Quantitative Proteomics in Development of Disease Protein Biomarkers - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 256

methylation of peptidyl amines is a commonly
practiced method for multiplexed proteomic
quantitation 33 e 37 due to the relatively simple
derivatization procedure, low cost, 35 and the
potential for automation. 34,58 The reductive meth-
ylation can be applied for differentially labeling
up to

stable cand

proteome-wide manner during cell culture or
small animal feeding. 66 This powerful labeling
method uses the natural metabolism of
a cell/animal to introduce heavy amino acids. 67
These heavy amino acids are incorporated into
every protein of the subject, which would nor-
mally contain the light amino acids. Up to

isotopes in a residue-speci

five different samples. This class of peptide
derivatization can be considered as the type II
active derivatization, 59 resulting from which the
derivatization group (dialkylated N-terminal
amine) facilitates the preferential cleavage of the

ve
samples can be pooled together for concurrent
proteomic quantitation. 68 The most signi cant

advantage of this method is the potential of intro-
ducing the labeled proteins at an earlier stage of
proteomic sample preparation work

first amine bond on the original peptide,
producing a 1 ions. These ions are peptide speci c

and good quantitation reporter ions, 60 consid-
ering the reduced possibility of co-eluting and
“

ow. Thus
protein-level reduction in the sample complexity
can be implemented for high accuracy and preci-
sion proteome quanti rst

amino acid. Because the quantitation is based on
fragment ions and the tagged peptides have
different masses, cautions need to be taken to
ensure the authentic sampling of precursor ions,
which differ by a few Daltons. Matrix-assisted
laser desorption/ionization (MALDI)

close-to-isobaric ” peptides having the same cation, with little differen-

tial protein loss. 69 By only supplying heavy
amino acid feed to an organism, organism-wide
labeling can be accomplished. Whole mouse
labeling has recently been applied to many areas
in proteomic research, including cardiac 70 and
aging changes. 71 The cost for the isotopically
labeled feed for the mouse can be expensive,
granting the ability to accomplish protein turn-
over organism-wide. 4 Therefore, proteomes of
individual organs, tissues, and cells can be quanti-

time-of-

flight (TOF)/TOF MS, which has large precursor
selection windows, can be used for quantifying
these types of tagged peptides.
Nicotinylation of amines on proteins, 61,62 using
isotope-coded protein label (ICPL), is another
mass-difference tagging approach. A commercial
kit of isotopic labeling reagents for 4-plexed pro-
teomic quantitation is available. These reagents
can also be used for introducing stable isotopes
topeptides after proteomedigestion. 63 Nicotinyla-
tion can be considered as a type I active derivatiza-
tion and the tagged peptides produce fragment
ions from the derivatizing groups relatively
easily. 59 The seminal chemical mass difference
tagging reagents,

fied with little differential protein loss during
multiple steps of the sample complexity reduction
at/above the protein level.
The SILAC technology has been applied for
investigating disease mechanisms and identi-
fying biomarkers in human samples. 72 e 76
However, a fundamental challenge for applying
this technology to the analysis of human tissues
and body

fluids is the inability to label human
subjects organism-wide like other species. Vari-
ations of the technology, however, have
provided the means to partially overcome this
challenge. They combine SILAC proteomes of
different cell lines to increase the total number
of labeled proteins and the cell lines can be
selected based on heterogeneous human tissues
or body

nity tag
(ICAT) reagents, 64,65 remain as an attractive
option for analyzing highly complex human and
animal samples, due to their capabilityof reducing
the sample complexity of proteome digests. 44

isotope-coded af

Metabolic Labeling
The method of stable isotope labeling with
amino acids in cell culture (SILAC) introduces

fluids. Two interesting methods are

known as stable isotope-labeled pro-

teome (SILAP) 73,74

and super-SILAC spike-in