Integrated Functional Analysis of the Nuclear Proteome of Classically and Alternatively Activated Macrophages

Figure 6

Summarized proteome profile of nuclear fractions of M1 and M2 macrophages. The nuclear protein samples from M1, M2, and M0 macrophages ( per group) were incubated with (Asc⁺) or without (Asc⁻) ascorbate, labeled with BODIPY FL N-(2-aminoethyl) maleimide (BD), and resolved by the 2DE approach. Gels were imaged for BD fluorescence, scanned for BD spillover in Pro-Q Diamond fluorescence detection range, and then labeled with Pro-Q Diamond and imaged for phosphorylation profile. All images were analyzed with SameSpots software; normalized spot volumes were used for comparison. Protein spots with -fold change in abundance, S-nitrosylation level, or phosphorylation level () in M1 or M2 macrophages (vs. M0 controls) were subjected to MALDI-TOF MS/MS analysis, and those identified with high confidence are highlighted in light brown color. Ratiometric calculation from BODIPY-fluorescence units in Asc⁺ aliquots (normal vs. experimental) was conducted for quantifying the differential abundance of protein spots (Δ protein M1 or M2/Asc⁺ M0). The ratio of ratios, i.e., /[Asc⁺ M1 or M2/Asc⁺ M0] was calculated to obtain the change in SNO levels normalized for protein abundance. Note that because SNO modification inhibits the Cys-BODIPY labeling, a negative RoR value indicates an increase in SNO levels (and vice versa). Ratiometric calculation from Pro-Q Diamond fluorescence units (after subtracting BD spillover) in normal vs. experimental gels was conducted for quantifying the differential phosphorylation of protein spots (Δ protein or M2 PQD/M0 PQD). The dark/light green and dark/light pink/orange colors indicate an increase and decrease in abundance, RoR values, and phosphorylation levels, respectively, in M1 and M2 (vs. M0) macrophages. The darker shade means the higher value.