Although SILAC internal standard and analyte detergent insoluble proteomes were equally loaded by protein weight, a remaining concern is whether the light and heavy detergent insoluble protein pools are directly comparable, since the condition promoting aggregation could expand or contract the total protein amount that is resistant to detergent extraction. This issue of mass balance underpins whether any quantitation as calculated is reliably indicating the specified change in protein. In the case of TDP-43 overexpression, mass balance is roughly maintained because the total protein in the detergent insoluble fraction containing aggregates does not grossly change as a percentage of total cellular protein in lysates of TDP-43 or TDP-S6 transfected vs. untransfected HEK-293 cells. However, if this experimental workflow is adapted for use in the case of another protein that more grossly alters bulk protein biochemistry, then mass balance would not be maintained and additional normalization would be necessary to provide accurate quantitation. In such a case, the number of cells from which protein is fractionated could be strictly controlled rather than the mixed protein amount, so that the target aggregate proteome amount would be allowed to reflect expansion or contraction. When protein quantity is properly controlled prior to mixing with internal standard, it should hold true, then they lie outside the “target aggregate proteome” of interest. This does not preclude the presence of aggregates in the background detergent-insoluble proteome of mock-transfected cells, but they are not identified by the method employed, because they present no quantitative change. Identifications of 299 proteins increasing moderately or better, at least 0.5 log2-ratio units in either the TDP-43 or TDP-S6 aggregate proteome, were input into Ingenuity Pathway Analysis. Selected output functional annotation categories for these enriched proteins are given in Table 2, and include a general RNA processing category, translation, splicing, and cell death or apoptosis. These categories are consistent with established roles of TDP-43, and further implicate protein biosynthesis or translation, as reported in the soluble TDP-43 interactome. Proteins associated with cell death or apoptosis increasing in the aggregate proteome with TDP-43 overexpression are also consistent with evidence that overexpression of wild type, full length TDP-43 leads to neuronal toxicity in particular. However, nuclear fragmentation was not apparent in the vast majority of HEK-293 cells transfected up to 72 h with either TDP-43 or TDP-S6 by immunocytochemistry, so it is not clear whether a shift in these proteins to the aggregate proteome represents a decrease in cellular survival mechanisms that progresses to apoptosis. The TDP-S6 model, which forms the largest TDP-nucleated or -associated aggregates, has less of an BU 4061T increase in detergent-insoluble death-associated proteins than the TDP-43 model, consistent with a possible protective effect.