Overview of Quantitative LC-MS Techniques for Proteomics and Activitomics - LC-MS/MS in Proteomics: Methods and Applications - page 22

Although not covered in this volume, we refer the reader
to several published protocols describing sample labelling with
ICAT reagents, chromatographic fractionation of labelled tryp-
tic peptides, protein identification and ICAT quantification using
MS/MS ( 15 - 17 ) . Variations to the original ICAT strategy have
also been reported. These include fluorescent-labelled ICAT
(FCAT), allowing absolute quantification by fluorescence mea-
surement and peptide enrichment using an anti-FITC antibody
or iminodiacetic acid-coated beads ( 18 ) ; visible isotope-coded
affinity tags (VICAT) that contain a visible probe for monitor-
ing chromatographic behaviour and a photo-releasable biotin
affinity tag for selective capture and release of labelled peptides
( 19 , 20 ) ; acid-labile isotope-coded extractants (ALICE), a class
of chemically modified resins that contain a thiol-reactive group
to capture cysteine-containing peptides and a heavy or a light
isotope-coded, acid-labile linker ( 21 ) ; and metal-coded affinity
tags (MeCATs), where different element-coded metal chelates
are used for cysteine labelling which can be purified on metal
chelate-specific affinity resins ( 22 ) . The compatibility and robust-
ness of the MeCAT technology for the relative quantification was
recently shown using standard LC-MS techniques and offered the
unique advantage of absolute quantification via inductively cou-
pled plasma mass spectrometry ( 23 ) .
The principle of ICAT has also been developed to exam-
ine differential protein post-translational modifications. In the
phosphoprotein isotope-coded affinity tag (PhIAT) approach,
phosphoserine and phosphothreonine residues are derivatised
by hydroxyl ion-mediated beta-elimination followed by Michael
addition of 1,2-ethanedithiol (EDT). Peptides are captured after
labelling the EDT moiety with an isotope-coded biotin affinity
tag ( 24 ) . This idea was further developed such that instead of
using biotin-avidin enrichment, EDT peptides were captured and
labelled on a solid-phase support in a single step using light ( 12 C 6 ,
14 N) or heavy ( 13 C 6 , 15 N) phosphoprotein isotope-coded solid-
phase tagging (PhIST) reagents and released from the solid-phase
support by UV photo-cleavage ( 25 ) . Similarly, a beta-elimination
and Michael addition-based approach for the relative quantifica-
tion of O -phosphate or O -GlcNAc-modified peptides used dif-
ferential isotopic labelling with normal or deuterated dithiothre-
itol ( 26 ) . Thiol chromatography was used for enrichment, whilst
the specificity of O -phosphate versus O -GlcNAc mapping was
achieved by enzymatic dephosphorylation or O -GlcNAc hydrol-
ysis. Finally, given the fact that cysteine thiols are the targets of
oxidative modifications relevant to biological function and dis-
ease, the ICAT reagents have been used for quantification of
oxidative PTMs by virtue of the fact that cysteine thiol oxidation
blocks ICAT labelling ( 27 , 28 ) .