Antibody-free approach for ubiquitination profiling by selectively clicking the ubiquitination sites.

Ubiquitination is a reversible post-translational modification that plays a pivotal role in numerous biological processes. Antibody-based approaches, as the most used methods for identifying ubiquitination sites, exist sequence recognition bias, high cost, and ubiquitin-like protein modification interference, limiting their widespread application. Here, we proposed an Antibody-Free approach for Ubiquitination Profiling, termed AFUP, by selectively clicking the ubiquitinated lysine to enrich and profile endogenous ubiquitinated peptides using mass spectrometry. Briefly, protein amines were blocked with formaldehyde, and then the ubiquitin molecules were hydrolyzed from the ubiquitinated proteins by non-specific deubiquitinases USP2 and USP21 to release the free ε-amine of lysine. Peptides containing free ε-amines were selectively enriched with streptavidin beads upon NHS-SS-biotin labeling. Finally, the enriched peptides were eluted by DTT and analyzed by LC-MS/MS, resulting in ubiquitination profiling. Preliminary experiment showed that 349 ± 7 ubiquitination sites were identified in 0.8 mg HeLa lysates with excellent reproducibility (CV = 2%) and high quantitative stability (Pearson, r ≥ 0.91) using our method. With the combination of AFUP and simple basic C18 pre-fractionation, approximately 4000 ubiquitination sites were identified in a single run of 293T cells. In addition, we showed that 209 ubiquitination sites were significantly regulated in UBE2O knockdown cells after normalized to protein abundance. In conclusion, our results demonstrated that AFUP is a robust alternative strategy for ubiquitomics research.

PrUb-EL: A hybrid framework based on deep learning for identifying ubiquitination sites in Arabidopsis thaliana using ensemble learning strategy.

Identification of ubiquitination sites is central to many biological experiments. Ubiquitination is a kind of post-translational protein modification (PTM). It is a key mechanism for increasing protein diversity and plays a vital role in regulating cell function. In recent years, many models have been developed to predict ubiquitination sites in humans, mice and yeast. However, few studies have predicted ubiquitination sites in Arabidopsis thaliana. In view of this, a deep network model named PrUb-EL is proposed to predict ubiquitination sites in Arabidopsis thaliana. Firstly, six features based on the protein sequence are extracted with amino acid index database (AAindex), dipeptide deviates from the expected mean (DDE), dipeptide composition (DPC), blocks substitution matrix (BLOSUM62), enhanced amino acid composition (EAAC) and binary encoding. Secondly, the synthetic minority over-sampling technique (SMOTE) is utilized to process the imbalanced data set. Then a new classifier named DG is presented, which includes Dense block, Residual block and Gated recurrent unit (GRU) block. Finally, each of six feature extraction methods is integrated into the DG model, and the ensemble learning strategy is used to gain the final prediction result. Experimental results show that PrUb-EL has good predictive ability with the accuracy (ACC) and area under the ROC curve (auROC) values of 91.00% and 97.70% using 5-fold cross-validation, respectively. Note that the values of ACC and auROC are 88.58% and 96.09% in the independent test, respectively. Compared with previous studies, our model has significantly improved performance thus it is an excellent method for identifying ubiquitination sites in Arabidopsis thaliana. The datasets and code used for the article are available at https://github.com/Tom-Wangy/PreUb-EL.git.

Construction of nano receptors for ubiquitin and ubiquitinated proteins based on the region-specific interactions between ubiquitin and polydopamine.

Ubiquitination is a prevalent post-translational modification that controls a multitude of important biological processes. Due to the low abundance of ubiquitinated proteins, highly efficient separation and enrichment approaches are required for ubiquitinome analysis. In this work, we disclose the region-specific interactions between the hydrophobic patch of ubiquitin and polydopamine. Taking advantage of this inherent binding property, we have constructed surface-imprinted magnetic nanoparticles (NPs) for ubiquitin by sequential dopamine polymerization and surface PEGylation. The obtained molecularly imprinted polymer (MIP) NPs showed a binding constant of 2.6 × 106 L mol-1 for the template ubiquitin. The bound ubiquitin could be quantitatively released by heating to 70 °C at pH 2.0 or 90 °C at neutral (pH 7.0) conditions. The MIP NPs exhibited nano receptor-like property which not only effectively blocked the formation of branched ubiquitin chains but also selectively separated ubiquitin from the bacterial cell lysates. By incubating the MIP NPs with the lysates of 293T cells, totally 529 ubiquitinated proteins were captured, among which 287 proteins were not identified by the anti-ubiquitin monoclonal antibodies (mAbs). With the distinct merits of low cost and high stability, the as-prepared MIP NPs may be utilized either separately or as an important complement to the mAbs for the purification and enrichment of ubiquitin and ubiquitinated proteins from complex biological samples. Furthermore, due to the flexibility in modification of the binding sites during or after the imprinting reactions, the results of this work also paved the way for generation of artificial receptors for branched ubiquitin chains and polyubiquitinated proteins with higher avidity and specificity.

Ubiquitination of G3BP1 mediates stress granule disassembly in a context-specific manner.

Stress granules are dynamic, reversible condensates composed of RNA and protein that assemble in eukaryotic cells in response to a variety of stressors and are normally disassembled after stress is removed. The composition and assembly of stress granules is well understood, but little is known about the mechanisms that govern disassembly. Impaired disassembly has been implicated in some diseases including amyotrophic lateral sclerosis, frontotemporal dementia, and multisystem proteinopathy. Using cultured human cells, we found that stress granule disassembly was context-dependent: Specifically in the setting of heat shock, disassembly required ubiquitination of G3BP1, the central protein within the stress granule RNA-protein network. We found that ubiquitinated G3BP1 interacted with the endoplasmic reticulum­associated protein FAF2, which engaged the ubiquitin-dependent segregase p97/VCP (valosin-containing protein). Thus, targeting of G3BP1 weakened the stress granule­specific interaction network, resulting in granule disassembly.

The 20S as a stand-alone proteasome in cells can degrade the ubiquitin tag.

The proteasome, the primary protease for ubiquitin-dependent proteolysis in eukaryotes, is usually found as a mixture of 30S, 26S, and 20S complexes. These complexes have common catalytic sites, which makes it challenging to determine their distinctive roles in intracellular proteolysis. Here, we chemically synthesize a panel of homogenous ubiquitinated proteins, and use them to compare 20S and 26S proteasomes with respect to substrate selection and peptide-product generation. We show that 20S proteasomes can degrade the ubiquitin tag along with the conjugated substrate. Ubiquitin remnants on branched peptide products identified by LC-MS/MS, and flexibility in the 20S gate observed by cryo-EM, reflect the ability of the 20S proteasome to proteolyze an isopeptide-linked ubiquitin-conjugate. Peptidomics identifies proteasome-trapped ubiquitin-derived peptides and peptides of potential 20S substrates in Hi20S cells, hypoxic cells, and human failing-heart. Moreover, elevated levels of 20S proteasomes appear to contribute to cell survival under stress associated with damaged proteins.

Monitoring protein ubiquitination and SUMOylation in real-time by NMR.

The covalent conjugation of ubiquitin (Ub), known as ubiquitination, is a multi-step reaction involving multiple enzymes. We report a real-time, tag-free method to monitor protein ubiquitination by NMR spectroscopy under physiological conditions. The approach is also applicable for monitoring other ubiquitin-like modifications, and the disassembly of Ub polymers.

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry.

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine ('K-ε-diglycine' or simply 'diGly') can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

Facile Semisynthesis of Ubiquitylated Peptides with the Ligation Auxiliary 2-Aminooxyethanethiol.

The posttranslational modification of cellular proteins by ubiquitin (Ub), called ubiquitylation, is indispensable for the normal growth and development of eukaryotic organisms. In order to conduct studies that elucidate the precise mechanistic roles for Ub, access to site-specifically and homogenously ubiquitylated proteins and peptides is critical. However, the low abundance, heterogeneity, and dynamic nature of protein ubiquitylation are significant limitations toward such studies. Here we provide a facile expressed protein ligation method that does not require specialized apparatus and permits the rapid semisynthesis of ubiquitylated peptides by using the atom-efficient ligation auxiliary 2-aminooxyethanethiol.

Anti-Tumor Efficacy of an Adjuvant Built-In Nanovaccine Based on Ubiquitinated Proteins from Tumor Cells.

BACKGROUND AND AIM: We have previously identified ubiquitinated proteins (UPs) from tumor cell lysates as a promising vaccine for cancer immunotherapy in different mouse tumor models. In this study, we aimed at developing a highly efficient therapeutic adjuvant built-in nanovaccine (α-Al2O3-UPs) by a simple method, in which UPs from tumor cells could be efficiently and conveniently enriched by α-Al2O3 nanoparticles covalently coupled with Vx3 proteins (α-Al2O3-CONH-Vx3). METHODS: The α-Al2O3 nanoparticles were modified with 4-hydroxybenzoic acid followed by coupling with ubiquitin-binding protein Vx3. It was then used to enrich UPs from 4T1 cell lysate. The stability and the efficiency for the UPs enrichment of α-Al2O3-CONH-Vx3 were examined. The ability of α-Al2O3-UPs to activate DCs was examined in vitro subsequently. The splenocytes from the vaccinated mice were re-stimulated with inactivated tumor cells, and the IFN-γ secretion was detected by ELISA and flow cytometry. Moreover, the therapeutic efficacy of α-Al2O3-UPs, alone and in combination with chemotherapy, was examined in 4T1 tumor-bearing mice. RESULTS: Our results showed that α-Al2O3-UPs were successfully synthesized and abundant UPs from tumor cell lysate were enriched by the new method. In vitro study showed that compared to the physical mixture of α-Al2O3 nanoparticles and UPs (α-Al2O3+UPs), α-Al2O3-UPs stimulation resulted in higher upregulations of CD80, CD86, MHC class I, and MHC class II on DCs, indicating the higher ability of DC activation. Moreover, α-Al2O3-UPs elicited a more effective immune response in mice, demonstrated by higher IFN-γ secretion than α-Al2O3+UPs. Furthermore, α-Al2O3-UPs also exhibited a more potent effect on tumor growth inhibition and survival prolongation in 4T1 tumor-bearing mice. Notably, when in combination with low dose chemotherapy, the anti-tumor effect was further enhanced, rather than using α-Al2O3-UPs alone. CONCLUSION: This study presents an adjuvant built-in nanovaccine generated by a new simple method that can be potentially applied to cancer immunotherapy and lays the experimental foundation for future clinical application.

Preparation and purification of mono-ubiquitinated proteins using Avi-tagged ubiquitin.

Site-specific conjugation of ubiquitin onto a range of DNA repair proteins regulates their critical functions in the DNA damage response. Biochemical and structural characterization of these functions are limited by an absence of tools for the purification of DNA repair proteins in purely the ubiquitinated form. To overcome this barrier, we designed a ubiquitin fusion protein that is N-terminally biotinylated and can be conjugated by E3 RING ligases onto various substrates. Biotin affinity purification of modified proteins, followed by cleavage of the affinity tag leads to release of natively-mono-ubiquitinated substrates. As proof-of-principle, we applied this method to several substrates of mono-ubiquitination in the Fanconi anemia (FA)-BRCA pathway of DNA interstrand crosslink repair. These include the FANCI:FANCD2 complex, the PCNA trimer and BRCA1 modified nucleosomes. This method provides a simple approach to study the role of mono-ubiquitination in DNA repair or any other mono-ubiquitination signaling pathways.

Lysosomal integral membrane protein LGP85 (LIMP-2) is ubiquitinated at the N-terminal cytoplasmic domain.

LGP85/LIMP-2 is a type III transmembrane glycoprotein of lysosomes, which traverses the membrane twice with an N-terminal uncleaved signal sequence and C-terminal hydrophobic domain. In addition to functioning as a receptor for a lysosomal enzyme ß-glucocerebrosidase and for several enteroviruses, LGP85 plays a key role in the biogenesis and maintenance of endosomal/lysosomal compartments (ELCs). Our previous studies have demonstrated that overexpression of rat LGP85 into COS cells results in the enlarged ELCs, from where membrane trafficking is impaired. We show here that rat LGP85 is polyubiquitinated at the N-terminal short cytoplasmic domain that comprises of only three amino acid residues, alanine, arginine, and cysteine. Replacement of either arginine or cysteine with alanine within the N-terminal cytoplasmic domain did not influence the ubiquitination of LGP85, thereby indicating that ubiquitin (Ub) is conjugated to the α-NH2 group of the N-terminal alanine residue. Furthermore, we were able to define a domain necessary for ubiquitination in a region ranging from the amino acids 156 to 255 within the lumenal domain of LGP85. This is the first report showing that the integral lysosomal membrane protein LGP85 is ubiquitinated.

Analysis of the topology of ubiquitin chains.

The small protein ubiquitin and its multiple polymers are encountered free in cells and as post-translational modifications on all proteins. Different polyubiquitin three dimensional structures are shown to correlate uniquely with different cellular functions as part of the diverse ubiquitin signaling. At the same time, this multiplicity of structures provides serious challenges to the analytical biochemist. Globally applicable strategies are presented here for the analyses of polyubiquitins and of ubiquitinated proteins, which take advantage of the speed, specificity and sensitivity of top-down tandem mass spectrometry. Particular attention is given to the supervised interpretation of fragmentation as revealed in the MS/MS spectra of these branched proteins. The strategy is compatible with any MS activation technology, is applicable to all polyubiquitin linkage and chain types, can be extended to ubiquitin-like proteins, and will be compatible with and enhanced by continuing advances in LC-MS/MS instrumentation and interpretation software.

[A biosensor study of protein interaction with the 20S proteasome core particle]. / Biosensornoe issledovanie vzaimodeistviia belkov s korovoi chast'iu proteasomy.

It becomes increasingly clear that ubiquitination of cellular proteins is not an indispensable prerequisite of their degradation in proteasomes. There are a number of proteins to be eliminated which are not pre-ubiquitinated for their recognition by regulatory subcomplex of 26S proteasome, but which directly interact with the 20S proteasome core particle (20S proteasome). The obligatory precondition for such interaction consists in existence of disordered (hydrophobic) fragments in the target protein. In this study we have investigated the interaction of a number of multifunctional (moonlighting) proteins (glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldolase, pyruvate kinase) and neurodegeneration-related proteins (a-synuclein, myelin basic protein) with 20S proteasome immobilized on the SPR-biosensor chip and stabilized by means of a bifunctional agent dimethyl pimelimidate (in order to prevent possible dissociation of this subcomplex). Only two of all investigated proteins (aldolase and pyruvate kinase) interacted with the immobilized 20S proteasome (Kd of 8.17´10-7 M and 5.56´10-7 M, respectively). In addition to earlier detected GAPDH ubiquitination, mass spectrometric analysis of the studied proteins revealed the presence of the ubiquitin signature (Lys-e-Gly-Gly) only in aldolase. Oxidation of aldolase and pyruvate kinase, which promotes elimination of proteins via their direct interaction with 20S proteasome, caused a 2-3-fold decrease in their Kd values as comparison with this parameter obtained for the intact proteins. The results of this study provide further evidence for direct interaction of both ubiquitinated proteins (aldolase), and non-ubiquitinated proteins (pyruvate kinase) with the 20S proteasome core particle (20S proteasome). The effectiveness of this interaction is basically equal for the ubiquitinated proteins and non-ubiquitinated proteins.

A Combined Chemical Derivatization/Mass Spectrometric Method for the Enhanced Detection and Relative Quantification of Protein Ubiquitination.

Mass spectrometry (MS) is a sensitive analytical technique with wide application across the sciences including for the detection of peptides and proteins in biological analysis. Ubiquitinated (Ub) proteins are typically analyzed by proteolytic digestion and subsequent chromatographic separation followed by MS detection of the resulting isopeptides. Here we describe a novel method which enables enhanced detection of this important posttranslational modification (PTM) by use of a simple chemical labeling strategy prior to Data-Independent Acquisition (DIA) using a SWATH-based acquisition approach on a suitable Quadrupole-Time-Of-Flight (Q-TOF) mass spectrometer.

Sequence and structure-based characterization of ubiquitination sites in human and yeast proteins using Chou's sample formulation.

Ubiquitination is an important post-translational event responsible for half-life and turnover of proteins inside the cell. Proteins are ubiquitinated by forming an iso-peptide bond between their lysine residue and C-terminal glycine residue of ubiquitin leading to rapid degradation of proteins by 26S proteosome complex. Deregulation of ubiquitination is manifested by aberrant expression of E3-ligase activity or mutation in the surroundings of ubiquitination sites. Many new experimentally validated ubiquitinated lysines have been recently identified that motivated the study of the environments surrounding the ubiquitinated lysines. With the help of known ubiquitinated proteins, here we present a comprehensive study of sequence and spatial environment of ubiquitination sites of human and yeast proteins. To identify position-specific features, this work distinguishes the spatial environments as proximity and distal regions. Certain amino acids specific to these regions, well differentiate the ubiquitination sites from non-ubiquitination sites are revealed. Additionally, amino acid signatures that contribute for protein disordered regions and solvent accessibility of amino acids are found to be contributing factors in ubiquitination sites. These results suggest that the ubiquitination site environment of the substrate determines the recognition and unfolding of substrate to facilitate the entry into 26S proteosomal complex. We believe that these findings will help in better prediction of ubiquitination sites using the sequence and spatial information.

DeepUbi: a deep learning framework for prediction of ubiquitination sites in proteins.

BACKGROUND: Protein ubiquitination occurs when the ubiquitin protein binds to a target protein residue of lysine (K), and it is an important regulator of many cellular functions, such as signal transduction, cell division, and immune reactions, in eukaryotes. Experimental and clinical studies have shown that ubiquitination plays a key role in several human diseases, and recent advances in proteomic technology have spurred interest in identifying ubiquitination sites. However, most current computing tools for predicting target sites are based on small-scale data and shallow machine learning algorithms. RESULTS: As more experimentally validated ubiquitination sites emerge, we need to design a predictor that can identify lysine ubiquitination sites in large-scale proteome data. In this work, we propose a deep learning predictor, DeepUbi, based on convolutional neural networks. Four different features are adopted from the sequences and physicochemical properties. In a 10-fold cross validation, DeepUbi obtains an AUC (area under the Receiver Operating Characteristic curve) of 0.9, and the accuracy, sensitivity and specificity exceeded 85%. The more comprehensive indicator, MCC, reaches 0.78. We also develop a software package that can be freely downloaded from https://github.com/Sunmile/DeepUbi . CONCLUSION: Our results show that DeepUbi has excellent performance in predicting ubiquitination based on large data.

Exploring the Rampant Expansion of Ubiquitin Proteomics.

The ubiquitin proteasome system can arguably affect all cellular proteins with few exceptions. In addition to regulating many pathways such as cell cycle progression, inflammation, gene expression, DNA repair, and vesicle trafficking-to just name a few-ubiquitination can occur to any nascent or newly translated protein that misfolds. In the past years, substantial progress has been achieved in advancing our global understanding of the ubiquitinome-the ensemble of ubiquitinated proteins within a cell-using mass spectrometry-based proteomics. Notably, over 50,000 conjugation sites have now been reported. In this review, we discuss recent proteomics methods used to expand our knowledge of the ubiquitin proteasome system through the identification of ubiquitination sites, poly-ubiquitin chain types, and E3 ubiquitin ligase substrates.

iAcet-Sumo: Identification of lysine acetylation and sumoylation sites in proteins by multi-class transformation methods.

MOTIVATION: Posttranslational modification (PTM) is a biological mechanism involved in the enzymatic modification of proteins after translation by ribosomes. Two or more modifications occurring at one residue can be transformed into a multi-label system. Two or more simultaneous modifications on a residue is more common than single PTMs. Lysine residues in proteins can be subjected to a variety of PTMs, such as ubiquitination, acetylation, sumoylation, methylation, and succinylation. Identification of uncharacterized sequences in proteins is a highly significant and state-of-the-art issue. Notably, in order to provide a method of processing multi-label sequences of lysine residues, it is highly desirable to develop computational methods to predict lysine acetylation and sumoylation modifications. RESULTS: In this paper, we first launched an integrated approach, known as the five-step prediction method (FSPM), to solve the problem effectively by (1) using one-sided selection (OSS) to deal with imbalanced data, (2) extracting binary features from protein sequences, (3) incorporating binary relevance, classifier chains and multi-class transformation methods to simplify multi-label problems, (4) constructing different classifiers, and (5) implementing cross-validation and evaluating these classifiers. In 10-fold cross-validation, FSPM achieved an accuracy of 61.49% and an absolute-true rate of 60.17%. The results showed that FSPM is accurate and could be used as a powerful engine in multi-label systems. We also conducted a variety of statistical analyses of the predicted results to discuss the biological functions of lysine acetylation and sumoylation.

Proteome-wide identification of ubiquitin interactions using UbIA-MS.

Ubiquitin-binding proteins play an important role in eukaryotes by translating differently linked polyubiquitin chains into proper cellular responses. Current knowledge about ubiquitin-binding proteins and ubiquitin linkage-selective interactions is mostly based on case-by-case studies. We have recently reported a method called ubiquitin interactor affinity enrichment-mass spectrometry (UbIA-MS), which enables comprehensive identification of ubiquitin interactors for all ubiquitin linkages from crude cell lysates. One major strength of UbIA-MS is the fact that ubiquitin interactors are enriched from crude cell lysates, in which proteins are present at endogenous levels, contain biologically relevant post-translational modifications (PTMs) and are assembled in native protein complexes. In addition, UbIA-MS uses chemically synthesized nonhydrolyzable diubiquitin, which mimics native diubiquitin and is inert to cleavage by endogenous deubiquitinases (DUBs). Here, we present a detailed protocol for UbIA-MS that proceeds in five stages: (i) chemical synthesis of ubiquitin precursors and click chemistry for the generation of biotinylated nonhydrolyzable diubiquitin baits, (ii) in vitro affinity purification of ubiquitin interactors, (iii) on-bead interactor digestion, (iv) liquid chromatography (LC)-MS/MS analysis and (v) data analysis to identify differentially enriched proteins. The computational analysis tools are freely available as an open-source R software package, including a graphical interface. Typically, UbIA-MS allows the identification of dozens to hundreds of ubiquitin interactors from any type of cell lysate, and can be used to study cell type or stimulus-dependent ubiquitin interactions. The nonhydrolyzable diubiquitin synthesis can be completed in 3 weeks, followed by ubiquitin interactor enrichment and identification, which can be completed within another 2 weeks.

Vx3-Functionalized Alumina Nanoparticles Assisted Enrichment of Ubiquitinated Proteins from Cancer Cells for Enhanced Cancer Immunotherapy.

A simple and effective strategy was developed to enrich ubiquitinated proteins (UPs) from cancer cell lysate using the α-Al2O3 nanoparticles covalently linked with ubiquitin binding protein (Vx3) (denoted as α-Al2O3-Vx3) via a chemical linker. The functionalized α-Al2O3-Vx3 showed long-term stability and high efficiency for the enrichment of UPs from cancer cell lysates. Flow cytometry analysis results indicated dendritic cells (DCs) could more effectively phagocytize the covalently linked α-Al2O3-Vx3-UPs than the physical mixture of α-Al2O3 and Vx3-UPs (α-Al2O3/Vx3-UPs). Laser confocal microscopy images revealed that α-Al2O3-Vx3-UPs localized within the autophagosome of DCs, which then cross-presented α-Al2O3-Vx3-UPs to CD8+ T cells in an autophagosome-related cross-presentation pathway. Furthermore, α-Al2O3-Vx3-UPs enhanced more potent antitumor immune response and antitumor efficacy than α-Al2O3/cell lysate or α-Al2O3/Vx3-UPs. This work highlights the potential of using the Vx3 covalently linked α-Al2O3 as a simple and effective platform to enrich UPs from cancer cells for the development of highly efficient therapeutic cancer vaccines.