OOPS-MS Service: Unbiased Total RNA-Binding Protein (RBPome) Discovery

Q: Does OOPS-MS identify which specific RNA is bound to the protein?

No. OOPS-MS identifies the global community of proteins bound to the total cellular RNA pool. It tells you 'which proteins have RNA-binding capacity.' To identify the specific RNA targets of a single discovered protein, you would need our CLIP-seq Service.

Q: How do you prove the proteins at the interphase are actually bound to RNA?

This is addressed through our strict RNase control. We collect the interphase and perform extensive denaturing washes. We then split the sample: one half receives RNase treatment, the other serves as a negative control. Only proteins that are specifically released into the soluble fraction dependent upon RNA degradation are classified as genuine RBPs.

Q: Can I use OOPS-MS on samples that were not UV cross-linked?

No. The orthogonal organic phase separation relies on the covalent bond created by UV cross-linking between the RNA and the protein. Without this covalent tether, the RNA will partition to the aqueous phase and the protein to the organic phase, leaving nothing at the interphase.

Q: Why is OOPS better than using Trizol extraction alone and analyzing the organic phase?

The organic phase contains all cellular proteins, regardless of whether they bind RNA. OOPS specifically isolates the interphase, which is highly enriched for the physical RNA-protein complexes. Analyzing the interphase provides the specificity required to map the RBPome.

Q: How long does a typical OOPS-MS project take?

From sample receipt to the delivery of the final bioinformatics report, a standard OOPS-MS project typically takes 6 to 8 weeks, depending on sample complexity, cross-linking optimization requirements, and mass spectrometry queue times.

Orthogonal Organic Phase Separation coupled with Mass Spectrometry (OOPS-MS) is a revolutionary, tag-free technique designed to capture the entire RNA-binding proteome (RBPome). By utilizing physical phase separation, OOPS-MS isolates proteins cross-linked to all RNA species—including mRNA, lncRNA, circRNA, and rRNA—enabling unbiased, total RNA-protein interaction discovery without the severe biases of poly(A) enrichment.

Whether you are charting the global RNA-protein interaction network, discovering novel non-coding RNA binding partners, or investigating dynamic RBPome shifts under drug stress, our comprehensive OOPS-MS Service delivers deep, highly specific proteomic data.

Universal Total RNA Coverage: Captures proteins bound to both coding and non-coding RNAs simultaneously.
Completely Tag-Free: Requires no genetic manipulation or custom probes, making it ideal for primary cells, tissues, and clinical models.
Stringent False-Positive Control: Employs rigorous RNase digestion controls and parallel negative controls to eliminate background noise.
Deep Proteomic Coverage: Powered by high-resolution Orbitrap LC-MS/MS coupled with advanced bioinformatics for structural domain mapping.

Consult Our Proteomics Experts

What is OOPS-MS? The Principle of Orthogonal Organic Phase Separation

Understanding global RNA-protein interactions requires a method that efficiently separates cross-linked RNA-protein complexes from the vast excess of free proteins and free RNAs in a cell. Orthogonal Organic Phase Separation (OOPS) achieves this through fundamental physical chemistry and exploiting the distinct biochemical properties of macromolecules.

Following in vivo UV cross-linking to freeze physiological interactions at zero distance, cells are lysed using a standard acidic guanidinium thiocyanate-phenol-chloroform mixture (commonly known as TRIzol reagent). Upon rigorous homogenization and high-speed centrifugation, the lysate resolves into three distinct phases based on solubility and polarity:

The Upper Aqueous Phase: Highly polar, uncross-linked, free RNAs (and some DNA depending on pH) partition into this upper water-based layer.
The Lower Organic Phase: Phenol denatures free, uncross-linked proteins, exposing their hydrophobic cores and causing them to dissolve entirely into this lower organic layer, along with cellular lipids.
The Interphase (Middle Layer): This is the crucial fraction. Covalently cross-linked RNA-protein complexes represent massive amphiphilic hybrid molecules. The highly polar RNA tail resists the organic phase, while the denatured hydrophobic protein mass resists the aqueous phase. Consequently, these complexes are trapped and accumulate exclusively at the interphase.

By carefully extracting this interphase, subjecting it to stringent denaturing washes to remove loosely associated contaminants, and utilizing specific RNases to release the covalently bound proteins, OOPS-MS elegantly enriches the Total RBPome for downstream high-resolution mass spectrometry analysis.

Diagram of Orthogonal Organic Phase Separation showing RNA, protein, and interphase layers in a centrifuge tube.
The OOPS Principle: Absolute enrichment of RNA-protein complexes at the aqueous-organic interphase following UV cross-linking.

Overcoming the Poly(A) Limitation: Why Choose Total RBPome Profiling?

Historically, global RNA-binding protein discovery relied heavily on RNA Interactome Capture (RIC), which utilizes Oligo(dT) magnetic beads to pull down transcripts. While highly effective for specific translational applications, RIC has a critical, fundamental blind spot: it exclusively captures polyadenylated (poly(A)+) transcripts, effectively ignoring up to 70% of the active transcriptome.

Our Total RBPome Profiling powered by OOPS-MS entirely eliminates this sequence bias. It is the essential method for researchers focused on the intricate world of the Non-coding RNA interactome. By abandoning poly(A) selection, OOPS-MS allows you to identify effector proteins interacting with:

Long non-coding RNAs (lncRNAs): Essential for epigenetic regulation and chromatin remodeling.
Circular RNAs (circRNAs): Crucial miRNA sponges and transcriptional regulators often implicated in oncology.
MicroRNAs (miRNAs) and small nucleolar RNAs (snRNAs): The core components of the RNA interference (RNAi) and splicing machineries.
Bacterial and Viral RNAs: Which naturally lack eukaryotic poly-A tails, making OOPS-MS indispensable for host-pathogen interaction studies.

Technical Advantages of Our Tag-Free OOPS-MS Platform

We have optimized the OOPS-MS protocol over hundreds of diverse sample types to deliver superior sensitivity and specificity, overcoming the traditional limitations of interactome capture.

Tag-Free and Antibody-Free

The protocol operates completely independent of artificial protein tagging (such as FLAG, HA, or GFP) or the need for highly specific antibodies. This eliminates artificial overexpression artifacts, prevents steric hindrance caused by large tags, and captures the true native interactome exactly as it exists in vivo.

Universal Species Compatibility

Because orthogonal phase separation relies on the universal physical properties of cross-linked macromolecules rather than specific sequence tags, our service is universally applicable. We successfully process human clinical biopsies, murine models, Arabidopsis plant tissues, yeast, and complex bacterial cultures.

Addressing the Specificity Concern (Strict RNase Controls)

A common critique of interphase collection is the potential co-precipitation of highly insoluble free proteins or aggregated cellular debris. We eliminate this false-positive risk by incorporating a strict RNase digestion step. Following interphase isolation, the sample is split. Only proteins that are liberated into the soluble supernatant specifically after targeted RNA degradation—and are absent in the undigested negative control—are considered genuine, high-confidence RBPs.

Technical Services

Service Scope Workflow MS Platforms Tech Comparison Case Study Bioinformatics Deliverables Sample Requirements FAQ Get a Custom Proposal

Comprehensive OOPS-MS Service Scope: Applications in Total RBPome Discovery

Our robust OOPS-MS platform supports a wide array of high-value research applications across basic biology and translational medicine:

Global RBPome Discovery

Unbiased identification of all RNA-binding proteins within a specific cell type, tissue, or developmental stage, significantly expanding the known universe of RNA-protein interactions beyond classical translation factors.

Spatial-Temporal RBPome Dynamics

Quantitatively profiling the global shift in RNA-binding proteins in response to viral infection, metabolic stress, oxidative damage, hypoxia, or targeted drug treatments.

Non-Classical RBP Identification

Discovering novel "moonlighting" proteins. Emerging research shows that many metabolic enzymes and kinases lack classical RNA-binding domains (like RRM or KH) but temporarily interact with RNA to regulate cellular homeostasis. OOPS-MS is the gold standard for discovering these hidden interactions.

Target Deconvolution

Complementing our RIP-seq Service and CLIP-seq Service by providing the comprehensive proteomic half of the interactome puzzle, linking uncharacterized non-coding RNAs to their effector protein complexes.

The Standardized OOPS-MS Workflow & Strict RNase Quality Control

Generating high-confidence RBPome data requires meticulous execution. Our end-to-end workflow ensures high reproducibility and deep proteome coverage while aggressively filtering false positives at multiple checkpoints.

Step-by-step workflow of OOPS-MS total RNA binding protein profiling service.

The OOPS-MS Workflow: From in vivo cross-linking to high-resolution mass spectrometry.

In Vivo UV Cross-linking

Live cells or cryo-pulverized tissues are irradiated with high-intensity UV light (typically 254 nm) on ice. This instantly freezes direct, zero-distance RNA-protein interactions via covalent bonds.

Cell Lysis & Phase Separation

Samples are homogenized in a specialized acidic phenol-chloroform reagent and subjected to high-speed centrifugation to establish the three-phase system.

Interphase Recovery & Purification

The dense RNA-protein-rich interphase is carefully extracted. Multiple stringent denaturing washes (using high salt and urea) are performed to strip away uncross-linked contaminating proteins and free RNA.

RNase Release (Critical QC)

The purified interphase is treated with optimized, high-efficiency RNase cocktails. This specifically digests the RNA backbone, liberating the cross-linked RBPs into the soluble fraction while leaving insoluble debris behind.

LC-MS/MS Identification

The released proteins are subjected to in-solution trypsin digestion, generating peptides that are analyzed using high-resolution mass spectrometers.

Mass Spectrometry Platforms & Analytical Parameters

Deep RBPome profiling demands ultimate analytical sensitivity and dynamic range. To detect both high-abundance ribosomal complexes and low-abundance transient regulatory RBPs, we utilize industry-leading instrumentation and rigorous data acquisition parameters.

Instrumentation: Nano-LC systems coupled to Orbitrap Exploris™ 480 or Orbitrap Eclipse™ Tribrid™ mass spectrometers. These systems provide exceptional resolution and mass accuracy (< 5 ppm), which is essential for confidently resolving complex interphase peptide mixtures.
Acquisition Modes: We offer both traditional Data-Dependent Acquisition (DDA) for maximum discovery depth in novel models, and advanced Data-Independent Acquisition (DIA) for highly reproducible, quantitative profiling across multiple treatment groups.
Quantification Strategy: Label-Free Quantification (LFQ) is standard for dynamic RBPome comparisons. For high-throughput, multi-condition experimental designs, TMT (Tandem Mass Tag) multiplexing is available to eliminate run-to-run variation.
Quality Control Parameters: Peptide and protein identifications are strictly filtered at a False Discovery Rate (FDR) of < 1%. Decoy database searching and strict RNase/No-RNase control subtractions are mandatorily applied to validate genuine RNA-binding capacity before data release.

Technology Comparison: OOPS-MS vs. Oligo(dT) Capture vs. ChIRP-MS

Selecting the right biophysical assay is critical for project success and budget optimization. Use this expanded guide to determine the best approach for your specific RNA-protein interaction question.

Analytical Parameter	OOPS-MS (Total RNA)	Oligo(dT) Capture (RIC)	ChIRP-MS Service
Primary Target	Total RNA (mRNA + all ncRNA)	mRNA only (Poly-A restricted)	One Specific Target RNA
Output Data	Global, unbiased Total RBPome	Global mRNA-bound RBPome	Proteins bound to one specific RNA of interest
Capture Mechanism	Physical phase separation (Interphase)	Oligo(dT) magnetic bead pull-down	Custom biotinylated antisense probes
Starting Material Need	Low (∼ 1 × 10⁷ cells)	High (∼ 1 × 10⁸ cells)	High (∼ 1 × 10⁸ cells)
Species Compatibility	Universal (All organisms)	Eukaryotes only	Universal (if RNA sequence is known)
False-Positive Risk	Low (Mitigated by RNase controls)	Medium (Bead non-specific binding)	High (Requires extensive scramble probe controls)
Dynamic Profiling	Excellent for whole-cell stress responses	Good for translational responses only	Targeted to the single RNA of interest
Best Used For	Discovering ncRNA interactomes, identifying non-classical RBPs, whole-cell mapping.	Profiling classic mRNA translational regulators.	Discovering the protein interactome of a single, well-characterized lncRNA.

Case Study: Comprehensive Identification of Total RNA-Protein Interactions

Research Objective:

To define the total RNA-binding proteome (RBPome) across highly divergent organisms (human, yeast, and bacteria) in a completely unbiased, tag-free manner, aiming to uncover proteins that bind to non-coding and non-polyadenylated RNAs that previous methods failed to detect.

How OOPS-MS Was Used:

Researchers applied in vivo UV cross-linking (254 nm) to living HEK293 cell lines, Saccharomyces cerevisiae, and Escherichia coli. This was followed by acidic phenol-chloroform extraction (the OOPS method). The interphase, heavily enriched for RNA-protein complexes, was isolated and subjected to stringent washes. Covalently bound proteins were specifically released via targeted RNase treatment and subsequently identified and quantified by high-resolution LC-MS/MS. Label-free quantification (LFQ) was used to rigorously compare the RNase-treated samples against no-RNase negative controls to establish true RBP status.

Key Findings from OOPS-MS:

As visualized in Figure 2 of the referenced study, the OOPS-MS platform recovered massive datasets of high-confidence RNA-binding proteins: 1,838 RBPs in human cells, 944 in yeast, and 926 in E. coli. Crucially, OOPS enriched a vast array of proteins completely missed by traditional poly(A) capture methods. Over 50% of the proteins identified in the OOPS-enriched fraction lacked classical RNA-binding domains (such as RRM, KH, or DEAD-box domains). This unveiled a massive hidden layer of "moonlighting" non-classical RBPs—including key metabolic enzymes like Enolase and GAPDH—that interact with RNA to dynamically regulate cellular metabolism and stress response pathways.

Why OOPS-MS Was Essential:

Traditional RNA Interactome Capture (RIC) relies on oligo(dT) beads, inherently failing to capture proteins bound to lncRNAs, circRNAs, or bacterial RNAs (which entirely lack poly-A tails). OOPS-MS provided an unbiased, organism-agnostic, and completely tag-free solution to map the true global RBPome across all domains of life.

Additional Techniques:

RNase/no-RNase false-positive filtering, quantitative proteomics (LFQ), GO/KEGG pathway enrichment analysis, cross-linking efficiency assays, and structural domain mapping using the Pfam database.

Reference:

Queiroz, R. M. L., et al. "Comprehensive identification of RNA–protein interactions in any organism using orthogonal organic phase separation (OOPS)." Nature Biotechnology 37.2 (2019): 169-178.

Venn diagram and bar chart demonstrating the superior RBP coverage of OOPS-MS across human, yeast, and bacterial models.

Comprehensive RBPome Discovery: OOPS-MS expands the known interactome by capturing non-polyadenylated RNA binders across multiple domains of life.

Advanced Bioinformatics for RBPome Analysis

Generating global proteome data is only the first step. Our dedicated bioinformatics team transforms complex mass spectrometry outputs into actionable, publication-ready biological insights.

High-Confidence RBP Filtering: Statistical deduction of background noise by comparing the RNase-released fraction against non-digested controls, ensuring only high-confidence RBPs are reported.
Differential RBPome Analysis: Identifying proteins with significantly altered RNA-binding affinity between experimental and control groups (e.g., wild-type vs. knockout, vehicle vs. drug treatment), visualized via customized Volcano plots.
RBD (RNA-Binding Domain) Mapping: Cross-referencing identified proteins with structural databases (Pfam, SMART) using Hidden Markov Models to systematically classify them into classical RBPs and novel, non-classical RBPs.
Protein-Protein Interaction (PPI) Networks: Mapping the physical and functional associations among the identified RBPs using the STRING database to reveal multi-protein regulatory complexes (e.g., spliceosome sub-complexes, exosome complexes).
GO & KEGG Pathway Enrichment: Determining the overarching biological processes and metabolic pathways governed by the enriched RNA-binding proteins.

Project Deliverables & Demo Results

We provide a comprehensive, transparent data package engineered to support seamless integration into your research narrative, IND applications, and high-impact journal submissions. Standard deliverables include the complete methodology report, raw LC-MS/MS spectra, normalized intensity matrices, and statistically filtered RBP lists.

Venn diagram comparing total RNA binding proteins identified by OOPS-MS versus traditional oligo-dT capture.

Technology Overlap Analysis

Demonstrating the superior coverage of OOPS-MS in capturing non-polyadenylated RNA interactors.

Volcano plot visualizing significant dynamic changes in the RBPome analyzed by OOPS-MS.

Differential Enrichment Plot

Highlighting significant shifts in RNA-binding dynamics following experimental treatment.

Pie chart showing the distribution of classical and non-classical RNA binding domains in proteins identified by OOPS.

Structural Domain Distribution

Revealing the high proportion of non-classical "moonlighting" proteins discovered within the global RBPome.

PPI network diagram showing functional clustering of RNA-binding proteins identified via OOPS-MS.

Protein-Protein Interaction (PPI) Network

Mapping functional clusters and multimeric regulatory complexes within the identified Total RBPome.

Sample Requirements for In Vivo Cross-Linking

Proper sample preparation and effective in vivo cross-linking are the most critical determinants of a successful OOPS-MS experiment.

Sample Type	Minimum Requirement	Recommended Amount	Notes for UV Cross-linking
Cultured Cell Lines	1 × 10⁷ cells	2 × 10⁷ to 5 × 10⁷ cells	Cross-link at 254 nm on ice (typically 150-400 mJ/cm²). Harvest immediately.
Animal Tissue	50 mg	100 mg - 200 mg	Tissue must be flash-frozen. Cryo-pulverization before UV cross-linking is required for uniform irradiation.
Plant Tissue	200 mg	500 mg	Requires careful optimization to penetrate cell walls during UV irradiation.
Yeast/Bacteria	OD₆₀₀ = 10 (∼50 mL)	OD₆₀₀ = 20	Rapid chilling before cross-linking is essential to freeze metabolic states.

*Note: If you lack the equipment for precise 254 nm UV irradiation, please consult our team. We can provide detailed protocols or discuss accepting live/frozen samples for cross-linking in our facility.

FAQs on OOPS-MS and Total RNA-Protein Interactions

1. Does OOPS-MS identify which specific RNA is bound to the protein?

No. OOPS-MS identifies the global community of proteins bound to the total cellular RNA pool. It tells you "which proteins have RNA-binding capacity." To identify the specific RNA targets of a single discovered protein, you would need our CLIP-seq Service.

2. How do you prove the proteins at the interphase are actually bound to RNA?

This is addressed through our strict RNase control. We collect the interphase and perform extensive denaturing washes. We then split the sample: one half receives RNase treatment, the other serves as a negative control. Only proteins that are specifically released into the soluble fraction dependent upon RNA degradation are classified as genuine RBPs.

3. Can I use OOPS-MS on samples that were not UV cross-linked?

No. The orthogonal organic phase separation relies on the covalent bond created by UV cross-linking between the RNA and the protein. Without this covalent tether, the RNA will partition to the aqueous phase and the protein to the organic phase, leaving nothing at the interphase.

4. Why is OOPS better than using Trizol extraction alone and analyzing the organic phase?

The organic phase contains all cellular proteins, regardless of whether they bind RNA. OOPS specifically isolates the interphase, which is highly enriched for the physical RNA-protein complexes. Analyzing the interphase provides the specificity required to map the RBPome.

5. How long does a typical OOPS-MS project take?

From sample receipt to the delivery of the final bioinformatics report, a standard OOPS-MS project typically takes 6 to 8 weeks, depending on sample complexity, cross-linking optimization requirements, and mass spectrometry queue times.