Recombinant Arabidopsis thaliana Ubiquitin-conjugating enzyme E2 34 (UBC34)

What is Ubiquitin-conjugating enzyme E2 34 (UBC34) and what is its basic function in Arabidopsis thaliana?

UBC34 is an E2 ubiquitin-conjugating enzyme that plays a crucial role in the ubiquitination pathway in Arabidopsis thaliana. As part of the E2 enzyme family, UBC34 catalyzes the transfer of ubiquitin from the E1 enzyme to target proteins, either directly or in conjunction with E3 ubiquitin ligases. The protein likely contains approximately 235 amino acids with a predicted molecular mass of around 27 kDa, similar to its poplar homolog PtoUBC34 . UBC34 functions in protein degradation, cellular signaling, and immune response regulation. Unlike the traditional view that E2 enzymes play merely auxiliary roles in ubiquitination, recent research indicates that E2 enzymes, including UBC34, have specific and essential functions in plant biological processes .

How does UBC34 compare to other E2 enzymes in the Arabidopsis genome?

The Arabidopsis genome contains approximately 37 E2 family members, comparable to the 40 identified in tomato . UBC34 belongs to a specific phylogenetic group (corresponding to Group III in tomato) characterized by a conserved UBC domain and C-terminal transmembrane domain. This structural arrangement is significant as it suggests membrane localization, likely in the endoplasmic reticulum, distinguishing it from cytosolic E2 enzymes. The presence of a conserved active cysteine residue (position may be similar to Cys87 in PtoUBC34) in the catalytic domain is essential for thioester bond formation with ubiquitin, a feature shared across most active E2 enzymes . Unlike some other E2s that operate broadly, UBC34 likely demonstrates specificity in its interactions with E3 ligases and substrate proteins, contributing to precise regulation of target protein stability.

What experimental approaches can validate the enzymatic activity of recombinant UBC34?

To validate the enzymatic activity of recombinant UBC34, researchers should employ the following methodological approaches:

• Thioester assay: This is the primary method to confirm E2 enzymatic activity. The assay detects the formation of a thioester bond between the active site cysteine of UBC34 and ubiquitin. The procedure involves incubating purified recombinant UBC34 with ubiquitin and an E1 enzyme in the presence of ATP, followed by SDS-PAGE analysis under non-reducing and reducing conditions. Active E2 enzymes will show a higher molecular weight band under non-reducing conditions (UBC34~Ub) that disappears upon treatment with reducing agents .

• In vitro ubiquitination assay: This approach tests whether UBC34 can transfer ubiquitin to substrate proteins. The reaction mixture contains E1, recombinant UBC34, ubiquitin, ATP, and potential substrate proteins. Western blot analysis using anti-ubiquitin antibodies can detect ubiquitinated products .

• Auto-ubiquitination assay: Some E2 enzymes exhibit auto-ubiquitination activity. This can be tested by incubating UBC34 with E1, ubiquitin, and ATP, followed by mass spectrometry to identify ubiquitination sites on UBC34 itself.

• E3-independent ubiquitination activity test: As some plant E2 enzymes display E3-independent activity, UBC34 should be tested for its ability to ubiquitinate model substrates without an E3 ligase present .

What are the key structural domains of UBC34 and their functions?

UBC34 contains several critical structural domains essential for its function:

• UBC domain (UBCc): Located at the N-terminus, this core catalytic domain is approximately 150 amino acids in length. It contains:

  • The active site cysteine residue that forms a thioester bond with ubiquitin

  • Several highly conserved residues that mediate E3 ligase interactions, including positions corresponding to 8K, 65P, 66Y, 100P, and 101M in PtoUBC34

  • Multiple ubiquitin thioester intermediate interaction residues that stabilize the E2~Ub intermediate, including positions corresponding to 79R, 80F, 82T, 86I, 87C, 88L, and others identified in PtoUBC34

• Transmembrane domain: Present at the C-terminus, this hydrophobic region anchors UBC34 to cellular membranes, particularly the endoplasmic reticulum membrane. This localization is crucial for its function, as it positions UBC34 to ubiquitinate specific membrane-associated or ER-localized proteins .

• Linker region: The region between the UBC domain and the transmembrane domain likely serves as a flexible linker, allowing the catalytic domain to access substrate proteins while remaining tethered to the membrane.

The conserved structural features of UBC34 reflect its specialized function in the plant ubiquitination system, enabling it to interact with specific E3 ligases and target proteins at particular cellular locations.

What techniques are most effective for determining the subcellular localization of UBC34?

The following methodological approaches are recommended for determining UBC34 subcellular localization:

• Fluorescent protein fusion and confocal microscopy:

  • Generate constructs expressing UBC34 fused to fluorescent proteins (GFP, YFP, etc.) at either N- or C-terminus

  • Transform plant protoplasts or tissues with these constructs

  • Co-localize with established organelle markers (e.g., BiP:RFP for ER localization)

  • Analyze using confocal laser scanning microscopy

  • This approach successfully demonstrated ER localization of PtoUBC34 when YFP:PtoUBC34 was co-expressed with BiP:RFP

• Subcellular fractionation and immunoblotting:

  • Fractionate plant tissues into membrane and soluble components

  • Perform additional separation of different membrane types (microsomal, plasma membrane, etc.)

  • Use western blot with anti-UBC34 antibodies to detect the protein in different fractions

  • Include markers for different organelles as controls

• Immunogold electron microscopy:

  • Fix and section plant tissue

  • Label with anti-UBC34 antibodies followed by gold-conjugated secondary antibodies

  • Visualize using transmission electron microscopy for high-resolution localization

• Biochemical validation of membrane integration:

  • Treat isolated membranes with various agents (high salt, alkaline pH, detergents)

  • Analyze UBC34 release patterns to determine the nature of membrane association

For UBC34, which likely contains a transmembrane domain, proper fixation and membrane preservation techniques are crucial for accurate localization results.

How can researchers effectively express and purify recombinant Arabidopsis UBC34?

Effective expression and purification of recombinant Arabidopsis UBC34 requires careful consideration of its structural features, particularly its transmembrane domain. Here is a detailed methodological approach:

Expression System Selection:

Optimized Protocol for E. coli Expression:

• Construct design:

  • Clone the UBC domain (N-terminal portion without transmembrane domain) into pET28a with an N-terminal His-tag

  • Alternatively, use full-length UBC34 with solubilizing fusion tags (SUMO, MBP, GST)

• Expression conditions:

  • Transform into E. coli BL21(DE3) cells

  • Grow at 37°C to OD600 of 0.6

  • Induce with 0.5 mM IPTG

  • Shift to 16-18°C for overnight expression

  • Add 0.1 mM ZnCl2 to the media to stabilize protein structure

• Lysis and purification:

  • Lyse cells in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM DTT, 10% glycerol, and protease inhibitors

  • Purify using Ni-NTA affinity chromatography

  • Further purify by size-exclusion chromatography

• Activity preservation:

  • Include 1 mM DTT in all buffers to maintain the active site cysteine in reduced form

  • Store in small aliquots at -80°C with 10% glycerol

For full-length UBC34 including the transmembrane domain:

• Use detergent solubilization (0.1% DDM or CHAPS)

• Consider membrane scaffold protein (MSP) nanodisc technology for native-like membrane environment

For researchers using E2 proteins in ubiquitination assays, enzymatic activity should be validated using thioester assays as described in Question 1.3 .

What are the specific E3 ligase partners of UBC34 and how can these interactions be characterized?

UBC34 likely interacts with specific E3 ligases to regulate target protein ubiquitination. Based on studies of related E2 enzymes, the following methodological approaches are recommended for characterizing these interactions:

• Yeast two-hybrid (Y2H) screening:

  • Use the UBC domain of UBC34 (excluding the transmembrane domain) as bait

  • Screen against an Arabidopsis cDNA library

  • Validate positive interactions with targeted Y2H assays

  • Note that membrane-based split-ubiquitin Y2H systems may be more appropriate for full-length UBC34

• Co-immunoprecipitation (Co-IP):

  • Express epitope-tagged UBC34 in Arabidopsis or a transient expression system

  • Perform IP with tag-specific antibodies

  • Identify interacting E3 ligases by mass spectrometry or western blotting

  • Use crosslinking agents to capture transient interactions

• In vitro binding assays:

  • Express and purify recombinant UBC34 and candidate E3 ligases

  • Perform pull-down assays or surface plasmon resonance (SPR)

  • Determine binding affinity and kinetics

  • Map interaction domains through truncation or mutagenesis

• In vitro ubiquitination assays:

  • Test functional cooperation between UBC34 and candidate E3 ligases

  • E2-E3 specificity can be determined using a reaction mixture containing E1, UBC34, the candidate E3 ligase, ubiquitin, and ATP

  • The presence of polyubiquitin chains indicates productive E2-E3 interaction

• Bimolecular Fluorescence Complementation (BiFC):

  • Fuse UBC34 and candidate E3 ligases to complementary fragments of fluorescent proteins

  • Co-express in plant cells to visualize interactions in vivo

  • This technique also provides spatial information about where interactions occur

Based on studies of homologous proteins, UBC34 may interact with RING-type E3 ligases and may have roles in ER-associated degradation (ERAD) or regulation of transcription factors, similar to how PtoUBC34 interacts with the transcription factor PtoMYB221 .

How does UBC34 contribute to plant immunity and stress responses?

Arabidopsis UBC34, like other Group III E2 enzymes, likely plays significant roles in plant immunity and stress response pathways. Based on studies of related E2 enzymes, the following functional aspects are relevant:

• Pattern-Triggered Immunity (PTI):

  • Group III E2 enzymes in tomato are essential for PTI responses

  • Silencing these E2 genes results in reduced PTI

  • UBC34 likely participates in similar immune signaling pathways in Arabidopsis

  • May regulate the stability of immune receptors or downstream signaling components

• Defense against bacterial pathogens:

  • Bacterial effectors like AvrPtoB from Pseudomonas syringae can hijack host E2 enzymes

  • Group III E2 enzymes specifically interact with AvrPtoB to promote the degradation of plant immunity components

  • UBC34 may be targeted by similar pathogen effectors

• Regulation of defense-related transcription factors:

  • Similar to how PtoUBC34 interacts with and regulates the transcription factor PtoMYB221

  • UBC34 may control the stability or activity of defense-related transcription factors in Arabidopsis

• Abiotic stress responses:

  • The ubiquitin system is involved in responses to various abiotic stressors

  • ER-localized E2s like UBC34 may participate in ER-associated degradation (ERAD) of misfolded proteins during stress

  • May regulate stress-responsive transcription factors

To experimentally investigate UBC34's role in immunity and stress responses, researchers should consider:

• Gene silencing or CRISPR-based knockout of UBC34 followed by pathogen infection assays

• Protein abundance analyses of immune components in UBC34 mutants

• Transcriptome analysis to identify genes affected by UBC34 dysfunction

• Stress tolerance tests of UBC34 mutant/overexpression lines

• Biochemical characterization of UBC34-dependent ubiquitination of candidate immune regulators

What is the relationship between UBC34 and hormone signaling pathways in plants?

UBC34's relationship with plant hormone signaling pathways represents an important area of research. Based on the known functions of E2 enzymes in plants, the following methodological approaches can elucidate these connections:

• Hormone response assays:

  • Compare wildtype and UBC34 mutant/overexpression lines for responses to various hormones (auxin, ABA, jasmonate, ethylene, etc.)

  • Measure root growth, hypocotyl elongation, germination rates, or other hormone-sensitive phenotypes

  • Document dose-response relationships for different hormones

• Protein stability analysis of hormone signaling components:

  • Monitor stability of hormone receptors or transcription factors in UBC34 mutants

  • Use cycloheximide chase assays to measure protein half-life

  • Perform in vivo and in vitro ubiquitination assays with specific hormone signaling proteins as substrates

• Transcriptome analysis:

  • Perform RNA-seq comparing wildtype and UBC34 mutants with and without hormone treatments

  • Identify hormone-responsive genes affected by UBC34 dysfunction

  • Use gene ontology enrichment to identify affected hormone pathways

• Protein-protein interaction studies:

  • Screen for interactions between UBC34 and components of hormone signaling pathways

  • Focus on E3 ligases known to regulate hormone signaling (e.g., TIR1/AFB for auxin, COI1 for jasmonate)

  • Test if UBC34 can function with these E3 ligases in ubiquitination assays

• Genetic interaction analysis:

  • Create double mutants between UBC34 and hormone signaling components

  • Analyze phenotypes for enhancement, suppression, or epistatic relationships

  • Test genetic interactions under various stress conditions

The transmembrane domain of UBC34 suggests it may be particularly involved in regulating membrane-associated hormone receptors or transporters. As observed with related E2 enzymes, UBC34 may work with specific E3 ligases to target components of hormone signaling pathways for degradation, thereby affecting plant development and stress responses.

What are the critical considerations for designing experiments to study UBC34 function in vivo?

When designing experiments to study UBC34 function in vivo, researchers should consider these critical methodological factors:

• Genetic manipulation approaches:

  • CRISPR/Cas9 for gene knockout: Design multiple sgRNAs targeting conserved regions of the UBC domain

  • RNAi for gene silencing: Consider potential off-target effects on related E2 genes

  • Overexpression studies: Use both constitutive (35S) and inducible promoters

  • Complementation assays: Test functional domains by expressing variants in knockout backgrounds

  • Function-blocking mutations: Target the active site cysteine (similar to Cys87 in PtoUBC34)

• Genetic redundancy considerations:

  • Identify close homologs that may mask phenotypes in single mutants

  • Create higher-order mutants of related E2 enzymes

  • Use artificial microRNAs to simultaneously target multiple family members

  • Group III E2 enzymes show functional redundancy in tomato , suggesting similar patterns in Arabidopsis

• Experimental design principles:

  • Include appropriate controls (wildtype, empty vector, inactive mutant proteins)

  • Use randomized complete block design with adequate replication

  • Control environmental variables that affect ubiquitination (temperature, light, stress)

  • Include time-course analyses to capture dynamic processes

  • Carefully select experimental units and sampling strategies

• Phenotypic analysis framework:

  • Assess multiple developmental stages and tissues

  • Test various stress conditions (biotic, abiotic)

  • Measure subtle phenotypes (e.g., root architecture, flowering time)

  • Use quantitative imaging methods for objective assessment

  • Consider conditional phenotypes that may only appear under specific conditions

• Sample size and statistical considerations:

  • Power analysis to determine appropriate replicate numbers

  • Use statistical methods appropriate for the experimental design

  • Consider nested designs when measuring multiple parameters

  • Account for variability in biological systems

The following table summarizes key experimental designs for UBC34 functional studies:

How can researchers effectively analyze the enzymatic activity of UBC34 in vitro?

To effectively analyze the enzymatic activity of UBC34 in vitro, researchers should employ a systematic approach focusing on ubiquitin transfer capabilities and specificity. The following methodological approaches are recommended:

• Thioester formation assay:

  • Reaction components: Purified E1 (50-100 nM), recombinant UBC34 (0.5-1 μM), ubiquitin (5-10 μM), ATP (2-5 mM), and buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 0.2 mM DTT)

  • Procedure: Incubate at 30°C for 10-30 minutes, then split sample and add either SDS loading buffer with or without reducing agent (β-mercaptoethanol)

  • Analysis: Run SDS-PAGE and western blot with anti-UBC34 antibodies. A higher molecular weight band (+8.5 kDa) in non-reducing conditions indicates thioester bond formation

  • Controls: Include catalytically inactive UBC34 (Cys→Ala mutation) as negative control

• E3-dependent ubiquitination assay:

  • Components: E1, UBC34, candidate E3 ligase, ubiquitin, ATP, and buffer

  • Detection methods:

    • Western blot with anti-ubiquitin antibodies

    • Use fluorescently labeled ubiquitin for quantitative assessment

    • Mass spectrometry to identify ubiquitination sites

  • Kinetic analysis: Sample reaction at multiple time points to determine reaction rates

  • E3 ligase screening: Test multiple E3 ligases to determine specificity

• Chain type specificity analysis:

  • Use ubiquitin mutants (K48R, K63R, etc.) to determine chain linkage preference

  • Employ linkage-specific antibodies to identify chain types

  • Mass spectrometry analysis of polyubiquitin chains

  • Compare activity with different E2 family members as controls

• Substrate specificity determination:

  • Candidate approach: Test known substrates of related E2 enzymes

  • Unbiased approach: Use protein arrays or mass spectrometry to identify substrates

  • Validation: Perform in vitro ubiquitination with purified substrates

  • Mapping: Determine ubiquitination sites using mass spectrometry

• Structural and biochemical parameters:

  • Determine Km and kcat values for ubiquitin and ATP

  • Assess pH and temperature optima

  • Evaluate effects of salt concentration and divalent cations

  • Test sensitivity to inhibitors

The following table summarizes typical reaction conditions for E2 enzyme assays:

These approaches provide a comprehensive analysis of UBC34's catalytic properties and help identify its specific roles in the ubiquitination cascade .

What statistical approaches are most appropriate for analyzing UBC34 experimental data?

The appropriate statistical approach for analyzing UBC34 experimental data depends on the experimental design, data types, and research questions. The following methodological framework provides guidance:

• For phenotypic comparison studies:

  • Two-group comparisons (wild-type vs. UBC34 mutant):

    • Student's t-test for normally distributed data

    • Mann-Whitney U test for non-parametric data

    • Report effect sizes (Cohen's d) alongside p-values

  • Multiple group comparisons (wild-type, UBC34 mutant, complementation lines):

    • One-way ANOVA followed by Tukey's HSD or Dunnett's post-hoc tests

    • Kruskal-Wallis test with Dunn's post-hoc for non-parametric data

    • Control family-wise error rate using Bonferroni or false discovery rate (FDR) correction

• For experimental designs with multiple factors:

  • Factorial designs (e.g., genotype × treatment × time):

    • Factorial ANOVA to test main effects and interactions

    • Mixed-effects models for repeated measures or nested designs

    • Post-hoc tests guided by specific research questions

  • Randomized complete block designs:

    • Block as random effect in mixed-effects models

    • Account for environmental variation across experimental units

• For biochemical and interaction data:

  • Enzyme kinetics:

    • Non-linear regression for Michaelis-Menten kinetics

    • Compare Km and Vmax parameters across conditions using extra sum-of-squares F-test

  • Dose-response relationships:

    • Four-parameter logistic regression for sigmoidal curves

    • Compare EC50/IC50 values using F-tests

  • Binding affinity measurements:

    • One-site or two-site binding models

    • Statistical comparison of Kd values

• For high-throughput data:

  • Transcriptomics:

    • DESeq2 or edgeR for differential expression analysis

    • FDR correction for multiple testing

    • GSEA or pathway enrichment with appropriate background controls

  • Proteomics:

    • Linear models with empirical Bayes methods (limma)

    • Proper normalization for sample loading differences

    • Adjustment for batch effects

• For time-course experiments:

  • Repeated measures ANOVA or mixed-effects models

  • Growth curve analysis using non-linear regression

  • Time-to-event analysis (survival analysis) for developmental transitions

The following table summarizes key statistical approaches for different experimental designs in UBC34 research:

Regardless of the statistical approach, researchers should:

• Clearly define hypotheses before data collection

• Conduct power analyses to determine appropriate sample sizes

• Report effect sizes and confidence intervals, not just p-values

• Consider biological significance beyond statistical significance

• Validate findings with independent experimental approaches

How can CRISPR/Cas9 be optimized for studying UBC34 function in Arabidopsis?

CRISPR/Cas9 technology offers powerful approaches for studying UBC34 function in Arabidopsis. The following methodological considerations will optimize CRISPR/Cas9 for UBC34 research:

• Guide RNA design strategy:

  • Target the conserved UBC domain, particularly regions encoding catalytic residues

  • Design multiple sgRNAs (3-4) targeting different exons to increase editing efficiency

  • Avoid regions with high homology to other E2 enzymes to prevent off-target effects

  • Use tools like CRISPOR or CRISPR-P2.0 for sgRNA design and off-target prediction

  • Prioritize sgRNAs with high on-target and low off-target scores

• CRISPR vector selection and optimization:

  • Use plant-optimized vectors with appropriate promoters:

    • Cas9: Use egg cell-specific promoters (EC1.2) for germline editing

    • sgRNA: U6 or U3 promoters work efficiently in Arabidopsis

  • Consider multiplex systems for targeting multiple sites simultaneously

  • For precise editing, include repair templates for homology-directed repair (HDR)

• Advanced CRISPR applications for UBC34 functional studies:

• Validation and characterization of CRISPR-edited lines:

  • PCR and sequencing to confirm edits

  • RT-qPCR and western blotting to verify transcript/protein loss

  • Enzyme activity assays to confirm functional disruption

  • Complementation with wildtype UBC34 to confirm phenotype specificity

  • Off-target analysis by whole-genome sequencing or Digenome-seq

• Addressing functional redundancy:

  • Multiplex CRISPR to target multiple E2 genes simultaneously

  • Create conditional knockouts if complete loss is lethal

  • Use tissue-specific or inducible CRISPR systems for spatiotemporal control

  • Combine with overexpression of dominant-negative UBC34 variants

By implementing these optimized CRISPR/Cas9 approaches, researchers can generate precise genetic tools for dissecting UBC34 function while avoiding limitations like lethality or redundancy that often complicate traditional genetic approaches.

What advanced proteomics approaches can identify UBC34 substrates and interacting partners?

Advanced proteomics approaches offer powerful methods to identify UBC34 substrates and interacting partners. The following methodological strategies are recommended:

• Affinity Purification-Mass Spectrometry (AP-MS):

  • Methodology:

    • Express epitope-tagged UBC34 (FLAG, HA, or TAP tag) in Arabidopsis

    • Include catalytically inactive mutant (C→A) as control

    • Perform immunoprecipitation followed by LC-MS/MS

    • Use SILAC or TMT labeling for quantitative comparison

  • Data analysis:

    • Compare with control purifications to identify specific interactions

    • Use CRAPome or similar tools to filter common contaminants

    • Apply statistical methods like SAINT for confidence scoring

• Proximity-based labeling combined with MS:

• Ubiquitinome analysis:

  • Global approach:

    • Express wild-type or mutant UBC34 in Arabidopsis

    • Enrich ubiquitinated peptides using K-ε-GG antibodies

    • Compare ubiquitinomes to identify UBC34-dependent changes

  • Targeted approach:

    • Perform in vitro ubiquitination with UBC34 and candidate substrates

    • Use mass spectrometry to map exact ubiquitination sites

    • Validate sites by mutagenesis and functional assays

• Crosslinking Mass Spectrometry (XL-MS):

  • Use chemical crosslinkers (DSS, BS3) to capture transient interactions

  • Identify crosslinked peptides by MS/MS

  • Generate structural models of UBC34-substrate complexes

  • Particularly useful for transient E2-E3 and E2-substrate interactions

• Protein Correlation Profiling:

  • Separate cellular extracts by size exclusion chromatography

  • Analyze fractions by quantitative MS

  • Identify proteins with similar elution profiles as UBC34

  • Reveals native protein complexes without tagging

• Targeted validation approaches:

  • Hydrogen-deuterium exchange MS to map interaction interfaces

  • Selected reaction monitoring (SRM) for quantitative validation

  • Parallel reaction monitoring (PRM) for improved specificity

  • AQUA peptides for absolute quantification of targets

For UBC34 as an ER-localized protein, special consideration should be given to membrane protein enrichment steps:

• Use specialized detergents (DDM, digitonin) for membrane solubilization

• Consider subcellular fractionation to enrich ER membranes

• Apply specific protocols for membrane protein crosslinking

• Optimize digestion conditions for transmembrane regions

These proteomics approaches should be complemented with orthogonal validation techniques such as co-immunoprecipitation, in vitro binding assays, and functional studies to confirm the biological relevance of identified interactions.

How can researchers integrate transcriptomics and proteomics data to understand UBC34-regulated pathways?

Integrating transcriptomics and proteomics data provides a comprehensive understanding of UBC34-regulated pathways. The following methodological framework enables effective multi-omics integration:

• Experimental design considerations:

  • Use matched samples for transcriptomics and proteomics

  • Include multiple time points to capture dynamic responses

  • Compare wild-type, UBC34 knockout, and UBC34 overexpression lines

  • Include appropriate controls and biological replicates

  • Consider diverse conditions (developmental stages, stresses) relevant to UBC34 function

• Data acquisition and preprocessing:

  • Transcriptomics:

    • RNA-seq with sufficient depth (30-50M reads per sample)

    • Quality control (FastQC, MultiQC)

    • Adapter trimming and read filtering

    • Alignment to reference genome using STAR or HISAT2

  • Proteomics:

    • Deep proteome coverage using fractionation techniques

    • Include phosphoproteomics and ubiquitinomics

    • Label-free or isotope labeling quantification

    • MS1 and MS2 level quality control

• Individual omics analysis:

• Multi-omics integration strategies:

  • Correlation analysis:

    • Calculate correlation between transcript and protein changes

    • Identify discordant pairs suggesting post-transcriptional regulation

    • Apply time-lagged correlation for dynamic studies

  • Pathway and network analysis:

    • Enrichment analysis using GO, KEGG, MapMan

    • Use integrated tools (MetaboAnalyst, PathwayConnector)

    • Network visualization (Cytoscape) with multi-omics data overlay

    • Causal network inference (WGCNA, ARACNE)

• Advanced integration methods:

  • Joint dimensional reduction:

    • MOFA (Multi-Omics Factor Analysis)

    • DIABLO (Data Integration Analysis for Biomarker discovery)

    • NMF (Non-negative Matrix Factorization)

  • Systems biology modeling:

    • Constraint-based modeling with omics data integration

    • Kinetic modeling of UBC34-regulated pathways

    • Bayesian network inference across data types

• Functional validation of integrated insights:

  • Target selection based on multi-omics convergence

  • Prioritize genes/proteins showing concordant regulation

  • Design validation experiments based on predicted pathway effects

  • Use genetic interaction studies to confirm network connections

• Data visualization and interpretation:

  • Integrated heatmaps showing transcript and protein changes

  • Multi-omics pathway visualization (e.g., PathVisio, IPath)

  • Customized Circos plots for multi-level data integration

  • Interactive dashboards for exploration (R Shiny, Tableau)

This integrated approach enables researchers to distinguish between transcriptional and post-transcriptional effects of UBC34, identify direct ubiquitination targets versus secondary effects, and construct comprehensive regulatory networks involving UBC34-mediated ubiquitination.