Hi Everyone:
I want to quantify the relative amount of a protein in 2 samples. I will first digest the sample with trypsin, and subject the resulting peptides to LC-MS/MS analysis. I will then identify the different peptide ions belonging to the protein, and quantify the areas under the different isotopic peaks of the peptide ions, in some sort of software. Lastly, I will determine the protein abundance by summing the different peptide ions belonging to the protein, and the relative amount of the protein in these 2 samples would be the ratio of the protein abundance in sample 1 over sample 2.
Does this sound like a good general procedure for quantifying the relative amount of a protein in 2 samples using MS? Are there any caveats in this approach?
Any answers would be appreciated!!
You might want to consider the isobaric tagging method (iTRAQ).
https://en.wikipedia.org/wiki/Isobaric_tag_for_relative_and_absolute_quantitation#:~:text=Isobaric%20tags%20for%20relative%20and,sources%20in%20a%20single%20experiment.
Based on your description, you may want to quantify one protein from two samples.
If you know the sequence of the protein, you can use MRM or PRM techniques to quantify the protein using a label-free method, as this method is very inexpensive.
What are MRM and PRM?
https://help.biognosys.com/help/what-are-mrm-and-prm
Mr. Zhang,
The method of mass spectrometrey provides exact (or absolute) quantitative and 3D structural molecular information about analytes, despite, their molecular weights and types. However, an absolute quantification in chemometric (or analytical quantitative chemical) terms can be carried out, currently, only within the framework our (mine and my co-author's according to the shown authorship, below) innovative stochastic dynamic theory and model formulas, which account precisely, accurately, selectively and sensitively for the fluctuations of the experimental measurable variables; for instance, the mass spectrometric 'intensity'-values.
Our method, has been tested, so far, on analytical pg.(mL)-1 and ng.(L)-1 concentrations of analytes in mixtures, showing coefficients of linear correlations between theory and experiment /r/ = 0.99997-1.
Please, consider, references [1-3].
The chemometrics of six replicates shows reproducibility /r/ = 1 or 100 %. (Consider detail on reference [3].)
[1] Steroids, 164 (2020) 108750
Stochastic dynamic mass spectrometric quantification of steroids in mixture — Part II; Bojidarka Ivanova, Michael Spiteller
[2] B.Ivanova, M. Spiteller, Stochastic dynamic mass spectrometric approach to quantify reserpine in solution, (2020) in press.
[3] Bioorganic Chemistry, 93 (2019) 103308
A mass spectrometric stochastic dynamic diffusion approach to selective quantitative and 3D structural analyses of native cyclodextrins by electrospray ionization and atmospheric pressure chemical ionization methods
Bojidarka Ivanova, Michael Spiteller
Since, we have a considerable experience in the analytical chemistry of peptides, please consider as well as reference [4], which not only provides information about the applicability of our new theory to study peptides, but also sheds light on the chemical processes of peptides with respect to experimental conditions, including data on among the most important phenomena of inter- and intramolecular proton transfer effects of peptides. These latter processes, as you know, affect crucially on the chemical reactivity and 3D conformations of the corresponding macromolecular peptide derivatives.
[4] B. Ivanova, M. Spiteller; EXPERIMENTAL MASS SPECTROMETRIC AND THEORETICAL TREATMENT OF THE EFFECT OF PROTONATION ON THE 3D MOLECULAR AND ELECTRONIC STRUCTURES OF LOW MOLECULAR WEIGHT ORGANICS AND METAL–ORGANICS OF SILVER(I) ION; pp. 1-182; in Book ''Protonation: Properties, Applications and Effect,'' A. Germogen (Ed.) NOVA Science Publishers, New York, (2019); ISBN: 978-1-5361-4886-2.
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