For LC-MS quantitation, you require a standard, since every substance has its own sensitivity of detection by LC-MS. You need a standard consisting of the same substance you wish to quantify. You use it to prepare a standard curve using a set of solutions of known concentrations.

Hello!

Peptides quantification is a whole subject, hard to summarize in 2 lines.

You will have pre-analytical issues because of a lack of stability, adsorption, matrix effect, problems with the LOQ...

I recommend to read a lot about your peptide and its physical, chemical and biological properties. Then, try to understand how it behaves when you work with it (adsorption, degradation, solubilization...), and understand what is the effects caused by the matrix you are using (plasma, urine, serum, etc.).

Finaly, you can try to extract your peptide by precipitation, solid-phase extraction, etc. depending on what you have read in the literature.

After that, you will indeed need the standard peptide to prepare the calibration curve, and you will need the internal standard (IS) to correct the areas. Indeed, in LC/MS, we want a ratio between the peptide and its IS to correct the fluctuations caused by the extraction and the instrument.

Maybe, if you ask more precise questions, I can be more specific.

I suggest:

Article Strategies to reduce aspecific adsorption of peptides and pr...

Article Recommendations for the Generation, Quantification, Storage,...

And for a practical example for the method validation in a clinical context: Article Proneuropeptide Y and neuropeptide Y metabolites in healthy ...

Jonathan Maurer Hi Jonathan - thank you very much! This is rather helpful. We are in our early stages, but we're looking to quantify three peptides (all around 1200 MW) in cosmetics.

You need to purchase a synthetic version of the intact for absolute quantification of the peptides. You may use spiked-in peptides labeled or analog for compensating the analytical bias.

Hi Tijana Vasiljevic,

I have never worked on cosmetics, but you will definitely have troubles with the matrix. You will have to optimize the extraction to get rid of the interferences caused by the other molecules in your sample. But first, you have to create an LC/MS method that allows to measure your peptides in a clean solution. Then you have to work on peptide stability, and finally you can improve the extraction. In summary:

1) Create a LC method to eluate your compounds (increase % organic in your flow, use various column…)

2) Found at least two MS transitions for each peptide and its corresponding internal standard by using MS in SCAN mode

3) Be sure that your peptides are properly solubilized in your solution, and change the solvents/pH to increase the solubilization

4) Check that your peptides are stable over time (at least few hours in the LC sample manager, and check the adsorption by serially transfer the peptides in multiple vials)

5) When you have a method that works to measure your peptides in a clean solution, then you can work on the extraction (watch out to not clog your instrument with your cosmetics!) à change the SPE type, volume of sample, washing methodology, elution solvent, reconstitution volume and solvent, etc.

I hope this will help :)

Jonathan Maurer Thank you - this is very helpful.

Dear Tijana Vasiljevic ,

It's been a while!

I don't know if you are still working on peptides, but we have recently published an article that summarizes many pre-analytical challenges in peptides analysis. It might be of interest for you or your team.

Article Tutorial review for peptide assays: An ounce of pre-analytic...

Best wishes,

Jonathan

Jonathan Maurer Thank you Jonathan! Peptides are still very much a thing, I'll take a look at your publication.