Figure 3. Flow Chart of CLL Phosphoproteomics Analysis.

Lysates were prepared from primary CLL B cells that had been stimulated over an hour time course with 30 nM CXCL12. Lysates were denatured, reduced and alkylated in preparation for trypsin digest. Tryptic peptides were then enriched for phosphopeptides by IMAC and LC-MS/MS was performed. Data was analyzed using the InsPecT database search algorithm for phosphorylations on Ser, Thr, or Tyr. A decoy database and manual validation of the spectra were used as quality control. Spectral count comparisons were made as a qualitative assessment of CXCL12 stimulation response and interesting target proteins were selected for follow-up studies if antibodies were available.