Compound detectability

Discussions about GC-MS, LC-MS, LC-FTIR, and other "coupled" analytical techniques.

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I’ve gleaned some useful tips from Chromforum in the past but this is my first post. I hope someone can help. I’ve got significant experience with LC-MS for small molecule stuff but am currently encountering a frustrating problem with detection of a large(ish) peptide(ish) species.

I’m using a Thermo LTQ XL for this work, coupled to an Accela LC, but I also have a Thermo TSQ available which I’ve tried out with essentially the same result. Not doing chromatography at this stage; either injecting from autosampler without column or direct infusion from syringe pump.

A post-doc is building a molecule which I can’t describe in detail because of IP issues but broadly it is a Si-based cage anchor with 8 arms/appendages each of 10 residues terminating in an amide, each of mass about 1000; the overall mass of the molecule is about 10kDa. The synthesis results in mostly 8 arms being attached but apparently with some with a smaller number of arms attached with decreasing number as the number of arms reduces. We’ve had this confirmed by MALDI-TOF but we don’t have this onsite and samples have been going out to an external commercial lab, which clearly has cost implications, hence the desire to be able to pick this up in the LTQ.

The solubility is not great and my solution is about 1.2mg/ml ie roughly 0.1uM. At this concentration, it’s invisible in the LTQ; the mass limit of the MS is 4000m/z, so I’m relying on 3+ charged ions being present. I’ve tried a range of MS conditions and gone through the compound optimisation built into the LTQ software aiming at the 3x charged ion. I’ve tried in water and with 0.1% formic acid and even played with DMSO and m-NBA in the mix to manipulate the charging behaviour. Wondering if there was a problem with the MS, I’ve run lysozyme in the same conditions at the same mass concentration (lower molar concentration) and see a nice clear multiply-charged envelope, quite sufficient to calculate the molecular mass. I can also see quite clearly the appendage peptide or the cage molecule run individually at the same molar concentration.

Can anyone tell me a) if there’s some obvious reason I don’t know about that explains why this is invisible or b) if there is something I should be trying to make it ionise?

Hopefully getting a delivery of a Q-TOF in a few weeks, which will be able to tell me if the singly-charged ion is visible, but I’d rather see something before that plus it’s aggravating not understanding what’s going on.
Let me know if there’s anything else I can share (short of details of the molecule) which would be helpful.
Would SEC solve the problem without having to worry about the finicky process of finding a way (with your locally available items) to float your lead balloon of a molecule into a MSD?
Would SEC solve the problem without having to worry about the finicky process of finding a way (with your locally available items) to float your lead balloon of a molecule into a MSD?

It's an interesting idea. I've previously done GPC SEC on big polymers but nothing on smaller molecules in aqueous environment. A bit of googling has failed to reveal whether it would be reasonable to expect an aqueous GPC column to resolve 1000Da differences at 10KDa, so advice welcome on that. A possible issue is that we don't know how the molecule is conforming and it may be that, due to the geometry of the Si cage, the hydrodynamic radius is only weakly dependent on the number of arms; it's distinctly 2-sided and each side may be sticking out in a group.

Still, might be worth a try if we can get a column at a reasonable price, or even better on loan.

Anyone care to advance a theory on why it's not ionising?
Hi Matt

an interesting observation, but frustrating for you having to get the instrument to deliver results.
The Thermo electrospray source often requires time to optimise parameters for ionisation, but with infusion, gas temperature, gas flows and voltage can be individually adjusted to improve response for specific analytes.
Two other factors... standard source with fused silica needle ? / or HESI with a steel needle ?

yes, I went though the ESI optimisation, adjusting the gases, voltage etc with no improvement.

I was using a standard ESI interface with fused silica capillary but also tried briefly on my triple quad with the H-ESI - same result.

I have since tried the same compound on my brand new Shimadzu 9030 QTOF and still detect none of the expected ions, despite its ~1000x better sensitivity. I haven't yet got enough experience to change the source settings on this instrument but I'll quiz the apps guy when we have our training.

It is strange.

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