Real-time monitoring and effector screening of APE1 based on rGO assisted DNA nanoprobe.

Human apurinic/pyrimidine endonuclease 1 (APE1) played a critical role in the occurrence, progress and prognosis of tumors through overexpression and subcellular localization. Thus, it has become an important target for enhancing the sensitivity of tumor cells to radiotherapy and chemotherapy. Therefore, detecting and imaging its intracellular activity is of great significance for inhibitor discovery, cancer diagnosis and therapy. In this work, using DNA-based nanoprobe, we developed a new method for monitor intracellular APE1 activity. The detecting system was consisted by single fluorophore labeled hairpin probe and reduced graphene oxide (rGO). The in vitro result showed that a liner response of the detection method ranged from 0.02 U/mL to 2 U/mL with a limit of detection of 0.02 U/mL. Furthermore, this strategy possessing high specificity was successfully applied for APE1-related inhibitor screening using intracellular fluorescence imaging. Panaxytriol, an effective inhibitor of APE1 activity, was screened from traditional Chinese medicine (TCM) and its effect on APE1 activity was monitored in real time in A549 cells. In summary, this sensitive and specific APE1 detection technology is expected to provide an assistance for APE1-related inhibitor screening and diseases diagnosis.

A Cooperatively Activatable, DNA-based Fluorescent Reporter for Imaging of Correlated Enzymatic Activities.

The dynamic variation of the expression profile and spatial landscape of multiple enzymes are crucial factors influencing tumor progression and drug treatment. However, the comprehensive analysis of these events has been hampered by the limitations of existing imaging technologies. Here we report a cooperatively activatable, DNA-based fluorescent reporter programmed to detect the correlated activity of dual enzymes, telomerase (TE) and apurinic/apyrimidinic endonuclease 1 (APE1), both in vitro and in vivo. The conformational change of the DNA probe can be orthogonally triggered through TE-induced DNA elongation and APE1-mediated specific cleavage, producing a fluorescent signal for imaging the activity of the two enzymes in an AND-gated manner. Furthermore, we demonstrate the capability of the system for specific tumor imaging through "dual lock-and-key" strategy, and visualizing the correlated enzymatic activities during drug treatment of cancer.

Organelle-Specific Photoactivation of DNA Nanosensors for Precise Profiling of Subcellular Enzymatic Activity.

Understanding of the functions of enzymes in diverse cellular processes is important, but the design of sensors with controllable localization for in situ imaging of subcellular levels of enzymatic activity is particularly challenging. We introduce herein a spatiotemporally controlled sensor technology that permits in situ localization and photoactivated imaging of human apurinic/apyrimidinic endonuclease 1 (APE1) within an intracellular organelle of choice (e.g., mitochondria or nucleus). The hybrid sensor platform is constructed by photoactivatable engineering of a DNA-based fluorescent probe and further combination with an upconversion nanoparticle and a specific organelle localization signal. Controlled localization and NIR-light-mediated photoactivation of the sensor "on demand" effectively constrains the imaging signal to the organelle of interest, with improved subcellular resolution. We further demonstrate the application of the nanosensors for the imaging of subcellular APE1 translocation in response to oxidative stress in live cells.

MGARP is ultrastructurally located in the inner faces of mitochondrial membranes.

Mitochondria, the centers of energy production, are highly organized with inner membranes, cristae and outer membranes. The mitochondrial architecture determines their functions in all cellular processes. Changes in the mitochondrial ultrastructure are tightly related to a wide variety of diseases. MGARP, a mitochondria-localized protein, was predicted by bioinformatics and confirmed by cellular and biochemical methods to be located in mitochondria, but there is no direct and clear evidence for its precise location. This report demonstrates the precise ultrastructural location of MGARP within mitochondria by the ascorbate peroxidase 2 (APEX2) system in combination with electron microscopy (EM). EM revealed that more MGARP is located in the inner/cristae membranes, with its C-terminus at the inner faces of the intramembrane spaces, than in the outer membranes. MGARP overexpression caused both mitochondrial remodeling and cristae shaping, leading to the collapse of the mitochondrial network. The mitochondrial morphologies in MGARP-overexpressing cells were diverse; the cells became round or short, and their cristae were deformed and became discontinuous or circular. An engineered MGARP mutant deficient in its transmembrane domain no longer localized to the mitochondria and lost its effects on mitochondrial structure, confirming that the localization of MGARP in the mitochondria depends on its structural integrity. Collectively, our findings define the location of MGARP within the mitochondria, which is associated with its functional implications for the architecture and organization of mitochondria.

A specific DNA-nanoprobe for tracking the activities of human apurinic/apyrimidinic endonuclease 1 in living cells.

Human apurinic/apyrimidinic endonuclease/redox effector factor 1 (APE1) is an essential DNA repair protein. Herein, we demonstrate that avidin-oriented abasic site-containing DNA strands (AP-DNA) on the surface of silica coated magnetic nanoparticles (SiMNP) can selectively respond to APE1 while resist the digestion by other nucleases. Mechanism studies have revealed that avidin may serve as an organizer protein and recruit APE1 to the DNA substrates on the nanoparticles via strong and specific interactions. Taking advantage of this newly disclosed property, we for the first time successfully displayed the intracellular activities of APE1 in living cells by fluorescence imaging. The avidin organized AP-DNA-SiMNP assembly holds great potential for enzyme-mediated release of drugs inside tumor cells which often contain higher levels of APE1 than normal cells.

Polyubiquitination of apurinic/apyrimidinic endonuclease 1 by Parkin.

Apurinic/apyrimidinic endonuclease 1 (APE1) is an essential protein crucial for repair of oxidized DNA damage not only in genomic DNA but also in mitochondrial DNA. Parkin, a tumor suppressor and Parkinson's disease (PD) associated gene, is an E3 ubiquitin ligase crucial for mitophagy. Although DNA damage is known to induce mitochondrial stress, Parkin's role in regulating DNA repair proteins has not been elucidated. In this study, we examined the possibility of Parkin-dependent ubiquitination of APE1. Ectopically expressed APE1 was degraded by Parkin and PINK1 via polyubiquitination in mouse embryonic fibroblast cells. PD-causing mutations in Parkin and PINK1 abrogated APE1 ubiquitination. Interaction of APE1 with Parkin was observed by co-immunoprecipitation, proximity ligation assay, and co-localization in the cytoplasm. N-terminal deletion of 41 amino acid residues in APE1 significantly reduced the Parkin-dependent APE1 degradation. These results suggested that Parkin directly ubiquitinated N-terminal Lys residues in APE1 in the cytoplasm. Modulation of Parkin and PINK1 activities under mitochondrial or oxidative stress caused moderate but statistically significant decrease of endogenous APE1 in human cell lines including SH-SY5Y, HEK293, and A549 cells. Analyses of glioblastoma tissues showed an inverse relation between the expression levels of APE1 and Parkin. These results suggest that degradation of endogenous APE1 by Parkin occur when cells are stressed to activate Parkin, and imply a role of Parkin in maintaining the quality of APE1, and loss of Parkin may contribute to elevated APE1 levels in glioblastoma. © 2016 Wiley Periodicals, Inc.

Placental aging and oxidation damage in a tissue micro-array model: an immunohistochemistry study.

To evaluate the expression of markers correlated with cellular senescence and DNA damage (8-hydroxy-2'-deoxy-guanosine (8-OHdG), p53, p21, APE1/Ref-1 (APE1), interleukin (IL-6 and IL-8) in placentas from healthy and pathologic pregnancies. This retrospective study considered a placental tissue micro-array containing 92 controls from different gestational ages and 158 pathological cases including preeclampsia (PE), HELLP syndrome (hemolysis, elevated liver enzymes, low platelet count), small for gestational age (SGA) fetuses, and intrauterine growth restriction (IUGR) occurring at different gestational ages. In this study, we demonstrated a significant influence of gestational age on the expression in the trophoblast of 8-OHdG, p53, p21, APE1, and IL-6. In placentas of cases affected by PE, HELLP, or IUGR, there was an increased expression of 8-OHdG, p53, APE1, and IL-6 compared to controls (only IL-8 was significantly decreased in cases). In both groups of pathology between 22- and 34-week gestation and after 34-week gestation, APE1 levels were higher in the trophoblast of women affected by hypertensive disorders of pregnancy than women carrying an IUGR fetus. The cytoplasmic expression of 8-OHdG was increased in placentas in IUGR cases compared to PE or HELLP pregnancies. In cases after 34-week gestation, p21 was higher in SGA and IUGR than in controls and late PE. Moreover, p53 was increased after 34-week gestation in IUGR pregnancies. Placentas from pathological pregnancies had an altered expression of 8-OHdG, p53, p21, APE1, IL-6, and IL-8. The alterations of intracellular pathways involving these elements may be the cause or the consequence of placental dysfunction, but in any case reflect an impaired placental function, possibly due to increased aging velocity in pathologic cases.

Extreme Expression of DNA Repair Protein Apurinic/Apyrimidinic Endonuclease 1 (APE1) in Human Breast Cancer As Measured by Liquid Chromatography and Isotope Dilution Tandem Mass Spectrometry.

Apurinic/apyrimidinic endonuclease 1 (APE1) is a DNA repair protein and plays other important roles. Increased levels of APE1 in cancer have been reported. However, available methods for measuring APE1 levels are indirect and not quantitative. We previously developed an approach using liquid chromatography and tandem mass spectrometry with isotope dilution to accurately measure APE1 levels. Here, we applied this methodology to measure APE1 levels in normal and cancerous human breast tissues. Extreme expression of APE1 in malignant tumors was observed, suggesting that breast cancer cells may require APE1 for survival. Accurate measurement of APE1 may be essential for the development of novel treatment strategies and APE1 inhibitors as anticancer drugs.

Development of APE1 enzymatic DNA repair assays: low APE1 activity is associated with increase lung cancer risk.

The key role of DNA repair in removing DNA damage and minimizing mutations makes it an attractive target for cancer risk assessment and prevention. Here we describe the development of a robust assay for apurinic/apyrimidinic (AP) endonuclease 1 (APE1; APEX1), an essential enzyme involved in the repair of oxidative DNA damage. APE1 DNA repair enzymatic activity was measured in peripheral blood mononuclear cell protein extracts using a radioactivity-based assay, and its association with lung cancer was determined using conditional logistic regression with specimens from a population-based case-control study with 96 lung cancer cases and 96 matched control subjects. The mean APE1 enzyme activity in case patients was 691 [95% confidence interval (CI) = 655-727] units/ng protein, significantly lower than in control subjects (mean = 793, 95% CI = 751-834 units/ng protein, P = 0.0006). The adjusted odds ratio for lung cancer associated with 1 SD (211 units) decrease in APE1 activity was 2.0 (95% CI = 1.3-3.1; P = 0.002). Comparison of radioactivity- and fluorescence-based assays showed that the two are equivalent, indicating no interference by the fluorescent tag. The APE1Asp148Glu SNP was associated neither with APE1 enzyme activity nor with lung cancer risk. Taken together, our results indicate that low APE1 activity is associated with lung cancer risk, consistent with the hypothesis that 'bad DNA repair', rather than 'bad luck', is involved in cancer etiology. Such assays may be useful, along with additional DNA repair biomarkers, for risk assessment of lung cancer and perhaps other cancers, and for selecting individuals to undergo early detection techniques such as low-dose CT.

Unimolecular Chemically Modified DNA Fluorescent Probe for One-Step Quantitative Measurement of the Activity of Human Apurinic/Apyrimidinic Endonuclease 1 in Biological Samples.

A novel DNA structure containing a 3' internal-loop modified abasic site has been constructed which enables effective differentiation between apurinic/apyrimidinic endonuclease (APE1) and nonspecific endonuclease (DNase I). When this unique substrate structure is employed, a double-loop frayed-end chimeric fluorescent probe is successfully developed for quantitative measurement of the activity of APE1 in biological samples without the need of additional cleanup or preconcentration steps. The method is simple and rapid and has a single-step with a linear working range between 0.1 and 5.0 U/mL and a lower limit of detection of 0.1 U/mL. It holds great potential in real-time monitoring of the variation of intracellular and extracellular APE1, which will be very useful for further understanding of the DNA repair pathways in different organisms.

Ultrasensitive apurinic/apyrimidinic endonuclease 1 immunosensing based on self-enhanced electrochemiluminescence of a Ru(II) complex.

An alternative "signal on" immunosensor for ultrasensitive detection of apurinic/apyrimidinic endonuclease 1 (APE-1) was designed utilizing the self-enhanced electrochemiluminescence (ECL) of a novel Ru(II) complex functionalized coil-like nanocomposite as signal labels. The desirable self-enhanced ECL luminophore was achieved by combining the coreactant of poly(ethylenimine) (PEI) and the luminophor of bis(2,2'-bipyridine)-5-amino-1,10-phenanthroline ruthenium(II) [Ru(bpy)2(5-NH2-1,10-phen)(2+)] to form one novel Ru(II) complex, which exhibited significantly enhanced ECL efficiency and stability. Moreover, the carbon nanotubes (CNTs) were employed as nanocarriers for self-enhanced Ru(II) complex loading via π-π stacking to obtain the coil-like nanocomposite to act as signal probe. Compared with traditional ECL immunoassay, our proposed strategy is simple and sensitive, avoiding the adding of any coreactant into testing solution for signal amplification, and shows a detection limit down to subfemtogram per milliliter level under the optimized experimental condition.

Deregulation of base excision repair gene expression and enhanced proliferation in head and neck squamous cell carcinoma.

Defects in the DNA damage repair pathway contribute to cancer. The major pathway for oxidative DNA damage repair is base excision repair (BER). Although BER pathway genes (OGG1, APEX1 and XRCC1) have been investigated in a number of cancers, our knowledge on the prognostic significance of these genes and their role in head and neck squamous cell carcinoma is limited. Protein levels of OGG1, APEX1 and XRCC1 and a proliferation marker, Ki-67, were examined by immunohistochemical analysis, in a cohort of 50 HNSCC patients. Significant downregulation of OGG1 (p<0.04) and XRCC1 (p<0.05) was observed in poorly differentiated HNSCC compared to mod-well-differentiated cases. Significant upregulation of APEX1 (p<0.05) and Ki-67 (p<0.05) was observed in poorly differentiated HNSCC compared to mod-well-differentiated cases. Significant correlation was observed between XRCC1 and OGG1 (r=0.33, p<0.02). Inverse correlations were observed between OGG1 and Ki-67 (r=-0.377, p<0.005), between APEX1 and XRCC1 (r=-0.435, p<0.002) and between OGG1 and APEX1 (r=-0.34, p<0.02) in HNSCC. To confirm our observations, we examined BER pathway genes and a proliferation marker, Ki-67, expression at the mRNA level on 50 head and neck squamous cell carcinoma (HNSCC) and 50 normal control samples by quantitative real-time polymerase chain reaction. Significant downregulation was observed in case of OGG1 (p<0.04) and XRCC1 (p<0.02), while significant upregulation was observed in case of APEX1 (p<0.01) and Ki-67 (p<0.03) in HNSCC tissue samples compared to controls. Our data suggested that deregulation of base excision repair pathway genes, such as OGG1, APEX1 and XRCC1, combined with overexpression of Ki-67, a marker for excessive proliferation, may contribute to progression of HNSCC in Pakistani population.

Signal-on electrochemical immunoassay for APE1 using ionic liquid doped Au nanoparticle/graphene as a nanocarrier and alkaline phosphatase as enhancer.

In this paper, the Au nanoparticles decorated graphene nanosheets (AuNPs/Gr) were prepared as nanocarriers using ionic liquid (IL) as linker reagent. Then the alkaline phosphatase (ALP) and the ferrocene tagged detection antibodies (Fc-Ab2) were loaded on the IL doped AuNPs/Gr as a trace label for ultrasensitive measurements of human apurinic/apyrimidinic endonuclease 1 (APE1), which is a multifunctional protein in the DNA base excision repair pathway relating to various types of cancer. Several labeling protocols were investigated for the determination of the APE1 protein concentration and improved analytical features were obtained with the proposed carriers of IL doped AuNPs/Gr which were labeled with Fc-Ab2 and ALP (ALP/Fc-Ab2/AuNPs/IL/Gr). The reason may be that the IL doped AuNPs/Gr carriers (AuNPs/IL/Gr) could not only enhance the immobilized amount of ALP and Fc-Ab2, but also promote the electron transfer rate. Thus, through the specific recognition of antigen-antibody, numerous ALP/Fc-Ab2/AuNPs/IL/Gr, which are captured onto every single immunocomplex, could further catalyze the ascorbic acid 2-phosphate (AA-p) reaction to amplify the electrochemical signal. Transmission electron microscopy (TEM) images of the AuNPs/IL/Gr nanocomposites revealed the formation of a functionalized surface network structure. The resulting immunosensor exhibited a linear response to APE1 in the concentration range of 0.1-80 pg mL(-1) with a detection limit of 0.04 pg mL(-1), indicating potential applications in clinical diagnostics.

In Vitro Assay to Measure APE1 Enzymatic Activity on Ribose Monophosphate Abasic Site.

APE1 (apurinic/apyrimidinic endodeoxyribonuclease 1) is a central enzyme of the base excision repair (BER) pathway playing a pivotal role in protecting mammalian cells against genotoxins and in safeguarding genome stability. Recently, we demonstrated the APE1 ability to process abasic ribonucleotides embedded in DNA. Here, we provide a pipeline of protocols to quantify endodeoxyribonuclease activity by APE1 on these substrates, by using recombinant protein and whole-cell extracts. The repair capacity is measured by using fluorescent oligonucleotide substrates, which are then separated by polyacrylamide gel electrophoresis and detected by imaging scanning. The specificity of APE1 action is demonstrated using specific APE1 enzymatic inhibitors.

Progress in high-throughput assays of MGMT and APE1 activities in cell extracts.

DNA repair activity is of interest as a potential biomarker of individual susceptibility to genotoxic agents. In view of the current trend for exploitation of large cohorts in molecular epidemiology projects, there is a pressing need for the development of phenotypic DNA repair assays that are high-throughput, very sensitive, inexpensive and reliable. Towards this goal we have developed and validated two phenotypic assays for the measurement of two DNA repair enzymes in cell extracts: (1) O(6)-methylguanine-DNA-methyltransferase (MGMT), which repairs the O(6)-alkylguanine-type of adducts induced in DNA by alkylating genotoxins; and (2) apurinic/apyrimidinic endonuclease 1 (APE 1), which participates in base excision repair (BER) by causing a rate-limiting DNA strand cleavage 5' to the abasic sites. The MGMT assay makes use of the fact that: (a) the enzyme works by irreversibly transferring the alkyl group from the O(6) position of guanine to a cystein residue in its active site and thereby becomes inactivated and (b) that the free base O(6)-benzylguanine (BG) is a very good substrate for MGMT. In the new assay, cell extracts are incubated with BG tagged with biotin and the resulting MGMT-BG-biotin complex is immobilized on anti-MGMT-coated microtiter plates, followed by quantitation using streptavidin-conjugated alkaline phosphatase and a chemiluminescence-producing substrate. A one-step/one-tube phenotypic assay for APE1 activity has been developed based on the use of a fluorescent molecular beacon (partially self-complementary oligonucleotide with a hairpin-loop structure carrying a fluorophore and a quencher at each end). It also contains a single tetrahydrofuran residue (THF) which is recognized and cleaved by APE1, and the subsequently formed single-stranded oligomer becomes a fluorescence signal emitter. Both assays are highly sensitive, require very small amounts of protein extracts, are relatively inexpensive and can be easily automated. They have been extensively validated and are being used in the context of large-scale molecular epidemiology studies.

Oxidative stress triggers the preferential assembly of base excision repair complexes on open chromatin regions.

How DNA repair machineries detect and access, within the context of chromatin, lesions inducing little or no distortion of the DNA structure is a poorly understood process. Removal of oxidized bases is initiated by a DNA glycosylase that recognises and excises the damaged base, initiating the base excision repair (BER) pathway. We show that upon induction of 8-oxoguanine, a mutagenic product of guanine oxidation, the mammalian 8-oxoguanine DNA glycosylase OGG1 is recruited together with other proteins involved in BER to euchromatin regions rich in RNA and RNA polymerase II and completely excluded from heterochromatin. The underlying mechanism does not require direct interaction of the protein with the oxidized base, however, the release of the protein from the chromatin fraction requires completion of repair. Inducing chromatin compaction by sucrose results in a complete but reversible inhibition of the in vivo repair of 8-oxoguanine. We conclude that after induction of oxidative DNA damage, the DNA glycosylase is actively recruited to regions of open chromatin allowing the access of the BER machinery to the lesions, suggesting preferential repair of active chromosome regions.

Regulation of base excision repair: Ntg1 nuclear and mitochondrial dynamic localization in response to genotoxic stress.

Numerous human pathologies result from unrepaired oxidative DNA damage. Base excision repair (BER) is responsible for the repair of oxidative DNA damage that occurs in both nuclei and mitochondria. Despite the importance of BER in maintaining genomic stability, knowledge concerning the regulation of this evolutionarily conserved repair pathway is almost nonexistent. The Saccharomyces cerevisiae BER protein, Ntg1, relocalizes to organelles containing elevated oxidative DNA damage, indicating a novel mechanism of regulation for BER. We propose that dynamic localization of BER proteins is modulated by constituents of stress response pathways. In an effort to mechanistically define these regulatory components, the elements necessary for nuclear and mitochondrial localization of Ntg1 were identified, including a bipartite classical nuclear localization signal, a mitochondrial matrix targeting sequence and the classical nuclear protein import machinery. Our results define a major regulatory system for BER which when compromised, confers a mutator phenotype and sensitizes cells to the cytotoxic effects of DNA damage.

SIRT1 deacetylates APE1 and regulates cellular base excision repair.

Apurinic/apyrimidinic endonuclease-1 (APE1) is an essential enzyme in the base excision repair (BER) pathway. Here, we show that APE1 is a target of the SIRTUIN1 (SIRT1) protein deacetylase. SIRT1 associates with APE1, and this association is increased with genotoxic stress. SIRT1 deacetylates APE1 in vitro and in vivo targeting lysines 6 and 7. Genotoxic insults stimulate lysine acetylation of APE1 which is antagonized by transcriptional upregulation of SIRT1. Knockdown of SIRT1 increases cellular abasic DNA content, sensitizing cells to death induced by genotoxic stress, and this vulnerability is rescued by overexpression of APE1. Activation of SIRT1 with resveratrol promotes binding of APE1 to the BER protein X-ray cross-complementing-1 (XRCC1), while inhibition of SIRT1 with nicotinamide (NAM) decreases this interaction. Genotoxic insult also increases binding of APE1 to XRCC1, and this increase is suppressed by NAM or knockdown of SIRT1. Finally, resveratrol increases APE activity in XRCC1-associated protein complexes, while NAM or knockdown of SIRT1 suppresses this DNA repair activity. These findings identify APE1 as a novel protein target of SIRT1, and suggest that SIRT1 plays a vital role in maintaining genomic integrity through regulation of the BER pathway.

Role of PCNA-dependent stimulation of 3'-phosphodiesterase and 3'-5' exonuclease activities of human Ape2 in repair of oxidative DNA damage.

Human Ape2 protein has 3' phosphodiesterase activity for processing 3'-damaged DNA termini, 3'-5' exonuclease activity that supports removal of mismatched nucleotides from the 3'-end of DNA, and a somewhat weak AP-endonuclease activity. However, very little is known about the role of Ape2 in DNA repair processes. Here, we examine the effect of interaction of Ape2 with proliferating cell nuclear antigen (PCNA) on its enzymatic activities and on targeting Ape2 to oxidative DNA lesions. We show that PCNA strongly stimulates the 3'-5' exonuclease and 3' phosphodiesterase activities of Ape2, but has no effect on its AP-endonuclease activity. Moreover, we find that upon hydrogen-peroxide treatment Ape2 redistributes to nuclear foci where it colocalizes with PCNA. In concert with these results, we provide biochemical evidence that Ape2 can reduce the mutagenic consequences of attack by reactive oxygen species not only by repairing 3'-damaged termini but also by removing 3'-end adenine opposite from 8-oxoG. Based on these findings we suggest the involvement of Ape2 in repair of oxidative DNA damage and PCNA-dependent repair synthesis.

Coupling APEX labeling to imaging mass spectrometry of single organelles reveals heterogeneity in lysosomal protein turnover.

Quantification of stable isotope tracers after metabolic labeling provides a snapshot of the dynamic state of living cells and tissue. A form of imaging mass spectrometry quantifies isotope ratios with a lateral resolution <50 nm, using a methodology that we refer to as multi-isotope imaging mass spectrometry (MIMS). Despite lateral resolution exceeding diffraction-limited light microscopy, lack of contrast has largely limited use of MIMS to large or specialized subcellular structures, such as the nucleus and stereocilia. In this study, we repurpose the engineered peroxidase APEX2 as the first genetically encoded marker for MIMS. Coupling APEX2 labeling of lysosomes and metabolic labeling of protein, we identify that individual lysosomes exhibit substantial heterogeneity in protein age, which is lost in iPSC-derived neurons lacking the lysosomal protein progranulin. This study expands the practical use of MIMS for cell biology by enabling measurements of metabolic function from stable isotope labeling within individual organelles in situ.