UMMS Affiliation

Department of Pathology

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Amino Acids, Peptides, and Proteins | Biological Factors | Investigative Techniques


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. For complete details on the use and execution of this protocol, please refer to Clement et al. (2021).


antigen processing and presentation, endosomes, MHC II

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Copyright 2021 The Author(s). This is an open access article under the CC BY-NC-ND license (

DOI of Published Version



Clement CC, Nanaware PP, Stern LJ, Santambrogio L. A protocol for qualitative and quantitative measurement of endosomal processing using hot spot analysis. STAR Protoc. 2021 Jul 6;2(3):100648. doi: 10.1016/j.xpro.2021.100648. PMID: 34278334; PMCID: PMC8264744. Link to article on publisher's site

Journal/Book/Conference Title

STAR protocols

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Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.