Neha Agrawal How about Lowry method, BCA method, or Bradford method?

Michitoshi Watanabe would this methods work properly for peptides?

Hi Neha,

in R. K. Scopes’ book “Protein Purification. Principles and Practice” there is a nice description how to estimate peptide concentrations in aqueous solutions, using the UV-absorption of the peptide bond as a chromophore. You need to dilute and dialyze your sample against a low-absorbing buffer (5 mM potassium phosphate pH 7.5, 50 mM potassium sulphate) and measure the absorbance at 205 nm in a quartz cuvette. The corresponding absorption coefficient can be calculated from the coefficient at 280 nm (and that one you can easily calculate from the sequence). You can easily look up the procedure in the book or in the Supporting Information of a study I link you here (https://onlinelibrary.wiley.com/doi/full/10.1002/bip.23069) in which I applied this technique myself.

All the best,

Joscha

Stjepan Krešimir Kračun

Joscha Breibeck I will surely check that book. thank you.

Hi Neha,

one more thing that I forgot to mention: the proposed absorbance measurement takes place in a low-concentrated phosphate buffer because a lot of organic and inorganic molecules strongly absorb at 205 nm in higher concentrations. In your case with phosphopeptides, a considerable absorbance contribution by the phospho-groups can be expected, and you could easily overestimate the peptide concentration. I recommend to compare with a sample of known concentration to account for this.

Good luck!

Interesting question

Following

Joscha Breibeck thank you for your advice.

Some Vendors sell assays specifically for peptide quantification (e.g. Thermo/Pierce #23275). I have used this for digests, but with phospho-peptides you may be pushing the detection limit. It should still give you a better chance than a regular BCA.

Great discussion. I have been using nanodrop, qubit and sds page for protein but did not think about determining peptide concentration. Thank you for a nice productive discussion.

Kou Hayakawa

Joscha Breibeck can you please share the soft copy of a book that you have suggested?

Hello Neha,

I do not have the book here, but I can provide you the SI document of a former study of mine where I applied the UV-Vis measurement. You can find the relevant section on page 6. My final dilution for the measurement resulted in about 10 µg/mL peptide samples. Hope this helps!

Best regards,

Joscha

Joscha Breibeck thank you so much for sharing this.

1. If Phenylalanine is present, use standard A254 absorbance.

2. If tryptophan is there use standard A280 absorbance.

3. For nonaromatic peptide: Waddell, W. J. (1956) A simple UV spectrophotometric method for the determination of protein. The Journal of Laboratory and Clinical Medicine,48, 311–314.

Hi Prakash Kishore Hazam

I am looking for the method which can help me to find out to peptide concentration of digested proteins from whole cell lysate.

Please go through the paper, you will get everything