I could hardly see any expected ions such as [M+nH]n+, [M+nNa]n+, [M+nK]n+, [M+NH4]+

If there is, just a very low intensity of [M+H]+ (1143) at positive mode and [M-H]- (1141)

The reason why I use acetonitrile is that this peptide is highly hydrophobic. When I make stock solution, I can not dissolve it in methanol or water so that I don't use methanol or water to make working solution and the MS infusion sample.

But as you say these two solvents could increase the ionization efficiency, I will try to use them in the sample to see the different.

In case the solvents are not the solution, do you think of any compound that could increase the ionization?

Try DMSO additive as supercharging reagent for both solubility improvement and mobile phase additive...This will increase the ionization efficiency in ESI-MS...

İsmail Emir Akyildiz "Cys, Met or Trp-containing peptides are susceptible to oxidation. Therefore, it is recommended to dissolve them in oxygen-free buffers. Moreover, DMSO should be avoided when handling oxidation-sensitive peptides"

My CCK8 peptide contains 2 Met in its structure. Could you recommend another supercharging reagent for my compound?

As I know, supercharging reagent could make the compound being protonated at more site leading to smaller m/z. Could it also increase the sensitivity?

Noel W Davies I have made 1000 ng/ml solution in pure water. It still doesn't work.

Even the MW is only 1142.35 Da (small peptide), could we use supercharging reagent to increase the sensitivity or the ionization efficiency of the compound in MS?

Dear Chau Phuong Bui

It seems that your peptide does not have any chemical moiety which can be positively charged. Thus, you cannot see it in ES +. Na and K adducts will not improve the situation (they cannot change the charge, they can only substitute a proton). MS will not "see" not charged molecules. Using ES - mode should work perfectly in this case. You should see up to three species here.

Regards

Here are the other points, you may lean on;

If the purpose is quantification or purity check, LC-UV would be nice to use since the octapeptide you have several aromatic rings and would be highly responsive,

secondly, 500 ppb may be a low conc. to conduct a full scan..especially when the ionization efficiency is low.

Third, I would prefer combined flow scanning in place of infusion...In this mode, you are not taking benefits of the mobile phases which present donors to improve ionization...You should combine the lc flow and infusion (acid and/or DMSO additives in phases for pos ESI in this case) and retest the response...

By solving the peptide in an alkaline condition you are directing the peptide to deprotonation and this makes the peptide more amenable to neg ESI...If it is soluble in ACN directly..prepare your stock in ACN and dilute it with the same solvent, I prefer not to use aggressive pH which is not convenient for most of the peptides to the unintended H exchanges...

Last but not least, If the peptide is hydrophobic and dissolves only in organic solvents this is susceptible to be efficiently ionized in APCI, APPI rather than ESI...You may look for these alternative ionization techniques if MS analysis is the bottleneck and the abovementioned suggestions are useless...

Good Luck...

Consider running it in negative mode, as the presence of RSO4H allows for easy deprotonation of the proton. Additionally, you can also run it in direct injecton.

Itay Pitussi

Tomasz Fraczyk

İsmail Emir Akyildiz

Noel W Davies

I have tried changing the solvent from acetonitrile to aqueous NH4OH for dilution, with the addition of 0.1% formic acid or 2mM ammonium acetate. I run both positive and negative mode but only detect a very low intensity of the unsulfated form CCK8 (my standard is the sulfated CCK8 from Sigma).

I don't understand why I could not see my sulfated form of standard but the unsulfated form instead. This is the same when I used acetonitrile as solvent for dilution.

Chau Phuong Bui

Is CCK8 detected in negative or positive mode?

Have you experimented with different voltage settings?

Itay Pitussi the unsulfated CCK8 could be detected in both neg and pos mode but the intensity is very low. But my standard I injected is sulfated form of CCK8 , not the unsulfated CCK8, and I could not detect anything.

I have try from 20-100 Voltage.

Chau Phuong Bui

Have you typically experimented with adjusting the capillary voltage within the range of 0-3 kV?