Fig. 4. Chromatographic separation of the major tryptic phosphopeptides from ³²P in vivo labeled vimentin. Four 20 cm plates of semi-confluent BHK-21 cells were preincubated for 4 hours with 0.5 mCi/ml [³²P]orthophosphate. In order to maximize the in vivo labeling, vimentin dephosphorylation was inhibited by addition of 50 nM cl-A to the cells. Vimentin was isolated by preparative SDS-PAGE as described in Materials and Methods and then subjected to tryptic cleavage. (A) The generated phosphopeptides were first isolated by reversed phase chromatography on a microbore C-18 column. The UV chromatogram (left panel) indicates the presence of a high number of tryptic peptides, among which seven ³²P labeled fractions were isolated (right panel; fractions a-g). (B) Peaks a-g obtained on the C18 chromatography were then separated a second time on a reversed phase microbore C-8 column, yielding one single purified peptide from fractions a, b and d-g, and two peptides from fraction c. The inserts show the elution profile of the ³²P label, which in all cases corresponded to a peak on the UV chromatogram. The isolated peptides were numbered according to their hydrophobicity on the C8 reversed phase HPLC. The obtained peaks were subjected to automatic sequencing and manual Edman degradation. Peptide masses were confirmed by mass-spectrometry (data not shown). The results are presented in Table 1.