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BAMS is just form of affinity purification (that's where the affirmers fit in) with MS detection. The last (MS) bit can be on different platforms but I think they are referring to using maldi ToF . The maldi bit, is how you get non volatile peptides to ionise and the MS is what you are using as a detector. If you do it this way then you do not need to separate the peptides first by LC which makes it a lot faster. (ToF = time of flight - a type of Mass Spec)
The problem with MS is the effect of the matrix - no not the film... but the other 'stuff' in the sample which can enhance or supress a signal. There are also several steps in the sample prep , it will need deglycosylating (unless the specific peptides being detected have no glysoylation sites) and digesting with trypsin to give pieces small enough for MS.
They will be working on which peptides are te best to use and optimising the other steps to get the best results.
IS or SIS peptide for the intended target is spiked into the protein digest at a known concentration. Affi-BAMS beads are used to enrich for the corresponding target peptides. Beads are washed and then assembled into an ordered array onto a MALDI slide. The affinity-captured peptide targets are eluted from the beads within each micro-well and deposited onto the microarray slide for MALDI MS acquisition. The MALDI MS spectrum is matched to a reference spectrum for identification of the target protein or protein modification. The MS intensity of the target is then used to determine relative quantitation based on the reference peptide (IS or SIS) or the heavy and light SILAC pairs.
So I guess as part of the BAMS package they sell to accompany each piece of kit (that was the plan I seem to recall), they will need to have elucidated and supply reference spectra.
Ophidian
Clip clop clip clop... in trots his mounted lordship, gives his ‘I don’t know’ answer, trots off again... clip clop clip clop
Can BAMS microwave beans?
Actually Monkhood - just found your answer in a different paper - IS is labelled peptides.
Ophidian
My understanding from our website is that the BAMS machines are used for specific testing and require "converting" for virus usage. This is what Adeptrix are working on. Hope this makes sense.
what use are you !!!! lol
Monkshood - I have no idea on the details - I just found and read a poster by Jeff Silva that gives a good introductory overview.
Ophidian
I went out with a Maldives Tof first awhile. Strange girl. Used to laugh like a donkey falling down a well
@Richob. Do hospitals have lots of spare capacity on their MS's? This is a genuine question .the triple quads used in most hospitals are working 24/7 from what I have heard but no idea about maldi tof's if that is what is proposed.
@Ophidian, what do they use as an standard (internal) to allow for extraction losses and ion suppression?
There is no argument about specificity. Only those who don't understand SENSITIVITY were embarrassing themselves - well up to now I suppose.
Ophidian
There are only half a million of the BAMS type machines around !
Ophidian's back, cue an argument about specificity ;)
@Monkshood - BAMS is quantitative and eliminates the need for an LC separation.
Ophidian
Genuinely top post. What a nice lady / fellah.
That’s good to hear, thanks. I think the BAMS news will arrive before any LFD update in line with the list of substantial newsflow. From Sir Al’s comments they’re in every hospital and there should be plenty capacity available to make use of them. Due to their throughout we could end up capturing a fair chunk of that market before the LFD gets its approval.
I'm smiling - please put yourself on standby. If we come out of this in one piece I owe you a pint (or two). Cheers
I think that they said that this ( BAMS method with Adeptrix) was intended mainly for hospitals - not sure how many have the 'toys ' or capacity on them to do this but it would be fairly quick and cheap if they do have. I think that the test strips will be the preferred option but there should be almost no problems getting the MS method to work and so is a more certain option.
Just be careful RD or you’ll end up with crude acetone precipitation everywhere
In the meantime I'm going time have a go at the upfront, pulldown method.
See you in a bit
Likewise, appreciate your input. Didn’t understand a technical word as this stuff isn’t my bag, but my understanding would be that we only care about a +/- result, rather than a reading of Covid infection levels.
Monkshood (I'm not even going to ask what that means :-)). Thanks for taking the trouble to talk me through it. Listening to the CEO and looking at their presentations I think the plan is to utilise any mass spec available - its a generic solution. Given a) you sound like you know a bit about it b) I have no idea do you think they are onto something that will be taken up - from there might be possible to do the commercial numbers. At moment PCR seems to be the alternative. As I say thanks for the info.
Sorry! it has been shown that you can detect specific sar cov 2 peptides by LC MS in crude acetone precipitated saliva preparations. Essentially the affirms will be used to pull down the virons which will give a faster, cleaner and more concentrated sample than a crude acetone precipitation.
There is no reason why this would not work. There are several options for how this is then analysed (for novocyte lovers you could even use pcr!) but I believe the plan is to use a type of MS (Maldi) although other types are possible. Depending on whether they want just a +/- result or a more quantitative result will determine the type of controls used in the analysis.
The best is to add peptides containing heavy isotopes and look at the ratio of these to the unlabelled ones in te samples.
You lost me at "as you can see..."
As you can see marker peptides for sars cov2 in crude ppts from saliva by tryptic digest and LCMS/MS there is no reason to expect that an upfront pull down method would cause any problems and should be quite straight forward. I believe that they were proposing using Maldi but they could use LC-MS/MS if they needed more sensitivity although throughput may be slower .
Not sure if they are just going for a +/- or will use labelled peptides as an IS to give a quantitative output, anyone know?