. Author manuscript; available in PMC: 2015 Mar 20.

Published in final edited form as: Nat Protoc. 2014 May 8;9(6):1301–1319. doi: 10.1038/nprot.2014.075

TABLE 1.

Examples of stable-isotope labeling strategies for MS-based quantitative proteomics

Labeling	Principle	Advantages	Disadvantages
Metabolic
SILAC^*	Incorporates heavy-labeled selected amino acid counterparts into proteins	Label incorporation to cultures and small organisms like C. elegans and Drosophila. Less variability between samples and incorporations	Not applicability to complex organisms. Labeled amino acids are expensive. Metabolic derivatization of Arg to Pro
¹⁴N/¹⁵N	Incorporates ¹⁴N/¹⁵N counterparts into the proteins	Incorporation into many microorganisms	Complexity during data analysis due to incorporation of label at backbone and side-chains. Expected mass difference is unknown before peptide identification
Chemical
ICAT^*	Labels cysteine residues at the protein/peptide level	Simplified sample mixture and analysis as it excludes cysteine lacking peptides. Incorporation at the protein level	Only for analysis to proteins/peptides containing cysteines. Expensive reagent. Potential variability as it relies on affinity beads. Increased sample analysis, and time on MS
iTRAQ^* or TMT^*	Labels lysine and N-terminal amines with tags of varying masses	Enables multiplexed sample analysis per MS experiment	Requires tandem MS acquisition; increased sample analysis, and time on MS. High variation
NEM^*	Labels cysteine residues at the protein/peptide level	Very mild reaction. Cheap reagent. Simple. Analysis at MS level. Applicable to any sample	Only cysteine containing peptides can be used for quantitation
SA^*	Labels lysine and N-terminal amines primarily	Cheap reagent. Applicable to broader sample nature. Simple	Very fast reaction, which can cause variability and a demand for MS² analysis
Dimethyl labeling	Dimethyl-labeling of lysine and N-terminal amines	Fast reaction. Cheap reagent. Simple. Applicable to any sample	Isotope effect in LC separation. Variability during modifications
¹⁶O/¹⁸O	Enzyme-facilitated ¹⁸O	Cheap reagent. Simple. Applicable to any sample	Incomplete labeling or slow back exchange of ¹⁶O and ¹⁸O. Incorporation at peptide level

Stable Isotope Labeling with Amino acids in Cell culture (SILAC); Isotope coded affinity tag (ICAT); Isotope Tags for Relative and Absolute Quantitation (iTRAQ); Tandem Mass Tag (TMT); N-Ethylmaleimide (NEM); and Succinic anhydride (SA).