GreenD
National Hazard
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Registered: 30-3-2011
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LC-MS Experience needed.
Hey
I am trying to find a molecule around 250MW in a protein extraction buffer. I just want to see if the compound is present using LC-MS.
Without going into extreme detail I have 1 question to anyone seasoned in LC-MS use.
Will NOT precipitating my protein cause very bad sensitivity for the instrument? Is precipitating proteins (huge macro molecules) from solution
necessary for the machine to pick up a small molecule in the mix?
Its an Agilent machine, relatively new, past 3 years or so.
I'm not doing the expt. yet, but soon to pick it up, but the current worker is having trouble:
Putting a sample in, we see nearly no signal, barely above the signal-to-noise threshold of our compound at any given time.
If we, however, extract the compound from the sample using chloroform or some other solvent, we see this HUGE peak, massive peak.
Again, I'm not running the expt. but it is my hunch that it is absolutely necessary to ppt. proteins to gain any decent sensitivity of the instrument
- without "overloading" it - but is that even an issue with LC/MS, or are they very reproducible even with high noise?
Note: These are fractions we are testing - fractionated from HPLC using various columns, so the fractions that get input into the LC/MS are fairly
dilute, but not anything that would generally be troublesome to detect on LC/MS.
ʃ Ψ*Ψ
Keepin' it real.
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phlogiston
International Hazard
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Generally, the reason to do a protein precipitation step is to prevent the proteins from precipitating inside your column, which would clog it and act
as a sort of unintended active surface yielding unreliable and poor quality chromatograms.
The chlororm extraction likely means that you get rid of -something- that interferes with the detection of the compound, which doesn't necessarily
have to be protein. You also remove polar metabolites and salts in that way, which could give ion suppression.
If you do a protein precipitation step that will leave metabolites/salts present (acetonitrile precipitation for instance), do you also get a large
peak?
The extraction with chloroform implies that the compound you are after is apolar. Often, such compounds stick to plastic quite persistently if you use
aquaeous buffers, sometimes to the extent that you lose them nearly completely. It is best then to use glass sample tubes and pasteur pipettes etc,
rather than plastic eppendorf tubes and plastic tips etc, and to keep the compound in more apolar solvents as much as possible (methanol, chloroform,
ethylacetate, ether etc).
As with any analytical method, if your signal is only barely above the noise, you will get only noisy data.
If, on the other hand, you overload the MS, expect to see very nonlinear calibration curves which does not necessarily need to be a huge problem but
is generally undesirable. If you are using an internal standard that coelutes with the peak of interest, the calibration curve may often still be
quite linear. Are you using tandem MS? I often circumvent saturating the detector in those cases where simple dilution is not an option by using
off-optimal settings for the MS parameters. For instance, I might use a lower collision energy for a collision cell, or change the cone voltage for an
electrospray ionization source.
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"If a rocket goes up, who cares where it comes down, that's not my concern said Wernher von Braun" - Tom Lehrer |
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GreenD
National Hazard
Posts: 623
Registered: 30-3-2011
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Mood: Not really high anymore
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"Generally, the reason to do a protein precipitation step is to prevent the proteins from precipitating inside your column, which would clog it and
act as a sort of unintended active surface yielding unreliable and poor quality chromatograms."
I believe our column is fine for proteins, but not sure, and we are using a protein-extraction buffer as solvent both in HPLC and LC/MS.
"The chlororm extraction likely means that you get rid of -something- that interferes with the detection of the compound, which doesn't necessarily
have to be protein. You also remove polar metabolites and salts in that way, which could give ion suppression."
Ok. Ion suppression... ok.
"If you do a protein precipitation step that will leave metabolites/salts present (acetonitrile precipitation for instance), do you also get a large
peak?"
•I don't know the prof doesn't think its necessary, but I'd like to try it. Our HPLC just broke though, so can't try anytime soon. Plus I'm a bit
intimidated at the moment to say "Hey lets try this" as I think he'd get defensive.
"The extraction with chloroform implies that the compound you are after is apolar. Often, such compounds stick to plastic quite persistently if you
use aquaeous buffers, sometimes to the extent that you lose them nearly completely. It is best then to use glass sample tubes and pasteur pipettes
etc, rather than plastic eppendorf tubes and plastic tips etc, and to keep the compound in more apolar solvents as much as possible (methanol,
chloroform, ethylacetate, ether etc)."
Well, we can't - we have to use protein buffers for the compound, however you are right - we should be using glass. I wish they had such things as 96
well plates that were glass... But test tubes would suffice."
"As with any analytical method, if your signal is only barely above the noise, you will get only noisy data.
If, on the other hand, you overload the MS, expect to see very nonlinear calibration curves which does not necessarily need to be a huge problem but
is generally undesirable. If you are using an internal standard that coelutes with the peak of interest, the calibration curve may often still be
quite linear. Are you using tandem MS? I often circumvent saturating the detector in those cases where simple dilution is not an option by using
off-optimal settings for the MS parameters. For instance, I might use a lower collision energy for a collision cell, or change the cone voltage for an
electrospray ionization source."
Not tandem MS, just lc/ms. Ok. I don't think saturation will be a problem, but thanks for the information.
ʃ Ψ*Ψ
Keepin' it real.
Check out my new collaborated site: MNMLimpact.com
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