Glycosylation is a prevalent, yet heterogeneous modification with a broad range of implications in molecular biology. This heterogeneity precludes enrichment strategies that can be universally ...beneficial for all glycan classes. Thus, choice of enrichment strategy has profound implications on experimental outcomes. Here we review common enrichment strategies used in modern mass spectrometry–based glycoproteomic experiments, including lectins and other affinity chromatographies, hydrophilic interaction chromatography and its derivatives, porous graphitic carbon, reversible and irreversible chemical coupling strategies, and chemical biology tools that often leverage bioorthogonal handles. Interest in glycoproteomics continues to surge as mass spectrometry instrumentation and software improve, so this review aims to help equip researchers with the necessary information to choose appropriate enrichment strategies that best complement these efforts.
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•Glycosylation is complex and often requires enrichment prior to analysis•Review of common enrichment strategies for mass spectrometry–based glycoproteomics•Enrichment methods have practical considerations and experimental implications•Appropriate enrichment strategies will complement developments in mass spectrometry
Interest in mass spectrometry–based glycoproteomics analysis is increasing because of recent advances in instrumentation and data analysis tools. Such studies can provide a wealth of information across a wide spectrum of glycan classes and biological systems. However, many studies require the choice of an enrichment strategy for glycosylated species prior to analysis to obtain the maximum amount of analytical information. Here, common enrichment strategies are reviewed with strengths and weaknesses, and the practical considerations for various methods are discussed.
Systematic searches for plasma proteins that are biological indicators, or biomarkers, for cancer are underway. The difficulties caused by the complexity of biological-fluid proteomes and tissue ...proteomes (which contribute proteins to plasma) and by the extensive heterogeneity among diseases, subjects and levels of sample procurement are gradually being overcome. This is being achieved through rigorous experimental design and in-depth quantitative studies. The expected outcome is the development of panels of biomarkers that will allow early detection of cancer and prediction of the probable response to therapy. Achieving these objectives requires high-quality specimens with well-matched controls, reagent resources, and an efficient process to confirm discoveries through independent validation studies.
MicroRNAs are critical regulators of cancer initiation, progression, and dissemination. Extensive evidence suggests that the inhibition of over-expressed oncogenic miRNA function can be a robust ...strategy for anticancer therapy. However, in vivo targeted delivery of miRNA therapeutics to various types of cancers remains a major challenge. Inspired by their natural synthesis and cargo delivery capabilities, researchers have exploited tumor cell-derived extracellular vesicles (TEVs) for the cancer-targeted delivery of therapeutics and theranostics. Here, we investigate a TEV-based nanoplatform for multimodal miRNA delivery and phototherapy treatments as well as the magnetic resonance imaging of cancer. We demonstrated loading of anti-miR-21 that blocks the function of endogenous oncogenic miR-21 over-expressed in cancer cells into and subsequent delivery by TEVs derived from 4T1 cells. We also produced Cy5-anti-miR-21-loaded TEVs from two other cancer cell lines (HepG2 and SKBR3) and confirmed their robust homologous and heterologous transfection efficiency and intracellular Cy5-anti-miR-21 delivery. Additionally, TEV-mediated anti-miR-21 delivery attenuated doxorubicin (DOX) resistance in breast cancer cells with a 3-fold higher cell kill efficiency than in cells treated with DOX alone. We then investigated TEVs as a biomimetic source for the functionalization of gold–iron oxide nanoparticles (GIONs) and demonstrated nanotheranostic properties of TEV-GIONs in vitro. TEV-GIONs demonstrated excellent T2 contrast in in vitro magnetic resonance (MR) imaging and resulted in efficient photothermal effect in 4T1 cells. We also evaluated the biodistribution and theranostic property of anti-miR-21 loaded TEV-GIONs in vivo by labeling with indocyanine green near-infrared dye. We further validated the tumor specific accumulation of TEV-GIONs using MR imaging. Our findings demonstrate that the distribution pattern of the TEV-anti-miR-21-GIONs correlated well with the tumor-targeting capability as well as the activity and efficacy obtained in response to doxorubicin combination treatments. TEVs and TEV-GIONs are promising nanotheranostics for future applications in cancer molecular imaging and therapy.
Chromatin looping is key to gene regulation, yet no broadly applicable methods to selectively modify chromatin loops have been described. We have engineered a method for chromatin loop reorganization ...using CRISPR-dCas9 (CLOuD9) to selectively and reversibly establish chromatin loops. We demonstrate the power of this technology to selectively modulate gene expression at targeted loci.
Staphylococcus aureus (S. aureus) is a highly pathogenic facultative anaerobe that in some instances resides as an intracellular bacterium within macrophages and cancer cells. This pathogen can ...establish secondary infection foci, resulting in recurrent systemic infections that are difficult to treat using systemic antibiotics. Here, we use reconstructed apoptotic bodies (ReApoBds) derived from cancer cells as “nano decoys” to deliver vancomycin intracellularly to kill S. aureus by targeting inherent “eat me” signaling of ApoBds. We prepared ReApoBds from different cancer cells (SKBR3, MDA-MB-231, HepG2, U87-MG, and LN229) and used them for vancomycin delivery. Physicochemical characterization showed ReApoBds size ranges from 80 to 150 nm and vancomycin encapsulation efficiency of 60 ± 2.56%. We demonstrate that the loaded vancomycin was able to kill intracellular S. aureus efficiently in an in vitro model of S. aureus infected RAW-264.7 macrophage cells, and U87-MG (p53-wt) and LN229 (p53-mt) cancer cells, compared to free-vancomycin treatment (P < 0.001). The vancomycin loaded ReApoBds treatment in S. aureus infected macrophages showed a two-log-order higher CFU reduction than the free-vancomycin treatment group. In vivo studies revealed that ReApoBds can specifically target macrophages and cancer cells. Vancomycin loaded ReApoBds have the potential to kill intracellular S. aureus infection in vivo in macrophages and cancer cells.
We investigated the potential of in-depth quantitative proteomics to reveal plasma protein signatures that reflect lung tumor biology. We compared plasma protein profiles of four mouse models of lung ...cancer with profiles of models of pancreatic, ovarian, colon, prostate, and breast cancer and two models of inflammation. A protein signature for Titf1/Nkx2-1, a known lineage-survival oncogene in lung cancer, was found in plasmas of mouse models of lung adenocarcinoma. An EGFR signature was found in plasma of an EGFR mutant model, and a distinct plasma signature related to neuroendocrine development was uncovered in the small-cell lung cancer model. We demonstrate relevance to human lung cancer of the protein signatures identified on the basis of mouse models.
► Organ-type specific protein signatures found in plasmas of mouse models of cancer ► Plasma protein signatures reflect molecularly distinct subtypes of lung cancer ► Networks of plasma proteins inform about genes that drive tumor development ► Concordant findings in human lung cancer blood samples based on ELISAs
Circulating tumor-derived extracellular vesicles (EVs) have emerged as a promising source for identifying cancer biomarkers for early cancer detection. However, the clinical utility of EVs has thus ...far been limited by the fact that most EV isolation methods are tedious, nonstandardized, and require bulky instrumentation such as ultracentrifugation (UC). Here, we report a size-based EV isolation tool called ExoTIC (exosome total isolation chip), which is simple, easy-to-use, modular, and facilitates high-yield and high-purity EV isolation from biofluids. ExoTIC achieves an EV yield ∼4–1000-fold higher than that with UC, and EV-derived protein and microRNA levels are well-correlated between the two methods. Moreover, we demonstrate that ExoTIC is a modular platform that can sort a heterogeneous population of cancer cell line EVs based on size. Further, we utilize ExoTIC to isolate EVs from cancer patient clinical samples, including plasma, urine, and lavage, demonstrating the device’s broad applicability to cancers and other diseases. Finally, the ability of ExoTIC to efficiently isolate EVs from small sample volumes opens up avenues for preclinical studies in small animal tumor models and for point-of-care EV-based clinical testing from fingerprick quantities (10–100 μL) of blood.
Post-translational modifications (PTMs) on proteins often function to regulate signaling cascades, with the activation of T cells during an adaptive immune response being a classic example. Mounting ...evidence indicates that the modification of proteins by O-linked N-acetylglucosamine (O-Glcnac), the only mammalian glycan found on nuclear and cytoplasmic proteins, helps regulate T cell activation. Yet, a mechanistic understanding of how O-Glcnac functions in T cell activation remains elusive, partly because of the difficulties in mapping and quantifying O-Glcnac sites. Thus, to advance insight into the role of O-Glcnac in T cell activation, we performed glycosite mapping studies via direct glycopeptide measurement on resting and activated primary human T cells with a technique termed Isotope Targeted Glycoproteomics. This approach led to the identification of 2219 intact O-linked glycopeptides across 1045 glycoproteins. A significant proportion (>45%) of the identified O-Glcnac sites lie near or coincide with a known phosphorylation site, supporting the potential for PTM crosstalk. Consistent with other studies, we find that O-Glcnac sites in T cells lack a strict consensus sequence. To validate our results, we employed gel shift assays based on conjugating mass tags to O-Glcnac groups. Notably, we observed that the transcription factors c-JUN and JUNB show higher levels of O-Glcnac glycosylation and higher levels of expression in activated T cells. Overall, our findings provide a quantitative characterization of O-Glcnac glycoproteins and their corresponding modification sites in primary human T cells, which will facilitate mechanistic studies into the function of O-Glcnac in T cell activation.
Protein glycosylation is of increasing interest due to its important roles in protein function and aberrant expression with disease. Characterizing protein glycosylation remains analytically ...challenging due to its low abundance, ion suppression issues, and microheterogeneity at glycosylation sites, especially in complex samples such as human plasma. In this study, the utility of three common N-linked glycopeptide enrichment techniques is compared using human plasma. By analysis on an LTQ-Orbitrap Elite mass spectrometer, electrostatic repulsion hydrophilic interaction liquid chromatography using strong anion exchange solid-phase extraction (SAX-ERLIC) provided the most extensive N-linked glycopeptide enrichment when compared with multilectin affinity chromatography (M-LAC) and Sepharose-HILIC enrichments. SAX-ERLIC enrichment yielded 191 unique glycoforms across 72 glycosylation sites from 48 glycoproteins, which is more than double that detected using other enrichment techniques. The greatest glycoform diversity was observed in SAX-ERLIC enrichment, with no apparent bias toward specific glycan types. SAX-ERLIC enrichments were additionally analyzed by an Orbitrap Fusion Lumos mass spectrometer to maximize glycopeptide identifications for a more comprehensive assessment of protein glycosylation. In these experiments, 829 unique glycoforms were identified across 208 glycosylation sites from 95 plasma glycoproteins, a significant improvement from the initial method comparison and one of the most extensive site-specific glycosylation analysis in immunodepleted human plasma to date. Data are available via ProteomeXchange with identifier PXD005655.
Abstract
Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer that rarely develops de novo in primary tumors and is commonly acquired during the development of treatment ...resistance. NEPC is characterized by gain of neuroendocrine markers and loss of androgen receptor (AR), making it resistant to current therapeutic strategies targeting the AR signaling axis. Here, we report that MCM2, MCM3, MCM4, and MCM6 (MCM2/3/4/6) are elevated in human NEPC and high levels of MCM2/3/4/6 are associated with liver metastasis and poor survival in prostate cancer patients. MCM2/3/4/6 are four out of six proteins that form a core DNA helicase (MCM2-7) responsible for unwinding DNA forks during DNA replication. Inhibition of MCM2-7 by treatment with ciprofloxacin inhibits NEPC cell proliferation and migration in vitro, significantly delays NEPC tumor xenograft growth, and partially reverses the neuroendocrine phenotype in vivo. Our study reveals the clinical relevance of MCM2/3/4/6 proteins in NEPC and suggests that inhibition of MCM2-7 may represent a new therapeutic strategy for NEPC.
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