative-ion mode or both?
If a compound has the same mass as a substrate or product, does this interfere with RapidFire® analysis?
If I get a hit with a RapidFire® assay, what next?
Can I use sealed plates with RapidFire® analysis?
Can plates be re-read by RapidFire®?
Why don't you need chromatographic separation of the reaction components?
Do I need to have a background in mass spectrometry to understand RapidFire® data?
Are there financing options available for the RapidFire instrument?
RapidFire® FAQ
RapidFire® is a technology that allows label-free, high-throughput measurement of biologically relevant reactions.
The technology resulted from a need to develop assays for otherwise intractable targets, such as those that are not amenable to screening using traditional methods such as fluorescence or radiolabels.
By utilizing mass spectrometry to quantitate conversion of unlabeled substrate to product, assays are developed that can directly measure attenuation of reactions using libraries of lead compounds.
RapidFire® technology has allowed the development of assays for a wide range of targets that have otherwise proven very difficult to study. These include triglycerides, lipids, fatty acids, prostaglandins, steroids, co-enzyme A derivatives, neurotransmitters, antifungals and antiinfectives.
RapidFire® is a "stand-alone" platform, which is easy to integrate due to the standard plate format and preparation protocols used.
What are the Applications of RapidFire®?
RapidFire® has proven valuable for:
- screening of compound libraries against target molecules for discovery of new drug candidates
BIOCIUS RapidFire® Lead Discovery provides label-free, microliter-scale assay development and screening of otherwise intractable targets for drug discovery research.
Mass spectrometry-based high throughput screening of drug targets averages five seconds per sample. A 96-well plate can be run in around 8 minutes and a 384 well plate in around 35 minutes.
Samples are taken straight from the plate in which the assay was run and there are no laborious sample prep or extra upstream steps needed.
Targets include: triglycerides, lipids, fatty acids, prostaglandins, steroids, co-enzyme A derivatives, neurotransmitters, antifungals and antiinfectives.
RapidFire® has been used to screen for lead compounds in areas including metabolism, obesity, oncology, cardiovascular disease, infective diseases, anti-inflammatories and to study modification of small molecules, peptides and proteins.
Unique to RapidFire® is the ability to study reactions that are not otherwise amenable to high throughput screening and the ability to screen lead compounds with characteristics such as auto-fluorescence.
- measurement of drug-drug interactions such as Cytochrome P450 inhibition
BIOCIUS RapidFire® high throughput mass spectrometry (RFMS) drug-drug interaction assays, using liver microsomes and drug probes, are orders of magnitude faster than LCMS at <10 seconds per sample.
Using liver microsomes and screening drug probes means there is no need for expensive recombinant enzyme preparations or fluorogenic substrates.
Mass spectrometry data quality is obtained at speeds approaching fluorescent assays, such that RFMS provides time-course data in the time that is required for end-point analysis by LCMS.
Pre-developed DDI assays and screening services are available, as well as technology transfer for those clients who prefer to own their own hardware to develop and run assays in-house.
Predeveloped assays are available for the following:
| 3A4/5 | midazolam / testosterone |
| 2D6 | dextromethorphan / bufuralol |
| 2C9 | tolbutamide / diclofenac |
| 2C19 | mephenytoin /omeprazole |
| 1A2 | phenacetin / melatonin / tacrine |
| 2E1 | chlorzoxazone |
| 2B6 | bupropion |
| 2C8 | amodiaquine / taxol |
RapidFire® technology utilizes micro-scale solid phase extraction, coupled to flow injection tandem mass spectrometry (MSMS), to quantitate amounts of specific substrate/s and product/s in a reaction mix.
Assays are developed where substrate to product conversion may be easily measured by optimizing the MSMS conditions for each compound.
In these assays, the amount of substrate and product is greatly in excess of test compound. By employing a proprietary in-line cleanup step, RapidFire® removes salts, detergents and other materials from the reaction mix. All other assay components are eluted onto the mass spectrometer and the amounts of substrate and product quantitated. Test compound is not measured.
The control reaction for conversion of substrate to product is optimized, then attenuation of the reaction is measured upon addition of test compounds.
RapidFire® analyses are performed serially. A computer-controlled fluidic robot takes each sample in turn from a microtiter plate and performs on-line purification. It then presents the samples to a standard mass spectrometer for analysis. Raw peak data is acquired from the mass spectrometer and transferred to proprietary RapidFire® software for analysis and presentation in plate-map format.
The RapidFire® system is compatible with sampling from standard 96 or 384-well microtiter plates and detection using any commercially available triple-quadrupole mass spectrometer.
Serial purification and analysis is performed at an average of 5-8 seconds per sample.
MRM transitions are used for very specific and sensitive quantitation of the substrate and product. The raw data is analyzed by proprietary RapidFire® software and displayed in an easy-to-read plate-reader style format.
What do I do if I don't want to send you my proprietary compound library?
You do not need to send a copy of your library to BIOCIUS in order to use RapidFire® technology. Once a robust assay has been developed we will transfer the assay protocol to you. Your enzymologists perform the assay in a 96 or 384 well plate, quench the reaction, and ship the frozen reaction plates to us on dry ice.
When we receive the assay plates we will thaw and spin them, and then perform RapidFire® analysis. We do not measure test compounds and do not even need to know the plate map.
Can RapidFire® quantitate trace compounds in plasma/urine?
RapidFire® technology utilizes a rapid clean-up step that allows serial measurement of reagents with minimal carryover. To develop a robust assay for screening, we need the reaction to be relatively free of background matrix.
Our clean up can remove the components needed in a standard in vitro enzyme assay. These include salts and buffers (up to 200 mM), detergents (up to 0.05%), proteins (BSA up to 0.02%) and cofactors (generally >1mM). It is not compatible with trace compound quantitation from plasma or urine samples.
What kind of mass spectrometer is compatible with RapidFire®?
Any high-end triple quadrupole mass spectrometer can be interfaced with RapidFire®. Raw time vs. intensity data is obtained for MRM transitions corresponding to the substrate and product and is transferred to proprietary RapidFire® software for analysis.
The mass spectrometer may be used with the RapidFire® system and a standard HPLC interchangeably, by simply connecting and disconnecting the pertinent peek tubing from the ionization source.
How many compounds can RapidFire® measure at once? If there are 2 or 3 substrates and products, can they all be accurately detected?
It is possible to monitor multiple channels per sample and quantitate numerous analytes, although this may require a compromise in sample throughput.
Generally we monitor up to three sets of MRM transitions during an analysis (substrate, product and internal standard) with a per-sample analysis time averaging five seconds.
In order to accurately quantitate analytes, we need to collect sufficient data points from the mass spectrometer for each species. To measure multiple analytes we can slow the flow rate for the purification, which broadens the elution peak for the sample as it is purified. Thus each sample takes slightly longer to analyze for each additional analyte that is quantitated.
We have experience with running robust screening assays whilst obtaining concurrent quantitation of multiple reaction components.
What kind of sample preparation does RapidFire® provide?
Sample preparation for RapidFire® Mass Spectrometry (RFMS) is akin to performing on-line solid phase extraction. There is no chromatographic separation of reaction components. Rather, all the analytes are introduced to the mass spectrometer together and they are individually quantitated using the very selective MRM transitions specific to each species.
The in vitro assays we monitor contain all reaction components required to assess enzyme activity and inhibition. These generally include salts, buffers, cofactors detergents and test compounds, as well as varying amounts of the substrate and product of the reaction.
Many of these reaction components are not compatible with mass spectrometric quantitation and must be removed. To do this the sample is aspirated from the assay plate (96 or 384 well) and is introduced to a clean-up cartridge. The cartridge is washed with buffer to remove unwanted material (salts etc.) and then the sample is eluted from the cartridge using buffers appropriate to the chemistry of the compounds.
Each sample is prepared and introduced to the mass spectrometer in a serial manner.
How do we ensure that substrate and product are not removed during RapidFire® sample preparation (where salts, detergents, etc. are removed)?
As part of our RapidFire® Assay Development Service the scientific team at BIOCIUS will determine the best clean-up and analytical conditions for your substrate and product.
We have access to numerous chemistries for our solid-supports and have years of experience developing assays to optimize sample clean-up.
What is needed for RapidFire® quantitation of substrate and product peaks?
As part of the RapidFire® Assay development Service we ask that you provide us with a small sample of pure substrate and (if available) product. Using these, we are able to determine the best analytical conditions to use.
Briefly, we need to know the MRM transitions to use to detect and quantitate each of the molecules. That is, the mass of each molecule and the mass of a fragment of that molecule that would be generated in the triple-quadrupole mass spectrometer.
Generally our clients are able to tell us what the substrate and product molecules are so we determine MRM transitions either from this information, or empirically, using the samples provided to us.
Once we know which MRM transitions to track, we monitor these same ones for each well as a measure of the amounts of substrate and product (and often internal standard) present. We never measure the test compounds in the wells.
What are the mass ranges of substrates/products than can be detected using RapidFire®?
We routinely assay over a wide range of masses including small molecule peptides up to about 40 amino acids in length. The mass range is determined by the mass spectrometer that is used for analyte quantitation. Most commercially available triple quadrupole mass spectrometers have an upper limit of about 3000 amu. Since these instruments can measure multiply charged species, we can look at larger molecules. Using RapidFire with a Time-of-Flight (TOF) instrument enables us to detect proteins of 150kb and larger.
Please contact us to discuss your assay needs as we have extensive experience quantitating various molecules.
Are only singly charged species detected by RapidFire, or also doubly, etc.?
We interface RapidFire® with commercially available triple quadrupole mass spectrometers. Thus, we can measure multiply charged molecules.
Does RapidFire® analyze samples in positive- or negative-ion mode or both?
We interface RapidFire® with commercially available triple quadrupole mass spectrometers. Thus, we are able to measure in either positive or negative ion mode. In order to maintain very high sample throughputs, we avoid switching between modes for a single analysis.
If a compound has the same mass as a substrate or product, does this interfere with RapidFire® analysis?
We use the highly selective MRM mode of triple quadrupole mass spectrometry to perform analyte quantitation. Thus, in order for a compound to interfere with RapidFire® quantitation it has to have the same mass as one of the analytes and also produce the same fragment upon dissociation within the mass spec. Whilst this is theoretically possible, in practice it happens extremely rarely.
If I get a hit with a RapidFire® assay, what next?
BIOCIUS is equipped to run primary screens and also provides services for follow up analysis. Once we have transferred a robust screening assay to you, you can prepare plates in whatever format you wish, including follow-up IC50 determinations and structure-activity relationship (SAR) analysis, mechanism of action and Ki determinations.
Just prepare the plates as the experiments are completed, ship them to us for analysis and you will receive your data electronically. We generally set up a monthly billing schedule for our screening services.
Can I use sealed plates with RapidFire® analysis?
Yes. Heat-sealed plates work well with RapidFire®. We do not recommend adhesive seals as the glue tends to be problematic for the plate-piercing device.
Can plates be re-read by RapidFire®?
Yes. Provided there is enough reaction volume remaining, there is no problem to go back to a well and perform additional analyses.
Why don't you need chromatographic separation of the reaction components?
We allow the selectivity of the tandem mass spectrometer to identify and specifically quantitate the compounds of interest.
The types of in vitro enzyme assays we are running comprise of substrate and product in vast excess of test compound and other assay components. We don't need extensive chromatography to remove matrix, so we just remove assay components that are incompatible with mass spectrometry, such as salts and detergents in the buffers.
The specific MRM transitions that we set up for the analysis ensure quantitation of the desired compounds only.
Do I need to have a background in mass spectrometry to understand RapidFire® data?
No. Raw data from the mass spectrometer is transferred to proprietary RapidFire® software for analysis. Final presentation of the data can be provided in plate-map format or as a simple text file for import into your LIMS system.
Are there financing options available for the RapidFire instrument?
Yes, BIOCIUS offers a variety of leasing programs for purchasing or upgrading your RapidFire instrument. Ask your sales representative or call or email us for details.
BIOCIUS. Faster Answers.
RapidFire® technology is described in patent numbers 6,932,939, 6,812,030 7,100,460, 7,588,725,