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Post-biosynthetic labeling strategies can be applied to any set of samples, including primary
cell culture and human, because they are performed post-lysis.The peptide amino-termini and lysine side-chains are targeted by the
amine-specific reactive group.Chemical labeling targets reactive groups on the side-chains of amino
acids or peptide termini.Improving on the use of isotope-coded affinity tags (ICAT) [51], isobaric mass tags are all the
same mass and it is only upon fragmentation that the different mass tags are observed [52].Accurately distinguishing the small mass
difference produced by enzymatic labeling requires an instrument with high mass accuracy
and high mass resolution.Consequently, comprehensive stable isotope incorporation is difficult
to achieve because labeling is sequence-dependent.A popular version of isobaric mass tags is the iTRAQ reagent
[53], which has recently expanded to incorporate up to eight reporter mass ions.This is of particular
relevance to biological experiments in which multiple conditions or multiple time-points are
being evaluated such as signaling networks [54].Enzymatic labeling is
performed using H2
18O during the peptide bond hydrolysis step of the digestion [50].The isobaric mass tag consists of an amine-specific reactive group, a balancer group and a
reporter mass group.
Post-biosynthetic labeling strategies can be applied to any set of samples, including primary
cell culture and human, because they are performed post-lysis. However, sample-processing
discrepancies can lead to the introduction of error with these methods. Enzymatic labeling is
performed using H2
18O during the peptide bond hydrolysis step of the digestion [50]. Carboxyl
oxygen exchange can result in the addition of a second 18O, although this reaction is
significantly less efficient and enzyme-dependent. Accurately distinguishing the small mass
difference produced by enzymatic labeling requires an instrument with high mass accuracy
and high mass resolution. Chemical labeling targets reactive groups on the side-chains of amino
acids or peptide termini. Consequently, comprehensive stable isotope incorporation is difficult
to achieve because labeling is sequence-dependent. Side reactions are also a common problem.
Improving on the use of isotope-coded affinity tags (ICAT) [51], isobaric mass tags are all the
same mass and it is only upon fragmentation that the different mass tags are observed [52].
The isobaric mass tag consists of an amine-specific reactive group, a balancer group and a
reporter mass group. The peptide amino-termini and lysine side-chains are targeted by the
amine-specific reactive group. A popular version of isobaric mass tags is the iTRAQ reagent
[53], which has recently expanded to incorporate up to eight reporter mass ions. Consequently,
the amount of run time required to analyze multiple samples can be reduced. This is of particular
relevance to biological experiments in which multiple conditions or multiple time-points are
being evaluated such as signaling networks [54].
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