A protocol for qualitative and quantitative measurement of endosomal processing using hot spot analysis

To trigger an immune response, protein antigens must be broken up into peptides and presented to the effector cells of the immune system. These peptides, along with the presentation components and cellular milieu, can determine whether the protein is recognised as friend or foe, eliciting either tolerance or immune activation, respectively. In this protocol paper, Clement et al. describe the method for isolating specific processing organelles (endosomes) from murine dendritic cells, exposing them to model antigens, then collecting and analysing the processed peptides. Clear and concise details in this publication, including step-by-step LC-MS/MS and bioinformatic instruction, make this paper an excellent guide for researchers interested in the identification and quantification of antigenic proteins and peptide epitopes.

How was PEAKS used?

The authors used the Database Search workflow in PEAKS 8.5 for data refinement, denovo peptide sequencing, and protein identification. Built-in filters, including false-discovery rate (FDR) and individual protein/peptide score cut-offs enabled the selection of high quality data. Extracted ion chromatograms from MS1 and fragmentation spectra from MS2, which are displayed directly in PEAKS software, allowed the authors to manually inspect and validate peptides near scoring thresholds. In addition, the PEAKS Q module was used for label-free quantification (LFQ) of peptides from the peptidomes generated under various experimental conditions. LFQ data were used to identify “hot-spots” or deferentially abundant epitopes in wildtype vs. leptin-deficient mice.

Clement, Cristina C., et al. “A Protocol for Qualitative and Quantitative Measurement of Endosomal Processing Using Hot Spot Analysis.” STAR Protocols, no. 3, Elsevier BV, Sept. 2021, p. 100648. Crossref, doi:10.1016/j.xpro.2021.100648.


A detailed quantification of antigen processing by endosomal compartments provides important information on the pattern of protein fragmentation. Here, we describe a protocol that combines gradient purified endosomes, incubated with antigens, followed by hot spot analysis of MS/MS-sequenced peptides. The analysis identifies differences in endosomal antigen processing by dendritic cells under diverse experimental conditions.