Ser76 is a predominant site of phosphorylation in resting T cells. CD43 immunoprecipitated from EL-4 thymoma T cells was trypsin digested and resulting peptides analyzed by mass spectrometry. (A) Full-scale MS mode spectrum with arrow indicating presence of the ion with m/z 730.7 corresponding to the monophosphorylated peptide SRQGSLVLEELKPGSGPNLK from CD43. (B) Observation of the ion with m/z 698, as indicated by the arrow, resulting from loss of H₃PO₄ from the ion with m/z 730.7 (in the 3+ charge state, this appears as a 32.6-Th shift). (C) Fully annotated tandem (MS³) mass spectrum of the fragment ion with m/z 698. (D) Expanded detail of the spectrum in panel C showing predominant production of a β-elimination (b5*) ion at m/z ratio of 498.2, with a very small signal correlating to the expected mass for the normal b-ion (b5) at 516.3. (E) Detail from the same spectrum as in panel C showing near-equal production of normal (b3) ion at 372.2 and β-eliminated (b3*) ion at 354.2, typical for an unphosphorylated serine (unphosphorylated serine will usually show some production of β-elimination ions). (F,G) DO.CD43^−/− T cells, transduced with (F) CD43-FL, CD43-Ser72Ala, or CD43-Ser76Ala or (G) CD43-FL or CD43-Ser72AlaSer76Ala were labeled with [³²P] orthophosphate. CD43 was immunoprecipitated and separated by SDS-PAGE, and phosphorylation was detected by autoradiography. Total CD43 was detected by Western blotting. Each lane represents CD43 immunoprecipitate from 10⁷ cells. Fold change was calculated as the ratio of [³²P]/total CD43 normalized to FL. Vertical lines have been inserted to show where a gel lane was cut. These bands came from the same experiment and the same gel.