Dear ResearchGate Community,
I am currently facing a challenge in the LC-MS analysis of a highly hydrophobic peptide (already purified to >98% by a manufacturer) on our 1260 LC-MS System.
The experimental setup involved running a gradient of 5-80% ACN over 30 minutes on an analytical column (C18, 95Å, 4.6 x 50 mm, 5 µm). I injected 1, 2, 5, and 50 µl from a sample with a concentration of approximately 0.5 µg/µl (prepared in either 100% Methanol or 80% ACN+20%H2O+0.1%FA).
However, I observed multiple peaks at retention times between 21-24 minutes with my expected m/z in the Total Ion Current (TIC) MS. Interestingly, there were no UV peaks at either 220 or 280 nm (see pic).
Has anyone else observed such behavior (i.e., broad peaks in MS with no UV signal) with any highly hydrophobic peptide or compound?
I would greatly appreciate any suggestions or tips to resolve this issue and achieve a single peak in both TIC and UV (220nm) for this highly hydrophobic peptide.
P.S.: I can observe a single peak in both MS (TIC) and UV (220nm) with my water-soluble hydrophilic peptide, indicating that the LC-MS System is functioning properly with the chosen mobile phases (A: 0.1% TFA/water, B: 0.1% TFA).
Thank you in advance for your assistance.
Hi,
You could consider spraying the peptide solution directly into your mass spec, so without the LC. This requires a solution that is fully volatile, of course. It seems that the mess eluting at the end of your gradient are contaminants from the sample.
What is the sequence/amino acid composition of your peptide: does it contain protonatable residues, such as lysine, arginine or histidine (is the N-terminus free, or capped)? And do you expect the peptide to absorb UV light, given the sequence?
Dear Eef,
Thank you very much for your input.
It's a pentapeptide (Carbobenzoxy-Leu-Gly-Leu-Ala-Leu-CHO) in which the C-terminal carboxy group has been reduced to the corresponding aldehyde, and the N-terminal amino group is protected as its benzyloxycarbonyl (Cbz/Z) derivative. This is a purely hydrophobic peptide that is only soluble in organic solvents.
This peptide has no protonatable residues (e.g., Lys, Arg, or His). Yes, I expect the peptide to absorb UV light and produce the expected m/z at its retention time.
Do you have any other suggestions on how to address the issue and obtain the correct MS spectra/UV chromatogram?
Increase the absorption wavelength (benzyl derivate),
Increase the initial gradient organic composition to efficiently transfer the substance,
Increase the total flow and inject less,
Use the C8 or C4 column to increase the elution efficiency,
Do not use TFA to omit ion suppression use FA instead,
Inject reagent blank/system blank to blank subtraction and pure signal of your target,
Try to get a sufficient signal at UV before the reliable result at MS analysis,
Consider the aldehyde moiety is highly reactive and take into account non-specific interaction conjugation derived loss during or prior to analysis,
Infusion mode analysis is a good option for divide-and-conquer troubleshooting as suggested by Eef Dirksen...
Good luck,
Emir
Increase the acetonitril concentration to at least 50% and run the gradient to100%. At 5% acetonitril you risk that your peptide precipitates and possibly does not dissolve quickly at higher concentrations of organic solvent.
You should definitely see a peak at UV 280 nm.
Let us assume that your polypeptide sample is pure, and therefore it can be said here that the sample was not dissolved sufficiently, and this is due to the presence of hydrogen bonds that were not broken for sufficient absorption to occur. In addition, I doubt that the sample medium is acidic, as free bonds of nitrogen have been occupied, and thus absorption does not occur. In this case, you have to enlarge the wavelength.
I would suggest an initial UV scan to figure out whether zhe peptide absorbs UV light, however I don’t believe that you see an obvious peak due to the missing aromatich aas. If it is almost pure, why do you need to load it on column? I think you should consider the infusion of the peptide directly to ms
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