RPS27A Human, Biotin

What is RPS27A and what are its key biological functions?

RPS27A is a small protein composed of 76 amino acids found only in eukaryotic organisms with strong sequence conservation across species. It serves multiple crucial cellular functions:

• As a component of the small ribosomal subunit (40S), participating in protein synthesis

• As ubiquitin, when cleaved from fusion proteins

• In protein degradation pathways, where multiple RPS27A copies target proteins for degradation by the 26S proteasome

• In cellular signaling and stress responses

RPS27A can be found either in free form or conjugated to proteins through a covalent bond between the glycine at the C-terminal end and lysine side chains . In humans, RPS27A (eS31) is synthesized as a ubiquitin-RP precursor protein that requires processing for proper functioning .

What is the significance of biotinylated RPS27A in experimental research?

Biotinylated RPS27A offers several experimental advantages:

• Enables specific detection using streptavidin-based assays due to the extremely high affinity between biotin and streptavidin

• Facilitates protein enrichment and purification from complex biological samples

• Allows for studying RPS27A interactions with other cellular components

• Provides a tool for tracking RPS27A localization and dynamics

• Serves as a model for studying ubiquitination processes

The NHS-biotin modification specifically targets primary amines without passing through cell membranes, making it ideal for cell surface protein labeling . This property enables researchers to distinguish between intracellular and surface-exposed populations of proteins.

How is recombinant biotinylated RPS27A produced and what are its specifications?

Recombinant human RPS27A protein biotinylated with NHS-biotin is typically produced in E. coli expression systems. The resulting product is:

• A single, non-glycosylated polypeptide chain containing 76 amino acids

• Has a molecular mass of approximately 8.6 kDa

• Supplied as a sterile filtered colorless liquid formulation in 1x PBS with 0.05% PBS

• Protein concentration is determined by 280nm absorbance

• Free biotin is eliminated by dialysis against PBS

• Biotinylation is verified by Western Blotting and ELISA analysis using streptavidin-HRP conjugate

What are optimal methods for handling and storing biotinylated RPS27A?

For optimal handling and storage of biotinylated RPS27A:

It is essential to prevent repeated freeze-thaw cycles as they can degrade the protein. The protein is typically supplied in 1x PBS with 0.05% PBS as a stabilizer . When working with the protein, maintain sterile conditions and briefly centrifuge the vial before use to collect all material at the bottom.

How does the biotinylation approach compare with hydrazide capturing for membrane protein enrichment?

Both approaches have distinct characteristics that make them suitable for different research questions:

What controls should be included when designing experiments with biotinylated RPS27A?

A comprehensive experimental design should include:

• Technical controls:

  • Non-biotinylated RPS27A to establish baseline behavior

  • Biotinylated control protein to distinguish RPS27A-specific effects

  • Free biotin control to assess non-specific binding

  • Streptavidin-only control for background binding evaluation

• Biological controls:

  • Wild-type cells/tissues for comparison

  • RPS27A knockdown/knockout to validate specificity

  • Mutant RPS27A variants (e.g., diglycine motif mutants) to study specific functional domains

• Process controls:

  • Input samples (pre-enrichment) to determine enrichment efficiency

  • Flow-through fractions to assess depletion efficiency

  • Mock treatment controls

• Specific controls for cell surface studies:

  • Cell viability assessment to ensure intact cells during labeling

  • Known surface and intracellular protein controls

How does the diglycine motif affect the processing and function of RPS27A in ribosome biogenesis?

The diglycine motif at the C-terminus of RPS27A plays a critical role in its processing and function:

• The diglycine motif (G73, G74) is essential for proper cleavage of the ubiquitin portion from the RPS27A fusion protein

• Mutations in this motif (G73,74A or G74V) render the fusion protein non-cleavable

• Non-cleavable mutants show altered sedimentation behavior in sucrose gradients, suggesting less efficient incorporation into 40S ribosomal subunits

• These mutants induce significant changes in pre-rRNA processing, with a strong increase in 26S and 18S-E pre-rRNAs and a decrease in 30S and 21S precursors

This pattern is similar to what occurs upon depletion of progression (p-)RPS that are important for 21S and 18S-E processing as well as cleavage at site 3. The requirement for processing of ubiquitin-RP fusion proteins is conserved from yeast to humans, indicating a fundamental role in coordinating ribosome assembly with ubiquitin production .

How can biotinylated RPS27A be utilized to study neurodegenerative disease mechanisms?

Biotinylated RPS27A offers several approaches to investigate neurodegenerative disease mechanisms:

• Studying ubiquitin-proteasome system dysfunction:

  • Many neurodegenerative diseases feature impaired protein degradation

  • Biotinylated RPS27A can track changes in ubiquitination patterns in disease models

  • Can identify abnormally accumulated ubiquitinated proteins

• Examining protein aggregates:

  • Can investigate if ubiquitin is incorporated into disease-associated aggregates

  • Helps study how the ubiquitin system responds to aggregate formation

  • Research has shown that in ALS-associated poly-dipeptide repeat aggregates, there is strong association with many proteins including a significant part of the ubiquitin/proteasome system

• Analyzing altered ribosomal function:

  • Ribosome dysfunction is emerging as a factor in neurodegenerative diseases

  • Biotinylated RPS27A can assess changes in ribosome biogenesis and function

• Studying sequestration mechanisms:

  • Can identify if RPS27A or ubiquitin is sequestered by disease-associated proteins

  • Helps characterize the composition and dynamics of pathological inclusions

What methodological challenges exist in distinguishing cell surface-specific RPS27A from intracellular pools?

Distinguishing cell surface-specific RPS27A from intracellular pools presents several challenges:

• Selective labeling:

  • Requires reagents that cannot permeate the cell membrane

  • Sulfo-NHS-SS-biotin is valuable as it selectively targets primary amines without crossing the cell membrane

  • Challenge: Ensuring absolute membrane impermeability of the labeling reagent

• Detecting low abundance surface pools:

  • RPS27A is primarily an intracellular protein, with potentially lower abundance at the cell surface

  • Requires methods with sufficient sensitivity for detecting smaller surface pools

• Avoiding cell lysis during labeling:

  • Any cell damage during labeling can expose intracellular RPS27A

  • Maintaining cell integrity throughout the experimental procedure is crucial

• Distinguishing specific binding from contamination:

  • RPS27A's abundance in cells means it can be a common contaminant

  • Requires appropriate controls to distinguish genuine surface localization from experimental artifacts

• Temporal dynamics:

  • Cell surface proteome composition can change rapidly

  • Capturing the dynamic nature of RPS27A surface localization requires careful experimental timing

How should mass spectrometry data from experiments using biotinylated RPS27A be analyzed?

A systematic approach to analyzing mass spectrometry data includes:

• Quality control and preprocessing:

  • Evaluate technical quality (identified peptides, mass accuracy)

  • Apply appropriate normalization methods

  • Filter contaminants and false positives using statistical thresholds

• Identification of biotinylation sites:

  • Search for mass shifts corresponding to biotin addition

  • Identify specific modified lysine residues

  • Compare observed sites with predicted accessible regions

• Quantitative analysis:

  • Use appropriate quantification methods (label-free, SILAC, TMT)

  • Apply statistical tests to identify significant changes

  • Normalize against internal standards

• Interaction analysis:

  • Compare against negative controls to identify specific interactors

  • Use statistical methods designed for interaction proteomics

  • Validate key interactions using orthogonal methods

• Pathway and network analysis:

  • Map identified proteins to biological pathways

  • Perform enrichment analysis to identify overrepresented functions

  • Construct protein-protein interaction networks

Modern proteomics technology can identify more than 10,000 proteins in individual cell types, but still benefits greatly from increased mass spectrometer performance for improved peptide identification and quantification accuracy .

How can researchers resolve contradictory data when comparing different enrichment methods for RPS27A?

When faced with contradictory data from different enrichment approaches:

• Technical validation:

  • Verify all methods were performed correctly

  • Ensure consistent sample handling

  • Check for potential technical artifacts

  • Validate findings using orthogonal techniques

• Methodological considerations:

  • Understand the inherent biases of each method:

    • Biotinylation targets primary amines

    • Hydrazide capturing targets glycosylated proteins

  • Consider that contradictions might reflect complementary rather than conflicting data

  • Determine if contradictions relate to identification, quantification, or both

• Biological explanations:

  • Investigate if RPS27A exists in different forms or locations

  • Consider post-translational modifications affecting detection

  • Evaluate if experimental conditions induced changes in RPS27A properties

• Reconciliation strategies:

  • Use a decision tree approach to evaluate the reliability of each finding

  • Weight results based on the proven reliability of each method

  • Consider developing hybrid approaches combining the strengths of multiple methods

Studies have demonstrated that the use of different approaches targeting various protein characteristics greatly improves coverage, suggesting that apparent contradictions may actually represent complementary aspects of complex biological systems .

What implications does RPS27A have for understanding cancer cell surfaceomes?

Biotinylated RPS27A studies offer insights into cancer cell surfaceomes:

• Identifying cancer-specific surface markers:

  • Cancer cells have altered surface protein expression

  • Cell surface biotinylation can reveal these differences

  • The hypothesis that cancer cells display detectable differences in membrane proteins due to genetic instability and different environmental needs is supported by research

• Method optimization:

  • Different enrichment strategies may reveal complementary aspects of the cancer cell surfaceome

  • Biotinylated RPS27A can help optimize these methods for maximum coverage

• Discovering unconventional surface proteins:

  • Some traditionally intracellular proteins may appear at cancer cell surfaces

  • Biotinylation approaches can identify these "moonlighting" proteins

• Developing targeted therapeutics:

  • Cancer-specific surface proteins represent potential targets for immunotherapy

  • Recent breakthroughs in immune therapies highlight the importance of distinguishing cancer cells from healthy cells through their surface proteins

• Understanding post-translational modifications:

  • Cancer cells may exhibit altered patterns of protein modifications

  • Comparing different enrichment strategies can reveal these differences