The Quantitative Nuclear Matrix
Proteome as a Biochemical
Snapshot of Nuclear Organization
- Publication date
- Publisher
Abstract
The nuclear matrix (NM) is an operationally
defined structure of
the mammalian cell nucleus that resists stringent biochemical extraction
procedures applied subsequent to nuclease-mediated chromatin digestion
of intact nuclei. This comprises removal of soluble biomolecules and
chromatin by means of either detergent (LIS: lithium diiodosalicylate)
or high salt (AS: ammonium sulfate, sodium chloride) treatment. So
far, progress toward defining <i>bona fide</i> NM proteins
has been hindered by the problem of distinguishing them from copurifying
abundant contaminants and extraction-method-intrinsic precipitation
artifacts. Here, we present a highly improved NM purification strategy,
adding a FACS sorting step for efficient isolation of morphologically
homogeneous lamin B positive NM specimens. SILAC-based quantitative
proteome profiling of LIS-, AS-, or NaCl-extracted matrices versus
the nuclear proteome together with rigorous statistical filtering
enables the compilation of a high-quality catalogue of NM proteins
commonly enriched among the three different extraction methods. We
refer to this set of 272 proteins as the NM central proteome. Quantitative
NM retention profiles for 2381 proteins highlight elementary features
of nuclear organization and correlate well with immunofluorescence
staining patterns reported in the Human Protein Atlas, demonstrating
that the NM central proteome is significantly enriched in proteins
exhibiting a nuclear body as well as nuclear speckle-like morphology