Recombinant Saccharomyces cerevisiae Unknown protein from spot 2D-000JYC of 2D-PAGE

What is 2D-PAGE and how does it contribute to the identification of unknown proteins?

2D-PAGE (Two-Dimensional Polyacrylamide Gel Electrophoresis) is a powerful separation technique that separates proteins based on two independent properties. In the first dimension, proteins are separated by their isoelectric point (pI) using isoelectric focusing (IEF), and in the second dimension, they are separated by molecular weight using SDS-PAGE . This technique creates a two-dimensional array of spots, each representing a unique protein or protein isoform. The technique maintains proteins in their intact states and enables the study of isoform distribution, which is not possible if the sample is proteolytically digested prior to separation . When analyzing a complex proteome like that of Saccharomyces cerevisiae, proteins can be isolated based on their position (spot) on a 2D gel and subsequently identified using mass spectrometry, which is why we refer to "Unknown protein from spot 2D-000JYC of 2D-PAGE" .

Why are some proteins from 2D-PAGE still labeled as "unknown" despite advanced identification techniques?

Proteins may be labeled as "unknown" when they have been visualized and isolated from a 2D gel but have not been fully characterized in terms of their sequence, structure, or function. This commonly occurs when a protein spot doesn't match any known protein in sequence databases, contains novel post-translational modifications, or represents a previously undocumented splice variant . For proteins like the one from spot 2D-000JYC, conventional database searching may be insufficient because no sequence database is available for the unknown synthetic protein . In such cases, de novo identification approaches become necessary, where search engines like Novor, DirecTag, and PepNovo+ are used to directly derive peptide sequences from MS/MS spectra based on mass differences between fragment ion peaks . The identification challenge is compounded when dealing with recombinant proteins that may have expression system-specific modifications or processing events .

What information can be derived from a protein's position on a 2D gel?

A protein's position on a 2D gel provides critical information about two fundamental properties: its isoelectric point (horizontal position) and molecular weight (vertical position) . This positional information serves as a first-pass characterization and can be used to verify subsequent identifications . Additionally, the intensity of the spot can indicate relative abundance, while the presence of multiple spots in a horizontal train might suggest post-translational modifications that alter charge but not size significantly . By comparing these experimental values with theoretical calculations based on an identified sequence, researchers can validate identifications or detect discrepancies that may indicate modifications or processing events . For the unknown protein from spot 2D-000JYC, its position in the 2D gel represents a unique combination of pI and molecular weight that distinguishes it from other proteins in the Saccharomyces cerevisiae proteome .

What are the optimal conditions for expressing recombinant Saccharomyces cerevisiae proteins in different host systems?

The choice and optimization of expression system are critical for successful production of recombinant Saccharomyces cerevisiae proteins. Based on available data, this unknown protein has been successfully expressed in multiple systems:

For this particular unknown protein, yeast expression (CSB-YP309881SAC) may provide the most native-like product, while E. coli systems offer higher yield but potentially incomplete post-translational modifications . Expression in baculovirus and mammalian systems should be considered if functional studies require specific post-translational modifications . Structural genomics laboratories often implement high-throughput protein expression and purification pipelines to test multiple constructs simultaneously, which increases the likelihood of obtaining soluble proteins .

How should researchers design experiments to characterize an unknown protein from 2D-PAGE?

Characterizing an unknown protein from 2D-PAGE requires a systematic experimental approach:

• Initial Spot Identification and Excision:

  • Visualize proteins using appropriate staining (Coomassie, silver, or fluorescent)

  • Document spot position, intensity, and pattern

  • Carefully excise the target spot (2D-000JYC) with minimal gel contamination

• Peptide Mass Fingerprinting:

  • Perform in-gel digestion with trypsin

  • Extract and analyze resulting peptides by MALDI-TOF MS

  • Search databases for matching peptide patterns

• Tandem MS Analysis:

  • If database searches are inconclusive, proceed to MS/MS analysis

  • Apply de novo sequencing using algorithms like Novor, DirecTag, and PepNovo+

  • Validate identifications manually after data reduction through spectral clustering

• Recombinant Expression Strategy:

  • Design multiple constructs with different tags and boundaries

  • Test expression in multiple systems (E. coli, yeast, baculovirus, mammalian)

  • Optimize conditions to maximize soluble protein yield

• Purification Optimization:

  • Develop multi-step purification protocols

  • Verify purity by SDS-PAGE and intact mass measurement

  • Assess homogeneity by size exclusion chromatography

• Structural Characterization:

  • Analyze secondary structure by circular dichroism (CD) spectroscopy

  • Perform limited proteolysis to identify domain boundaries

  • Attempt crystallization for X-ray crystallography

• Functional Analysis:

  • Design assays based on predicted functions or structural features

  • Test interaction with potential binding partners

  • Analyze subcellular localization if expressed in eukaryotic cells

This comprehensive approach combines traditional proteomics methods with recombinant protein techniques to fully characterize the unknown protein from spot 2D-000JYC .

What sample preparation steps are critical when working with unknown proteins for 2D-PAGE analysis?

Critical sample preparation steps for unknown proteins in 2D-PAGE analysis include:

• Protein Extraction:

  • Use buffers containing chaotropes (urea, thiourea), detergents (CHAPS), and reducing agents (DTT)

  • Maintain sample temperature below 30°C to prevent carbamylation

  • Include protease inhibitors to prevent degradation

• Contaminant Removal:

  • Eliminate salts through dialysis or precipitation (TCA/acetone)

  • Remove nucleic acids with nucleases or precipitation

  • Clean up lipids and polysaccharides with appropriate solvents

• Protein Solubilization:

  • Complete solubilization in IEF-compatible buffer is essential

  • Include carrier ampholytes to enhance protein solubility

  • Extend solubilization time for complex samples (2-3 hours minimum)

• First Dimension (IEF) Preparation:

  • Select appropriate IPG strip length and pH gradient for desired resolution

  • Choose optimal sample loading method (active rehydration recommended)

  • Program voltage ramping to prevent sample burning

• Equilibration Between Dimensions:

  • Two-step equilibration with DTT followed by iodoacetamide

  • Complete reduction and alkylation to prevent streaking

  • Temperature control during equilibration (room temperature, 15 minutes each step)

• Second Dimension (SDS-PAGE):

  • Select appropriate gel percentage based on target protein size

  • Ensure bubble-free contact between IPG strip and gel

  • Control separation conditions for reproducibility

• Protein Visualization:

  • Choose staining method based on sensitivity requirements and downstream applications

  • Document gel images under standardized conditions

  • Excise spots promptly to minimize diffusion and degradation

These critical steps ensure the successful separation, visualization, and subsequent identification of unknown proteins like that from spot 2D-000JYC . Proper documentation of all procedural details is essential for reproducibility and troubleshooting.

What mass spectrometry approaches are most effective for identifying unknown proteins from 2D gels?

For identifying unknown proteins from 2D gels like the protein from spot 2D-000JYC, several mass spectrometry approaches have proven effective:

• Peptide Mass Fingerprinting (PMF):

  • First-line approach due to simplicity and speed

  • Involves comparing experimental peptide masses against theoretical digests

  • Limited utility for truly unknown proteins not in databases

  • Typical workflow: in-gel digestion → MALDI-TOF MS → database searching

• Tandem Mass Spectrometry (MS/MS):

  • More definitive identification through peptide sequencing

  • LC-MS/MS analysis provides both retention time and fragmentation data

  • Higher sensitivity for low-abundance proteins

  • Critical for unknown proteins to generate sequence information

• De Novo Sequencing:

  • Essential for truly novel proteins not in databases

  • Directly interprets fragment spectra to determine peptide sequences

  • Software tools include Novor, DirecTag, and PepNovo+

  • Spectral clustering can reduce data complexity before analysis

• Spectral Networking:

  • Detects relationships between spectra and common mass differences

  • Useful for discovering post-translational modifications

  • Can provide insights into protein variants

• Top-down Proteomics:

  • Analyzes intact proteins rather than peptides

  • Preserves valuable information about proteoforms

  • Particularly useful for characterizing modification patterns

  • Requires specialized high-resolution instruments

For the truly unknown protein from spot 2D-000JYC, the optimal approach involves combining de novo sequencing with spectral clustering to manage data complexity, as demonstrated in the YPIC Challenge case study . This methodology reduced 110,234 spectra to 380 consensus spectra, making manual validation feasible and enabling identification of protein sequence fragments even without a reference database .

How can structural and functional predictions be made for an unknown protein identified from 2D-PAGE?

Making structural and functional predictions for an unknown protein from 2D-PAGE involves multiple complementary approaches:

• Sequence-Based Predictions:

  • Homology detection using sensitive algorithms (PSI-BLAST, HHpred)

  • Domain identification through databases like Pfam, SMART, InterPro

  • Functional motif detection for catalytic sites, binding regions

  • Secondary structure prediction (PSIPRED, JPred)

• Experimental Structural Analysis:

  • Circular dichroism (CD) spectroscopy to determine secondary structure content

  • Limited proteolysis to identify domain boundaries

  • Size exclusion chromatography to assess oligomeric state

  • Thermal stability assays to identify buffer conditions

• Computational Structure Prediction:

  • Template-based modeling if distant homologs exist

  • Ab initio prediction for novel folds

  • AI-based approaches like AlphaFold2 or RoseTTAFold

  • Validation against experimental data

• PTM Analysis for Functional Insights:

  • Phosphorylation site prediction (NetPhos, GPS)

  • Glycosylation site identification (NetNGlyc, NetOGlyc)

  • Mass spectrometry verification of predicted modifications

  • Assessment of modification impact on function

• Cellular Localization Prediction:

  • Signal peptide detection (SignalP)

  • Subcellular localization algorithms (PSORT, DeepLoc)

  • Transmembrane region prediction (TMHMM)

  • Experimental verification using tagged constructs

• Functional Screening:

  • Enzymatic activity assays based on structural predictions

  • Protein-protein interaction screening

  • Phenotypic analysis of knockout/knockdown models

  • Expression pattern analysis from public databases

For the unknown protein from spot 2D-000JYC, combining these approaches can generate testable hypotheses about structure and function, even in the absence of clear homologs . The CD spectroscopy approach mentioned in the YPIC Challenge provides valuable insights into secondary structure that can guide further functional predictions .

What are the recommended workflows for characterizing post-translational modifications in unknown proteins?

Characterizing post-translational modifications (PTMs) in unknown proteins requires a systematic workflow:

• Initial PTM Screening:

  • Intact mass analysis to determine total mass shifts

  • Specialized staining methods (Pro-Q Diamond for phosphorylation)

  • Western blotting with modification-specific antibodies

  • Comparison of observed vs. predicted molecular weight/pI

• MS-Based PTM Mapping:

  • Enrichment for specific modifications (IMAC for phosphopeptides)

  • Application of complementary fragmentation techniques (CID, ETD)

  • Use of neutral loss scanning for phosphorylation

  • Specialized data acquisition methods for labile modifications

• Analytical Workflow for Unknown Proteins:

• Bioinformatic Analysis:

  • Apply PTM-specific search engines and algorithms

  • Utilize site localization scoring (Ascore, ptmRS)

  • Map modifications onto predicted structures

  • Assess evolutionary conservation of modification sites

• Biological Significance Assessment:

  • Compare modifications between native and recombinant forms

  • Evaluate impact of expression system on modification patterns

  • Determine modification stoichiometry

  • Assess functional consequences through mutagenesis

For truly unknown proteins like from spot 2D-000JYC, establishing a reliable protein sequence through de novo approaches is the crucial first step before comprehensive PTM characterization . The selection of expression system for recombinant production significantly impacts PTM patterns, with yeast systems providing more native-like modifications for yeast proteins compared to bacterial systems .

How can researchers integrate multiple analytical techniques to comprehensively characterize an unknown protein?

Comprehensive characterization of an unknown protein like that from spot 2D-000JYC requires integration of multiple analytical techniques in a strategic workflow:

• Proteomic Identification Strategy:

  • Initial 2D-PAGE separation and spot isolation

  • De novo sequencing via tandem MS to establish primary sequence

  • Spectral clustering to manage data complexity and improve consensus spectra quality

  • Validation of sequence fragments through multiple search engines (Novor, DirecTag, PepNovo+)

• Recombinant Expression and Purification:

  • Multi-system expression testing (yeast, E. coli, baculovirus, mammalian)

  • Comparison of expression yields and solubility across systems

  • Optimization of purification strategies

  • Verification of recombinant protein identity through intact mass analysis

• Structural Characterization Pipeline:

  • Secondary structure analysis via circular dichroism spectroscopy

  • Domain boundary determination through limited proteolysis

  • Tertiary structure prediction through computational methods

  • Experimental structure determination attempts (crystallography, NMR)

• Functional Analysis Workflow:

  • Activity assays based on structural features

  • Protein-protein interaction studies (pull-downs, Y2H)

  • Cellular localization studies if appropriate

  • Phenotypic impact of protein depletion/overexpression

• Integrated Data Analysis:

  • Correlation between sequence, structure, and function

  • Comparison with related proteins in databases

  • Development of functional hypotheses for experimental testing

  • Documentation of all findings in standardized formats

The power of this integrated approach is demonstrated in studies like the YPIC Challenge, where combining spectral clustering, de novo sequencing, and circular dichroism spectroscopy allowed researchers to characterize a completely unknown synthetic protein . For the protein from spot 2D-000JYC, similar integration of techniques would provide a comprehensive profile even without initial database matches .

What strategies should be employed to study protein-protein interactions involving this unknown protein?

To study protein-protein interactions involving an unknown protein from spot 2D-000JYC, multiple complementary strategies should be employed:

• Affinity-Based Methods:

  • Recombinant expression with affinity tags (His, GST, FLAG)

  • Pull-down assays followed by mass spectrometry identification

  • Co-immunoprecipitation if antibodies become available

  • Surface plasmon resonance for quantitative binding analysis

• Yeast-Based Interaction Screens:

  • Yeast two-hybrid (Y2H) screening against genomic libraries

  • Split-ubiquitin assays for membrane-associated interactions

  • Protein-fragment complementation assays

  • Advantages: native expression environment for yeast proteins

• Proximity Labeling Approaches:

  • BioID or TurboID fusion constructs

  • APEX2 enzyme fusions for peroxidase-based labeling

  • In situ analysis of protein neighborhoods

  • Ability to detect weak or transient interactions

• MS-Based Interaction Analysis:

  • Crosslinking mass spectrometry (XL-MS) to map interaction interfaces

  • Hydrogen-deuterium exchange MS to identify binding regions

  • Protein correlation profiling across chromatographic fractions

  • Native MS to preserve non-covalent complexes

• Computational Prediction and Validation:

  • Docking simulations with potential partners

  • Coevolution analysis to identify interacting regions

  • Network-based prediction of functional associations

  • Experimental validation of top predictions

For the unknown protein from spot 2D-000JYC, leveraging the native yeast system provides a particular advantage, as potential interaction partners are in their natural cellular context. A systematic approach starting with tagged recombinant expression followed by affinity purification-mass spectrometry would provide an initial interactome map, which could then be validated and expanded using complementary techniques .

How can researchers investigate the functional role of an unknown protein in cellular processes?

Investigating the functional role of an unknown protein like that from spot 2D-000JYC requires a multi-tiered approach:

• Comparative Expression Analysis:

  • Examine expression under different growth conditions

  • Analyze protein abundance changes during stress responses

  • Compare expression patterns with functionally related proteins

  • Integration with existing transcriptomic/proteomic datasets

• Localization Studies:

  • Fluorescent protein tagging for live-cell imaging

  • Subcellular fractionation followed by western blotting

  • Immunofluorescence if antibodies are available

  • Correlation of localization with potential functions

• Genetic Manipulation Strategies:

  • CRISPR/Cas9 knockout or knockdown approaches

  • Overexpression studies to identify gain-of-function phenotypes

  • Complementation assays with mutant variants

  • Synthetic genetic interaction screening

• Biochemical Function Characterization:

  • Activity assays based on structural predictions

  • Substrate screening using metabolite libraries

  • Cofactor requirement determination

  • Post-translational modification analysis

• Systems Biology Integration:

  • Network analysis to identify functional modules

  • Metabolic profiling in knockout/overexpression strains

  • Transcriptomic analysis to identify regulated genes

  • Comparison with related species (evolutionary perspective)

The choice of recombinant protein expression system becomes crucial for functional studies. For a yeast protein like that from spot 2D-000JYC, expression in yeast systems may preserve native folding and activity better than bacterial systems . Cell culture experiments with recombinant proteins can help determine whether a protein can recover signaling after pathway inhibition, providing insights into its position in signaling networks . The integration of genetic approaches with biochemical characterization of the recombinant protein offers the most comprehensive understanding of function.

What are common challenges in expressing and purifying recombinant unknown proteins and how can they be addressed?

Common challenges in expressing and purifying unknown recombinant proteins include:

• Low Expression Yields:

  • Challenge: Insufficient protein production

  • Solutions:

    • Optimize codon usage for expression host

    • Test multiple expression strains/conditions

    • Add solubility-enhancing fusion tags (MBP, SUMO)

    • Consider alternative expression systems (yeast, baculovirus, mammalian)

    • Use stronger promoters or optimize induction parameters

• Protein Insolubility:

  • Challenge: Formation of inclusion bodies or aggregates

  • Solutions:

    • Lower expression temperature (16-20°C)

    • Reduce inducer concentration

    • Co-express with molecular chaperones

    • Add solubilizing agents (arginine, detergents)

    • Develop refolding protocols if necessary

• Protein Instability:

  • Challenge: Degradation during expression/purification

  • Solutions:

    • Include protease inhibitors throughout purification

    • Use protease-deficient host strains

    • Optimize buffer conditions (pH, salt, additives)

    • Maintain cold temperatures during processing

    • Add stabilizing ligands or cofactors

• Purification Difficulties:

  • Challenge: Poor separation from host proteins

  • Solutions:

    • Test multiple affinity tags and positions

    • Implement multi-step purification strategies

    • Optimize washing conditions

    • Consider ion exchange or hydrophobic interaction chromatography

    • Validate purity by mass spectrometry

• Incorrect Post-Translational Modifications:

  • Challenge: Missing or incorrect PTMs

  • Solutions:

    • Select expression systems capable of desired modifications

    • Co-express modifying enzymes

    • Verify modification status by mass spectrometry

    • Compare with native protein if available

For the unknown protein from spot 2D-000JYC, the availability of multiple expression options (CSB-YP309881SAC, CSB-EP309881SAC, CSB-BP309881SAC, CSB-MP309881SAC) allows researchers to select the system that provides the best balance of yield, solubility, and native-like properties . The yeast-based expression system may be particularly appropriate for this Saccharomyces cerevisiae protein to maintain its native folding and modifications .

How should researchers interpret discrepancies between predicted and observed properties of unknown proteins?

Interpreting discrepancies between predicted and observed properties of unknown proteins requires systematic analysis:

• Molecular Weight Discrepancies:

  • Post-Translational Modifications:

    • Glycosylation can add 1-50+ kDa

    • Phosphorylation adds ~80 Da per site

    • Other modifications (acetylation, methylation)

  • Proteolytic Processing:

    • N-terminal methionine removal (-131 Da)

    • Signal peptide cleavage

    • Internal processing events

  • Technical Factors:

    • SDS-PAGE migration anomalies

    • Calibration issues with molecular weight markers

• Isoelectric Point (pI) Differences:

  • Post-Translational Modifications:

    • Phosphorylation adds negative charge

    • Acetylation neutralizes positive charge

  • Conformational Effects:

    • Buried charged residues not contributing to surface charge

  • Technical Factors:

    • pI calculation algorithms using different pKa values

    • Non-equilibrium conditions during IEF

• Structural Discrepancies:

  • Secondary Structure Content:

    • Differences between CD spectroscopy measurements and predictions

    • Environment-dependent structural changes

  • Domain Organization:

    • Unexpected domain boundaries revealed by limited proteolysis

    • Presence of intrinsically disordered regions

• Investigation Approaches:

  • Mass Spectrometry Analysis:

    • Intact mass measurement to confirm total mass

    • Peptide mapping to identify modifications

    • De novo sequencing to verify primary structure

  • Protein Sequencing:

    • N-terminal sequencing to confirm processing

    • MS/MS sequencing for sequence verification

  • Structural Analysis:

    • CD spectroscopy for secondary structure

    • Limited proteolysis to probe domain organization

For the unknown protein from spot 2D-000JYC, these discrepancies may provide important clues about its unique features and biological role. The approach demonstrated in the YPIC Challenge case study, combining de novo sequencing with spectral clustering and structural analysis, provides a framework for resolving such discrepancies even for completely novel proteins .

What quality control measures are essential when working with recombinant versions of unknown proteins?

Essential quality control measures for recombinant versions of unknown proteins include:

• Identity Verification:

  • SDS-PAGE comparison with expected molecular weight

  • Western blotting with tag-specific antibodies

  • Peptide mass fingerprinting or MS/MS sequencing

  • N-terminal sequencing to confirm correct start site

• Purity Assessment:

  • Densitometric analysis of SDS-PAGE (target >90% purity)

  • Size exclusion chromatography to detect aggregates/oligomers

  • Reverse-phase HPLC for additional purity verification

  • Mass spectrometry to identify contaminants

• Structural Integrity Analysis:

  • Circular dichroism spectroscopy for secondary structure

  • Thermal shift assays to assess stability

  • Dynamic light scattering for homogeneity

  • Limited proteolysis to verify domain folding

• Functional Validation:

  • Activity assays based on predicted function

  • Binding studies with expected partners

  • Comparison with native protein when available

  • Multiple batch consistency testing

• Post-Translational Modification Verification:

  • Intact mass measurement to detect modifications

  • Site-specific MS/MS analysis of modified residues

  • Specialized staining for specific modifications

  • Comparison across expression systems

• Documentation and Reporting:

  • Comprehensive expression conditions recording

  • Detailed purification methodology

  • Storage stability monitoring

  • Batch-to-batch variation analysis