Conclusion
Immunoprecipitation afforded high-purity isolation of NPC sub-complexes from mitotic cells. Analysis of subcomplex constituents revealed the expected proteins and some surprises, such as abundant myosin 9 in the Nup107-160 complex IP, and Nup133 associated with FG-domain complexes. Phosphopeptide analysis revealed seventeen sites of mitotic phosphorylation, eight of which were wholly novel sites. MALDI analysis using an LTQ Orbitrap mass spectrometer provided rapid, high mass accuracy (<3 ppm) identification of the sub-complex protein constituents and sites of phosphorylation. The MALDI format, in combination with the sensitive MSn performance of the LTQ ion trap, allowed for manual, prolonged ‘revisiting’ of phosphopeptides that were not successfully identified in an automated first pass, thus permitting additional identifications.

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