Peptides or proteins are labeled with chemical groups that have nominally identical mass (isobaric), but vary in terms of distribution of heavy isotopes in their structure.
The samples are mixed in typically equal ratios and analyzed simultaneously in one MS run.
During the MS2 analysis and upon fragmentation, each isotopic variant of the tag produces sequence-specific product ions.
[4] A key benefit of isobaric labeling over other quantification techniques (e.g. label-free) is the multiplex capabilities and thus increased throughput potential.
The current available isobaric chemical tags facilitate the simultaneous analysis of 2 to 11 experimental samples.
A schematic of isobaric labeling: proteins are extracted from different conditions or cell types, digested into peptides, and labeled with isobaric stable isotope tags. These tags consist of reporter, balance, and reactive regions. Lighter reporter regions are paired with heavier balance regions, such that the entire tag attached to the peptide adds the same mass shift. Therefore, after mixing, in MS
1
, the peptides appear as a single precursor. However, when fragmented during MS
2
, in addition to the normal fragment ions, the reporter regions dissociate to produce ion signals that provide quantitative information regarding the relative amount of the peptide in the samples.
Isobaric labeling proteomic workflow with 4 unique reagents in a set, and 7 different biological samples combined into 2 labeling groups (plexes). As the number of samples is higher than the number of reagents, the labeling should be performed in two batches.
