Quantitative Proteomics

Isobaric labeling quantification

Isobaric reagents such as iTRAQ and TMT are set of chemical tags that are used for quantitative proteomics experiments.

iTRAQ reagents are available as 4-plex and 8-plex. TMT reagents are available as TMT0, TMT 2plex, TMT 6-plex, and TMT 10-plex.   

Isobaric tags of an isobaric reagent set have the same chemical structure and molecular weight.

The general chemical structure of an isobaric reagent consists of three groups: amine-reactive, mass balancer, and reporter ion. The amine-reactive group reacts with N-terminal amine and lysine ε-amine in a peptide to form a complex. The balancer group normalizes the mass difference created by reporter ions group so that molecular weight of all isobaric tags in an isobaric reagent set remains the same. The reporter ion group is linked with labile bond with balancer and is liberated during tandem mass spectrometry. 

For isobaric labeling-based quantification, samples are labeled with different isobaric tags available in an isobaric reagent set. The number of samples that can be analyzed together depends on the type of isobaric reagent kit used for labeling. For example, proteins from up to 8 and 10 samples can be identified and quantified simultaneously using iTRAQ 8-plex and TMT 10-plex reagents.

Relative quantification of proteins among samples is achieved by comparing the relative intensities of the reporter ion peaks. 

Factors affecting isobaric quantification (1): 

a) Isotope purity: Correct for isotopic impurities to achieve accurate quantification.
b) Intensity: Peptides with intense reporter ion peaks provide accurate quantitative ratios.
c) Interference from co-eluting peptides: Co-eluting peptides from non-changing background proteins can interfere with quantification.

Label free quantification

Larger proteins generate more peptides and therefore more MS/MS spectra compared to smaller proteins. 
For relatively accurate quantification, spectral counting-based label free method uses normalized spectral abundance factors (NSAF) for data normalization. NSAF is calculated by dividing the total number of MS/MS spectra (SpC) of peptides generated from protein (let's say) k by the protein's length (L), then dividing by the sum of SpC/L for total N proteins identified in the sample.  

NSAF accounts for protein size and variability between runs. It can be used to compare individual runs and determine relative protein abundance among samples. 

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