Nuclear Proteome Isolation With Detergent and Salt Extraction

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Solution Overview

Problem

Existing methods struggle to effectively solubilize and isolate nuclear proteins for proteomic characterization due to their low abundance, sequestration by insoluble chromatin, and extensive post-translational modifications.

Innovation Solution

A method involving suspension solutions without polycations, detergent treatment, and salt extraction is used to isolate nuclear proteins, followed by quenching and pelleting insoluble chromatin, enabling data-independent acquisition (DIA) for enhanced sensitivity and label-free quantification.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If conventional proteomics methods are used to characterize nuclear proteins, then the analysis can be performed with standard procedures, but the sensitivity is insufficient for detecting low abundance nuclear proteins

Engineering Contradiction:

Improvesensitivity of proteomic characterizationVSAvoidabundance of nuclear proteins

Core Design Contradiction:

Measurement precisionVSQuantity of substance

Solution Approach 1:

The patent extracts nuclear proteins from cells through a multi-step isolation process involving detergent treatment (NP-40) to remove membranes, salt extraction (NaCl) to release chromatin-associated proteins, and pelleting insoluble chromatin. This extraction isolates the target nuclear proteins from the complex cellular environment, enabling sensitive detection by mass spectrometry despite their low abundance in total cell lysates.

Inventive Principle:

Principle #2Taking out (Extraction)

2Ease of operation

If standard cell lysis methods are used, then the process is simple, but nuclear proteins remain sequestered by insoluble chromatin and are not adequately isolated

Engineering Contradiction:

Improvesimplicity of isolation procedureVSAvoidcompleteness of nuclear protein isolation

Core Design Contradiction:

Ease of operationVSReliability

Solution Approach 1:

The patent segments the isolation process into distinct sequential steps: (1) detergent treatment to remove membranes and cytoplasmic components, (2) salt extraction to release proteins from chromatin, (3) pelleting of insoluble chromatin, and (4) collection of supernatant containing isolated nuclear proteins. This segmentation transforms a complex isolation challenge into manageable steps, each optimizing for a specific function while maintaining overall procedural simplicity.

Inventive Principle:

Principle #1Segmentation

3Stability of the object's composition

If polycations are present in the suspension buffer, then chromatin structure is maintained, but nuclear proteins remain bound to chromatin and cannot be effectively isolated

Engineering Contradiction:

Improveintegrity of chromatin structureVSAvoidefficiency of protein isolation

Core Design Contradiction:

Stability of the object's compositionVSEase of manufacture

Solution Approach 1:

The patent applies preliminary action by first treating cells with detergent to remove membranes and cytoplasmic contaminants before performing salt extraction. This preliminary step prevents interference from other cellular components during the subsequent chromatin extraction, ensuring that the isolation process specifically targets nuclear proteins while maintaining chromatin structure integrity during the transition.

Inventive Principle:

Principle #10Preliminary action

4Adaptability or versatility

If extensive post-translational modifications are present on nuclear proteins, then the proteins have diverse functional states, but proteomic characterization becomes more difficult

Engineering Contradiction:

Improvefunctional diversity of nuclear proteinsVSAvoidcomplexity of proteomic analysis

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

The patent employs parameter changes in the form of controlled salt concentration variations during extraction. By adjusting NaCl concentration, the method optimizes the release of proteins with different chromatin association strengths, effectively separating proteins based on their binding characteristics regardless of post-translational modifications. This parameter optimization enables comprehensive detection of diverse nuclear proteins including those with extensive modifications.

Inventive Principle:

Principle #35Parameter changes

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

This approach significantly enhances the sensitivity and accuracy of proteomic characterization of nuclear proteins, allowing for comprehensive analysis of chromatin-associated protein complexes and systems-wide nuclear protein dynamics.

Implementation Method 1

adding a detergent solution to isolate the cell nuclei of the cells

Methodology Applied

Scientific EffectDetergent solubilization: Surfactant

Implementation Method 2

preparing a first suspension solution with buffer A not comprising polycations

Methodology Applied

Scientific EffectPolycation removal:

Implementation Method 3

preparing a second suspension solution comprising the first suspension solution and a salt

Methodology Applied

Scientific EffectIonic extraction: Ion Exchange

Implementation Method 4

pelleting insoluble chromatin from the second suspension solution

Methodology Applied

Scientific EffectSedimentation: Sedimentation

Data Source

PatentUS20250216400A1Isolation and Characterization of the Nuclear Proteome

Publication Date: 2025.07.03 ALTIUS INST FOR BIOMEDICAL SCI

AI summary

Disclosed herein are methods for solubilizing and isolating nuclear proteins from cells.