TSA-MS ratio. (A) Nuclear speckles or centromeres TSA-MS workflow. Nuclear speckles or centromeres were labeled using TSA with tyramide coupled to FITC (FITC-tyramide). FITC-labeled proteins were affinity purified and identified using tandem MS. The abundance of each protein in nuclear speckles or centromeres was estimated using LFQ, and the nuclear speckle versus centromere LFQ ratio for each protein was determined. (B) U2OS cell showing centromeres or nuclear speckles after combined immunofluorescence and TSA labeling, as indicated, and DNA (blue, DAPI) staining. Boxed areas are insets shown magnified. Scale bars: main panel, 5 µm; inset, 2 µm. (C) Silver-stained gels after TSA labeling (Input) and affinity pull-down of FITC-labeled proteins (Pulldown) from nuclear speckles or centromeres. Primary antibodies, including no primary antibody (No1), for TSA labeling are indicated at top of gel. (D) Western blot comparing SON and HP1α levels in the nuclear speckle and centromere fractions after pull-down of FITC-labeled proteins, as in C. (E and F) Numbers of the top 100 (E) and top 250 proteins (F), sorted by their nuclear speckle versus centromere (SPK-CEN) ratios, located in nuclear speckles (NS), nucleoplasm (NP), or cytosol (Cyt). See also Table S1.