Overview of Quantitative LC-MS Techniques for Proteomics and Activitomics - LC-MS/MS in Proteomics: Methods and Applications - page 20

give a peptide ratio of the 'heavy'-labelled versus 'light'-labelled
peptide. In a similar manner, differential isotopic labelling in vivo
allows quantification of peptides following incorporation of 'light'
( 12 C, 14 N, 1 H) and 'heavy' stable isotope-labelled ( 13 C, 15 N, 2 H)
amino acids and is exemplified by the stable isotope labelling of
amino acids in culture (SILAC) strategy ( 4 ) .
In all of these methods, the ratios of detected 'reporter' frag-
ments or isotopically labelled peptides are computed and inte-
grated into protein ratios which can then be evaluated statisti-
cally. Multiple software solutions for the analysis of quantitative
information using these labelling strategies are available and have
been recently reviewed ( 5 ) ( see also Chapter 4 ). Although many
of these solutions are instrument, data or tag dependent, they all
work on the same principle whereby isotopically labelled peptide
pairs (or reporter ions) are extracted on the basis of their char-
acteristic mass differences and successful MS/MS peptide assign-
ments. Ratios of the extracted isotopic pairs are then computed
and statistical evaluation performed. It is important to note that
the smaller the mass difference of the tags is, then the more diffi-
cult it becomes to interpret the data and perform accurate quan-
tification, since the isotope envelopes of the differentially labelled
peptides may overlap.
When using any labelling approach for LC-MS, the labels are
best introduced at the earliest point in the workflow to minimise
differences introduced into the samples by handling or quan-
titative differences between LC-MS runs. In this sense, the in
vivo labelling strategies outperform the chemical and enzymatic
labelling strategies in terms of accuracy of quantitation; however,
as will be discussed below, multiplexing using in vivo labelling
is presently more limited than chemical labelling, where 12-plex
strategies have been reported ( 6 ) . Another important difference
in these tagging strategies is in the analysis of primary tissues and
clinical samples such as tissues, body fluids and urine, which are
only amenable to the in vitro labelling approaches.

Although stable isotope labelling for protein quantitation had
been previously reported, ICAT was the first robust and univer-
sal differential labelling strategy to be developed for quantitative
LC-MS and is based on cysteine thiol group modification using
iodoacetamide tags. In the first report, ICAT was used to exam-
ine the expression profiles of yeast growing on either galactose
or ethanol in a single analysis ( 2 ) . Stable isotopes were incorpo-
rated into intact proteins after lysis by selective alkylation of cys-
teines with either a heavy (deuterium D 8 ) or a light (deuterium
D 0 ) reagent bearing a biotin tag. Prior to LC-ESI-MS/MS anal-
ysis, the protein mixture was digested with trypsin and the ICAT-
labelled (cysteine-containing) peptides enriched on monomeric
avidin-agarose. This had the advantage of simplifying the peptide

2.2. ICAT
Isotope-Coded
AffinityTagsand
Variations