https://pages.htgaa.org/2026a/louisa-zhu/labs/week-10-lab-mass-spectrometry/index.htmlThis laboratory module introduces Liquid Chromatography-Mass Spectrometry (LC-MS) as a premier tool for protein characterization, focusing on enhanced Green Fluorescent Protein (eGFP). The experiment follows a logical progression from “minimally perturbative” measurements to increasingly disruptive techniques, allowing students to determine three critical pieces of information: molecular weight, protein folding/structure, and primary amino acid sequence. By moving from intact protein analysis under both native and denaturing conditions to bottom-up peptide mapping, the lab demonstrates how a protein’s physical state directly influences its behavior in a mass spectrometer.Beyond standard eGFP analysis, the curriculum highlights the biochemical nuances of the protein, such as the autocatalytic self-cyclization of its fluorophore which involves a specific $20\text{ Da}$ mass loss. As a specialized bonus, the lab introduces Charge Detection Mass Spectrometry (CDMS) to analyze macromolecular structures that are too large for conventional MS. Students will use this technology to investigate the quaternary structure of Keyhole Limpet Hemocyanin (KLH), a massive complex that exists in multiple oligomeric states within the megadalton range.Hugoen