Mass spectrometry analysis for relative and absolute quantification of proteins

An introduction to the basic principles for quantitative mass-spectrometry analysis of proteins. Learn more about this class slavovlab.net/teaching/index....
This introduction uses figures from the following review articles:
Benjamin F. Cravatt, Gabriel M. Simon & John R. Yates III
The biological impact of
mass-spectrometry-based proteomics
www.nature.com/articles/natur...
Ruedi Aebersold & Matthias Mann
Mass-spectrometric exploration of
proteome structure and function
www.nature.com/articles/natur...

[@gitanjaliroy6235]

Hello Dr. Slavlov,
In the labeling section you mentioned that TMT labels are incorporated at the MS2 level.
The TMT labeling is done during sample preparation stage. SO what is meant by incorporating them at the MS2 level?

[@NikolaiSlavovResearch]

When labeled peptides are fragmented, they generate reporter ions, which are quantified at the MS2 level. Sometimes, fragments are further isolated and fragmented for MS3-level quantification.

[@Ceren_A498]

what is the difference between relative protein quantification and absolute protein quantification?

[@NikolaiSlavovResearch]

Absolute quantification is when a protein is quantified in units of molecules / cell or mol / L.
Relative quantification is when a protein in sample A is quantified relative to its abundance in another samples, e.g. samples B, so we aim to estimate the ratio (fold change) between the two samples. For example, we can accurately estimate that sample A has 2-fold more of a specific protein than sample B, without having a very accurate estimate of the number of protein molecules in either sample.

[@Ceren_A498]

@@NikolaiSlavovResearch thank you so much!! this is very helpful