We did protein identification by in-solution digestion coupled with LC-MS/MS analysis. The identified proteins having the corresponding protein areas as its abundance. We understand that the protein area (numerical values) were calculated based on desired peptide spectral matches for specific proteins. But we didn't find any units for protein area. Kindly if someone having more updates in mass spectrometry is being requested for clarification to find out the unit for protein area?
Dear Somashekar,
The unit of protein area is dimensionless and has no unit.
Since you performed a protein digest, you identified peptides by MS/MS and quantified them by MS. Depending upon the software you have used to perform the analysis, it will most likely have resulted in the appropriate peptide intensities (or areas) being added together (and then normalised perhaps) to produce an intensity value for the parent protein.
In addition, the area curve (at the peptide level) is generated by measuring discrete intensities at time points to produce points which are then joined by a curve-fitting software.
I hope this information is of use to you.
Kind regards,
Peter
Dear Somashekar,
The Area under the curve (AUC) has no units. Usually, in MS analysis, one can inject standards with 5-6 different concentrations and then plot conc vs. AUC 2D plot. Using this plot you can calculate the exact concentration of your analyte in an unknown sample.
Regards
Charan
Dear Somashekar,
If you are referring to eg., summed intensities of all peptides that have identified a protein, than the unit for the protein will be relative intensity. This is the summed intensities of all the peptides (MS1) that have been matched to amino acid sequences of a peptides, post the LC-MS/MS experiment.
This kind of analysis is done by publicly available softwares such as MaxQuant.
http://www.coxdocs.org/doku.php?id=maxquant:start
MaxQuant will identify each MS/MS spectrum, list the intensity of MS1 and in the results tables, will give you the total intensity of all the matched peptides that identify a particular protein. If you do not have MaxQuant software, post database search eg., Mascot, you will have to go to your raw mass spec file and manually integrate AUC for each of the peptides that are listed to identify a particular protein and then, add all the relative intensities of these peptides to arrive at the protein intensity.
Best,
Hediye
Dear Peter,
Thank you very much for your informative explanation on generation of MS protein area through area under the curve. It adds more confidence to our research findings.
Best regards
Somashekar
Dear Charan,
Thank you very much and we will take up your suggestions for our future experiments.
Best regards
Somashekar
Dear Hediye,
Thank you very much for your response,
We understand that the protein areas or intensities are generated from area under the curve (AUC) from the peptide spectral matches (PSM) as also explained by Peter and Charan and there is no standard unit.
In our analysis, we used Proteome discover or Scaffold software to obtain the protein identities. The species of interest taxonomy database was downloaded from NCBI or ENSEMBL genome browser. So far we have not used MaxQuant software. After referring to your explanation, we are interested to use MaxQuant software for our MS results.
Best regards
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