Figure 3.

Identification of proteins of the assembly by mass spectrometry. (A) pol I present in fraction PA600 was further purified on BioRex70 and separated on a Sephacryl S-300 column in the presence of 300 mM potassium acetate as described previously (Milkereit et al. 1997). The peak fraction in terms of protein content, which also contained most of pol I (data not shown), was separated by SDS-PAGE. Single bands of silver-stained gels were excised, in-gel digested with trypsin, and analyzed by MALDI mass spectrometry followed by database searching with the resulting peptide masses. Due to high mass accuracy of MALDI peptide maps, proteins were identified unambiguously. (B) MALDI mass spectrum of peptides recovered after tryptic in-gel digestion of single protein band. A search with detected masses in nonredundant protein database showed that 18 peaks (P) matched to calculated tryptic peptide masses of yeast FK506-binding nuclear protein (Fpr3) within an accuracy better than 50 ppm and covered 33% of the sequence. The peaks marked by M correspond to tryptic autolysis products.