What Does the Olink NPX Value Represent? Guidelines for Accurate Interpretation of Protein Expression Data
In Olink proteomics analysis based on the Proximity Extension Assay (PEA), the primary quantitative metric obtained is the NPX (Normalized Protein eXpression) value. NPX is Olink’s proprietary unit that reflects the relative abundance of target proteins. Following rigorous normalization, background subtraction, and log₂ transformation, NPX values enable valid comparisons across samples and assay batches. A precise understanding of NPX and its interpretation is essential for ensuring the reliability of biomarker discovery and subsequent studies.
Basic Concept of NPX Value
1. Definition of NPX
(1) NPX is a proprietary data unit developed by Olink to indicate the relative abundance of a specific protein.
(2) It is derived from the raw measurement signal, Cq values from qPCR or read counts from NGS, after normalization, background correction, and log₂ transformation.
(3) Values are expressed on a log₂ scale. Higher NPX values indicate higher relative protein abundance.
2. NPX: Not an Absolute Concentration
(1) NPX captures relative changes between samples but cannot be directly converted to absolute concentrations (e.g., pg/mL or ng/mL).
(2) Absolute quantification requires calibration using external standards or complementary methods such as ELISA or mass spectrometry.
NPX Value Calculation Workflow
1. Raw Signal Acquisition
(1) In qPCR-based assays, the cycle threshold (Cq) is recorded; in NGS-based assays, the number of sequencing reads is recorded.
(2) Blank wells and outliers in internal controls are removed to ensure data quality.
2. Normalization and Log Transformation
(1) Batch effects are corrected using Olink’s internal controls, including reference proteins and amplification controls.
(2) The normalized data are then log₂-transformed to produce stable NPX values, which typically range from approximately 5 to 15.
3. Quality Control Procedures
(1) Signals below the limit of detection (LOD) are flagged as missing or low-confidence.
(2) Cross-batch consistency is verified using standard samples or technical replicates, with coefficients of variation (CV) generally maintained below 10%.
Interpreting NPX Data
1. Relative Change Analysis
(1) The focus should be on NPX differences between experimental groups rather than absolute values.
(2) Common statistical approaches include t-tests, ANOVA, or non-parametric tests, with false discovery rate (FDR) correction applied to identify differentially expressed proteins.
2. Biological Interpretation
(1) Candidate proteins should be selected based on both fold change (2^Δ) and p-values.
(2) Functional insights can be obtained through pathway enrichment analysis (e.g., KEGG, Reactome) and integration with multi-omics datasets such as transcriptomics and metabolomics.
3. Important Considerations
(1) NPX values from different studies are not directly comparable due to differences in normalization strategies and batch effects.
(2) For borderline expression or low-abundance proteins, LOD flags should be carefully considered to avoid overinterpretation.
Support from MtoZ Biolabs
1. Data Processing and Analysis Services
(1) NPX normalization, differential protein analysis, pathway annotation, and generation of comprehensive visualization reports.
(2) Guidance for selecting robust candidate biomarkers based on NPX results.
2. Integrated Validation Solutions
(1) ELISA or mass spectrometry validation for key proteins, enabling conversion of NPX values to absolute concentrations.
(2) Delivery of complete data packages meeting publication standards and clinical research requirements.
NPX serves as the central metric for interpreting Olink proteomics data, representing rigorously standardized relative protein abundance. A clear understanding of both the statistical and biological implications of NPX allows researchers to more accurately identify candidate biomarkers and advance subsequent investigations. Leveraging extensive project experience and dual-platform validation capabilities, MtoZ Biolabs supports research teams throughout the entire proteomics workflow, from NPX interpretation to the translation of research findings into tangible outcomes.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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