Global Proteomic Profiling to Unravel Lipofuscin's Protein Repertoire

Lipofuscin, a yellow-brown complex mainly found in cellular waste, accumulates in cells as a byproduct of cellular degradation processes and is commonly associated with aging and oxidative stress. Lipofuscin's molecular "signature" comprises a diverse repertoire of oxidized proteins, lipids, and metals, yet the detailed protein composition of this unique complex remains undetermined. Toward this end, high-throughput proteomic analyses of lipofuscin are pivotal for elucidating its global protein content, the protein interactome, as well as biological mechanisms related to senescence-associated lipofuscin accumulation. Existing proteomic profiling approaches of lipofuscin primarily rely on "in-gel" digestion methods, often leading to incomplete protein digestion, suboptimal peptide recovery, and other analytical pitfalls leading to "loss" of significant molecular information. In this chapter, we introduce a global proteomic profiling methodology for lipofuscin preparations derived from cell cultures, based on FASP (Filter-Aided Sample Preparation) method coupled with nano-liquid chromatography-tandem mass spectrometry (nano-LC/MS-MS). This methodology aims to deliver robust characterization of the lipofuscin proteome, elucidate its complex interactions, and introduce a superior alternative to traditional "in-gel" digestion technique.