Systems Analysis of Arrestin Pathway Functions - Progress in Molecular Biology and Translational Science - page 435

MS-based functional interactomic analysis could yield an enormously
important body of information for the elucidation of GPCR signaling net-
works; however, the necessary specific isolation of small protein complexes
will likely yield a minimal mass of starting protein. Unfortunately, MS-based
protein identification techniques possess no ability to amplify peptide signals;
therefore, there are significant issues of appropriate experimental scale to
deal with when narrowing proteomic analyses down to the physical inter-
actomic level. Clearly, the primary focus of most interactomic studies is the
initial physical interactome enrichment process. Current interactomic stud-
ies rely upon antibody purification processes, in low detergent near-native
conditions, for the initial target protein isolation. 126 One major consider-
ation for antibody-based interactome purification and analysis is the likeli-
hood that the antigenic epitope of the target protein could be
idiosyncratically presented to the purifying antibody among distinct inter-
actomic protein composition populations. Therefore, for a single protein
physical interactome, biologically mediated changes in both the qualitative
or stoichiometric protein composition are likely to affect the global charge/
hydrophobicity profile of the purified entity. Identification of the biologi-
cally relevant changes in functional interactome composition can be mon-
itored using microfluidic separation devices with subsequent SILAC-based
protein identification. The physical interaction of an antibody-isolated
interactome (composed of multiple diverse proteins) with an analytical
fluidic phase will be related to the expression of hydrophilic or hydro-
phobic interfaces on the accessible regions of the isolated interactome.
In a manner similar to ion-mobility MS, 127 microfluidic separation and
immunopurification devices that can alter both the ambient electrical poten-
tial and solvent conditions 128 may represent a facile mechanism for the selec-
tive isolation of specific interactome species from any immunopurified
heterogenous pool. The ability to distinguish diverse, biologically relevant,
interactome variants will no doubt represent a major step forward in the
understanding of cellular signaling architecture.
As we have stated, the functional direct interactome of a specific signal-
ing protein, for example, b -arrestin, is likely to be highly variable and
dynamically coordinated. To assist the analysis of these rapid interactome
variations, the SILAC labeling approach can be used to great effect.
To observe treatment-induced alterations in interactomes, cellular proteins
can be initially labeled with nonradioactive (nitrogen-15) Arg or Lys isoto-
pic variants, and then interactomes can be isolated from distinct isotopically
labeled cellular pools (native nitrogen-14 or nitrogen-15) and then mixed