Systems Analysis of Arrestin Pathway Functions - Progress in Molecular Biology and Translational Science - page 430

(isobaric tag for relative and absolute quantitation) or isotope-coded affinity
tags, as well as protein labeling approaches such as SILAC (stable incorpo-
ration of labeled amino acids in culture) and SILAM (stable incorporation of
labeled amino acids in mammals) allow the rapid ratiometric analysis of pep-
tides/proteins in a complex biological sample. In both of these processes,
peptides are usually separated by multidimensional cation-exchange liquid
chromatography (LC) and eventually are identified with either TOF or
LC-MS 2 MS, with modified dissociation techniques such as pulsed
Q collision-induced dissociation 111 or higher energy collisional dissocia-
tion 112 . These diverse protein quantitation techniques provide an ability
to generate an unbiased appreciation of multiple ratiometric proteomic
alterations occurring within multiple samples. The ability to multiplex sev-
eral diverse experimental samples within a specific MS run, when using var-
ious iTRAQ mass tag label sizes (eight distinct mass tag labels are currently
available), provides two important advances compared to nonquantitative
workflows. First, multiplexing allows direct intraexperiment sample com-
parison and also reduces the run-to-run MS identification variability.
iTRAQ workflows are designed to provide relative quantitation of as many
proteins from the input sample as possible. This global detection process is
assisted by multiple levels of prefractionation and orthogonal separation as
well as the employment of MS scanning modes designed to detect as wide
an array of ionized peptides as possible. However, when a specific protein
identity is sought from a complex sample, alternative quantitation proce-
dures can be applied, for example, selective reaction monitoring
(SRM 113 ). SRM workflows restrict the analytical MS scanning parameters
to those that will likely detect the most characteristic peptide fragments for a
prespecified protein. SRM can then be converted into a quantitative process
if the target protein is readily available in a purified form. 114 Such specific
monitoring modes of MS may considerably slow down the rate of data
retrieval and may only be suitable for experiments in which high levels of
starting extract material are available. In contrast to array technology though,
the detection through SRM is still dependent on the ability of the MS to
physically detect the specified peptides. This detection reliability is often
more likely to demonstrate experiment-to-experiment variability than gene
array platforms.
While the accurate and unbiased collection of quantitative protein data is
paramount, one important caveat, with respect to data retrieval, is the need
to physically retain both significantly and nonsignificantly regulated protein
data. The nature of the “nonsignificantly regulated” data may yet yield