FIGURE 2.

In vivo identification of sumoylation sites by cysteine tag peptide enrichment and LC-MS/MS. His-SUMO-1C was expressed in HeLa cells, and the resulting sumoylation sites were identified by His tag protein purification, cysteine tag peptide enrichment, and LC-MS/MS analysis. A, MS/MS analysis of RanGAP1 sumoylated peptide ⁵¹⁶LLVHMGLL(GG-)KSEDK⁵²⁸ (LTQ Orbitrap XL). This peptide shows a known consensus sumoylation site, Lys⁵²⁴. (GG-)K, diglycyl lysine (sumoylation site). B, MS/MS analysis of CKAP2L sumoylated peptide ¹⁹⁸(GG-)KPDPKLpYTR²⁰⁶ (QSTAR Pulsar). This peptide shows a non-consensus sumoylation site, Lys¹⁹⁸. pY, phosphotyrosine. C, biological validation of the CKAP2L sumoylation site. HA-CKAP2L WT and HA-CKAP2L K198R were expressed together with WT His-SUMO-1 in HeLa cells. Samples were immunoprecipitated (IP) with anti-HA antibody, and sumoylated forms were detected by blotting with SUMO-1 antibody. Asterisks indicate sumoylated bands that are lost upon mutation of Lys¹⁹⁸. The full-length HA blot is presented in supplemental Fig. 1. WB, Western blot.