Fluorescent derivatization of a protease antigen to track antigen uptake and processing in human cell lines

Fluorescein labelled subtilisin conjugates had 0.06 to 2 moles of fluorescein per subtilisin molecule. High performance liquid chromatography and mass spectrometry (NanoESI-LC/MS/MS) analysis identified fluorescein conjugated to K141, K256, and the N terminus. Conjugates retained antigenic specificity to subtilisin specific antibodies and could be processed by whole cell extracts into low molecular weight fragments at pH 5.2. Maximal antigen uptake and processing occurred when PMSF (phenylmethylsulfonyl fluoride) inhibited subtilisin conjugate was incubated with cells at 100–200 μg/ml for 16 to 24 hr. Once optimal uptake conditions were established, processed subtilisin peptides were isolated and identified from human cell lines.Our studies show that FITC-conjugation provides an efficient tool to track the uptake and processing of this protease antigen and to facilitate identification of processed antigenic peptides from human cell lines.Antigen presentation by MHC class I and class II molecules is a key aspect of an effective immune response against infections, cancer, certain allergic responses and autoimmune disease. Several good reviews cover capture and processing of exogenous antigens [1,2]. The detection and identification of antigen-derived peptides is important to understand the mechanisms involved in the immune response and to be able to modulate it. Though some studies have employed synthetic peptides to detect peptide epitopes, this approach is unable to identify naturally processed and presented peptides. Some of the early methods employed for identification of naturally processed endogenous epitopes were HPLC and Edman degradation, which led to the identification of hen egg lysozyme (HEL) 52–61 on the MHC of murine B lymphoma cells expressing HEL [3]. Hunt et al. showed the power of joining immunological procedures and mass spectrometry to identify self-peptides presented on cell surfaces in picomole or less concentrations from mouse and human cell lin