Mass spectrometry identification of NASP binding partners in HeLa cells.
Nuclear autoantigenic sperm protein (NASP) is a linker histone binding protein that is cell-cycle regulated. Synchronized HeLa cells are delayed in progression through the G1/S border when transiently transfected to overexpress full-length NASP, but not the histone-binding site (HBS) deletion mutant (NASP-DeltaHBS). The purpose of the current study was to identify possible NASP-associated proteins in HeLa cell nuclei that could elucidate NASP's influence on the cell cycle and chromatin remodeling. For this purpose, we employed a new approach: mass spectrometry identification of initially cross-linked proteins after their separation in a second dimension by reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Of the twelve proteins identified, three appear to be relevant to NASP's function: heat shock protein 90 (HSP90), DNA-activated protein kinase, and ATP-dependent DNA helicase II (70-kDa subunit). Individual protein-protein interactions were tested by immunoprecipitation techniques. This new method can be used for expedited identification of binding partners of different proteins in enriched fractions and as a complementary or alternative strategy to the yeast two-hybrid system and immunoprecipitation methods.
Alekseev
Oleg M
OM
Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, 27599-7090, USA. oalex@med.unc.edu
Richardson
Richard T
RT
Pope
Marshall R
MR
O'Rand
Michael G
MG
D001324:Autoantigens / Q000737:chemistry* / Q000378:metabolism*; D015153:Blotting, Western; D003432:Cross-Linking Reagents; D015180:Electrophoresis, Gel, Two-Dimensional; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D008297:Male; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013388:Succinimides
D001324:Autoantigens; D003432:Cross-Linking Reagents; D018841:HSP90 Heat-Shock Proteins; C489390:NASP protein, human; D009687:Nuclear Proteins; D013388:Succinimides; C035759:3,3'-dithiobis(sulfosuccinimidyl propionate)