Hankum Park

Degradation and recycling of plasma membrane proteins occurs via the endolysosomal system, wherein endosomes bud into the cytosol from the plasma membrane and subsequently mature into degradative lysosomal compartments. While methods have been developed for rapid selective capture of lysosomes (Lyso-IP), analogous methods for isolation of early endosome intermediates are lacking. Here, we develop an approach for rapid isolation of early/sorting endosomes through affinity capture of the early end

Herein we report molecular shape-dependent nonspecific labeling of photoaffinity linkers (PLs) in the cellular proteome. Linear PLs have a greater tendency to engage in nonspecific binding than branched PLs. Exploiting this property, we discovered a smaller branched diazirine-based PL as the best photoaffinity probe with minimal nonspecific binding characteristics from among 5 probes with different PLs.

A label-free method for proteome-wide target identification was developed using in-gel fluorescence difference caused by thermal stability shift.

Phenotypic screening can not only identify promising first-in-class drug candidates, but can also reveal potential therapeutic targets or neomorphic functions of known proteins. In this study, we identified target proteins of SB2001, a cytotoxic agent that acts specifically against HeLa human cervical cancer cells. Because SB2001 lacks chemical modification sites, label-free target identification methods including thermal stability shift-based fluorescence difference in two-dimensional gel elect

Previous studies have developed methods for isolation of lysosomes, mitochondria, and peroxisomes from non-denaturing extracts. Here we describe an approach for purification of early/sorting endosomes, providing a means by which to examine early aspects of the endolysosomal system and to combine this with lysosome purification using Lyso-IP. We refer to this new method as Endo-IP. This allows us to examine the proteome and lipidome of endosomes, and to perform electron microscopy imaging of endo

We present a protocol for sample preparation for LC-MS analysis of whole cell lysates and for lysosomal and endosomal fractions purified by Lyso-IP and Endo-IP. Protocols for purification of lysosomes and endosomes is provided in protocol dx.doi.org/10.17504/protocols.io.byi9puh6 using cells that express endogenously tagged TMEM192-HA and stably expressing FLAG-EEA1 as descrbed in dx.doi.org/10.17504/protocols.io.byi7puhn.

Lyso-IP is a method that allows for the isolation of lysosomes for proteomics and metabolomics (dx.doi.org/10.17504/protocols.io.bybjpskn; dx.doi.org/10.17504/protocols.io.bx9hpr36). We have developed an analogous approach for purification of early/sorting endosomes (Endo-IP). In addition, we have found that endolysosomal purification via Lyso-IP and Endo-IP can be coupled with a quantitative proteomics workflow to obtain snapshots of Amyloid Precursor Protein (APP) processing to its Aβ products

Here we present a general protocol for immunological detection by Western blotting of APP and proteins of the endolysosomal system, including EEA1, RAB5, PSEN1, LAMP1, LAMP2, TMEM192, and BACE1.

Source datasets for Western blot quantification in Figures S1d and S5a for Park et al. "Spatial snapshots of amyloid precursor protein intramembrane processing via early endosome proteomics".

Selective purification of early endosomes can be achieved through affinity capture of the early endosome-associated protein EEA1 (termed Endo-IP) (Park et al. in submission). These purified endosomes can be used for proteomic and lipidomic studies to obtain snapshots of early endosomes. Here we present an immunofluorescence protocol to assess the extent of colocalization between FLAG-EEA1 and RAB5 with and without the Dynamin-1 and -2 (DNM1/2) inhibitor Dyngo4a.

The ability to detect processing of APP to the Ab amyloid peptide is challenging. This protocols describes methods for analysis of Ab "half-tryptic" peptides from purified organelles (endosomes and lysosomes). The targeted proteomics approach using TOMAHAQ coupled with Tomahto, which is an API for use on a Thermo orbitrap instrument that facilitates detection of trigger peptides and fragmentation of target peptide reporter ions.

TOMAHAQ-based targeted proteomics relies on heavy-labeled reference peptides for multi-plexed quantification of peptides of interest within a set of samples. The APP amyloid precursor protein is thought to be proteolytically processed within the endolysosomal system by β-secretase and ϒ-secretase to yeild various forms of Aβ. To quantitatively track APP products, we describe a protocol for design and generation of synthetic reference peptides for TOMAHAQ analysis. We also include reference pepti

Previous studies have developed methods for isolation of lysosomes, mitochondria, and peroxisomes from non-denaturing extracts. Here we describe an approach for purification of early/sorting endosomes, providing a means by which to examine early aspects of the endolysosomal system and to combine this with lysosome purification using Lyso-IP. We refer to this new method as Endo-IP. This allows us to examine the proteome and lipidome of endosomes, and to perform electron microscopy imaging of endo

TOMAHAQ-based targeted proteomics relies on heavy-labeled reference peptides for multi-plexed quantification of peptides of interest within a set of samples. The APP amyloid precursor protein is thought to be proteolytically processed within the endolysosomal system by β-secretase and ϒ-secretase to yield various forms of Aβ. To quantitatively track APP products, we describe a protocol for design and generation of synthetic reference peptides for TOMAHAQ analysis. We also include reference pepti