Use of MEDUSA-based data analysis and capillary HPLC-ion-trap mass spectrometry to examine complex immunoaffinity extracts of RBAp48.

To examine the Jurkat cell interaction partners of RbAp48, we digested entire immunoaffinity extracts with trypsin and identified potential interacting proteins using one- and two-dimensional microcapillary HPLC-ion-trap mass spectrometry. An Oracle-based automated data analysis system (MEDUSA) was used to compare quadruplicate anti-RbAp48 antibody affinity extracts with two sets of quadruplicate control extracts. The anti-RbAp48 extracts contained over 40 difference 1D gel bands. We identified all known proteins of the NuRD/Mi-2 complex including human p66. Three potential homologues of members of this complex were also found, suggesting that there may be more than one variant of this complex. Eleven proteins associated with RNA binding or pre-mRNA splicing were observed. Four other proteins, including a putative tumor suppressor, were identified, as were 18 ribosomal proteins. There was little overlap with RbAp48-interacting proteins defined by yeast two-hybrid methods. These results demonstrate the analysis of a complex immunoaffinity extract and suggest a more complex cellular role for RbAp48 than previously documented.