- Quantitative Proteome 25
label-free method is quantification by comparing the intensity of mass
spectrometric signal of each peptide from a given protein.^7 Isotope-based
methods determine protein abundance by incorporating specific molecules
into peptides by metabolic or chemical labeling. Samples are labeled with
different versions of a chemical reagent and can be combined, resulting in
paired peptides with a defined mass difference. The ratio of signal intensities
from the paired peptides with different masses can be determined after the
combined samples are analyzed.^9 Stable isotope labeling by amino acids
in cell culture (SILAC)^10 is one of the powerful approaches by metabolic
labeling. The cells are cultured in media containing stable^13 Cor^15 Nisotope-
labeled arginine and lysine, so the labeled amino acids are incorporated into
each protein in the cells. In chemical labeling, the isotope-coded affinity tag
(ICAT)^11 labeled the cysteine residue in peptides with a reagent containing
eight deuteriums and a biotin group for purification of labeled peptides.
Several other strategies of chemical labeling targeting on amino acid are
also available: methylation,^12 esterification, the isotope-coded protein label
(ICPL),^13 and the tandem mass tags (TMT).^14
The isobaric tags for relative and absolute quantitation (iTRAQ)^15 is
one of the most popular chemical labeling methods. The iTRAQ reagent
is amide reactive and can link to the N-terminus and lysine side chains
of peptides (Fig. 1). The term “isobaric” describes the characteristic that
different iTRAQ reagents labeling different samples have equal mass. The
mass is not distinguished in the survey scan of the first MS. The iTRAQ
reagents are fragmented by CID before the second MS and reporter
ions of m/z 114, 115, 116 and 117 in MS/MS spectra for four-plexed
reagent set are generated. In the MS/MS spectra, the intensities of the
reporter ions are used for peptide quantification while the remaining
peaks are used for peptide sequence identification. One of the strengths
of iTRAQ strategy is the enhanced signals of peptides by combining all
labeled-samples into a single analysis, greatly increasing the accuracy of
quantification.^16
The iTRAQ quantitative proteomics is a well-established method suit-
able for a multiplex experiment. The procedures of preparing samples for
iTRAQ quantitative proteomic analysis include protein extraction, reduc-
tion, alkylation, digestion, iTRAQ-labeling, strong cation exchange (SCX)
chromatography and ZipTip desalting (Fig. 2). The detailed protocol of
sample preparation for LC-MS/MS analysis is described later in this chapter.
The methods of gel-assisted digestion and gel extraction^17 areappliedinthis
protocol for the protein digestion step. Please also refer to the reference guide
of the iTRAQ Reagents to note the substances that may interfere with the