Mass Spectrometry in Systems Biology - Systems Biology: Networks, Models, and Applications - page 16

separation in proteomics research. Tong et al. [40] interfaced RP and CE
for the analysis of ribosomal proteins. Beside multidimensionality, CE
might be used as a stand-alone separation method for metabolites, pos-
sibly by providing faster separation than GC.
GC-MS is the method of choice for analysis of metabolites, either
with a quadrupole or a TOF detector. An attractive feature of GC is the
ability to predict relative retention time of bioanalytes. For a predefined
chemical structure, mass measurement ( m / z ) and relative retention
time produced by a GC-MS experiment are enough for compound
identification [41]. Separation techniques contribute to MS analytical
platforms by increasing peak capacities, in other words by increasing
the number of resolvable biomolecular components.
QUANTIFICATION OF BIOANALYTES BY MS
Quantification of bioanalytes is a key experiment in systems biology
studies. Quantification of compounds by MS is traditionally performed
by using a stable isotope internal standard to correct differences in ion
current profiles due to ionization efficiencies, chromatographic repro-
ducibility, sample handling errors, or matrix effects. If the concentration
of the stable isotope analog is known, an absolute measurement of
quantity can be obtained rather than a relative measure. We will pres-
ent here predominantly quantitative proteomics studies.
Quantitative proteomics follows either the relative expression levels
or absolute amounts of proteins. Quantitation of relative expression
levels for proteome analysis has its roots in differential display two-
dimensional electrophoresis (2DE). However, 2DE shows limitations
both in identification (e.g., low-abundance proteins, membrane pro-
teins, etc.) and in quantitation (i.e., posttranslational modifications
presented as multiple spots of the same protein) experiments. Solution-
phase proteomics methods using LC-MS/MS are more sensitive and
specific than traditional 2DE separations, but relative quantities cannot
be achieved without some form of stable isotope labeling or well-
controlled chromatography (see below).
Bottom-up proteomics emerged as the method of choice for analysis of
relative expression levels of proteins by stitching quantitative information
at the peptide level back to the parent proteins. Two different strategies
offer high-throughput capabilities for quantification of peptide levels by
mass spectrometry: (i) isotope labeling and (ii) stable isotope free.
Isotope labeling methods are composed of a growing number of
variants. When manipulation of the biological system is possible, meta-
bolic labeling provides an accurate measurement of relative quantities
of peptides by one of the following strategies:
1. Incorporation of 15 N into all amino acids [42,43]. 15 N incorpo-
ration has been applied to organism level by metabolic
labeling of C. elegans [44] and rats [45].