The discovery and validation of biomarkers in body fluid samples is an important means of achieving precise diagnosis and treatment of diseases, understanding the mechanisms of disease occurrence, discovering drug targets, and exploring their potential mechanisms of action. Taking neurodegenerative diseases as an example, this article compiles the currently available body fluid proteomics detection technologies, which mainly fall into two major categories: mass spectrometry-based and immunoassay-based.
01 Mass Spectrometry-Based Analytical Methods
Label-free Mass Spectrometry Detection
Label-free mass spectrometry detection (LF-MS) adopts a "bottom-up" analytical strategy to perform non-targeted protein content analysis on various body fluid or tissue samples. This method is peptide-centric: it first performs protease digestion to generate peptides, then infers proteins at the peptide level, and quantifies proteins based on accumulated signals. It requires no chemical labels or internal standards and is typically used for large-scale proteomics research. It can be performed on a variety of mass spectrometry platforms, such as electrospray ionization and high-resolution mass analyzers, including Orbitrap, quadrupole, and time-of-flight. Mass spectrometry data acquisition modes include DDA and DIA.
Targeted Quantitative Mass Spectrometry Detection
Mainly includes multiple reaction monitoring (MRM) and parallel reaction monitoring (PRM)
MRM: low-resolution triple quadrupole (QQQ) mass spectrometer
PRM: high-resolution mass analyzer, such as quadrupole-Orbitrap or Q-TOF systems
By adding isotope-labeled proteins/peptides to samples and simultaneously monitoring endogenous peptides and isotope-labeled peptides, quantitative detection of target proteins is achieved. Therefore, before performing targeted MS, the target proteins and their specific peptides must be determined in advance to create a specific targeted method.
Technical Characteristics
MS detection does not depend on antibodies, and has a broad range of protein identification with high specificity. However, the technology is complex and requires specialized personnel and laboratory-provided dedicated equipment and instruments.
LF-MS and its bioanalytical workflow can unbiasedly identify protein profiles under specific disease states and can be used for biomarker discovery.
Advantages: Simple experimental setup, no labeling or subsequent complex experimental design required, applicable to a variety of sample types (cell cultures, biological fluids, tissue extracts, etc.).
Disadvantages: Poor reproducibility, especially when using the DDA method, with coefficients of variation between 15%-20% and a large proportion of missing values. Low throughput, and differential expression analysis is less accurate than label-based methods.
MRM-MS/PRM-MS can be used for cohort validation and detect and quantify low-abundance proteins with higher sensitivity and in a repeatable manner.
PRM allows retrospective selection of relevant ion pairs for quantification, without the need to select them prior to analysis, and has high specificity.
Prospects
Research shows that quantitative targeted MS methods outperform immunoassays in discovering some key blood biomarkers for Alzheimer's disease (AD), such as amyloid and tau proteins. Therefore, the application of mass spectrometry in clinical settings has certain prospects, but significant improvements in technology and cost are needed to make it compatible with routine applications. Using the highly efficient parallel performance of MS to quantify a panel of biomarkers and combining different biomarkers to build effective and reliable algorithms—for example, for neurodegeneration, neuroinflammation, synaptic function, and co-pathology—is also highly promising.
02 Antibody-Based Detection Methods
Proteomics Based on Proximity Extension Assay
The proximity extension assay (PEA) technology is a multiplex antibody-based proteomics method that can currently detect more than 3,000 proteins. It combines antibody-based and DNA-based methods to measure different body fluid...
Technical Characteristics
Advantages: Simultaneous detection of thousands of proteins, high detection sensitivity, requiring only a small amount of sample.
Disadvantages: Protein selection is biased, possibly overestimating proteins with good antibody-binding capacity while underestimating those without antibodies or with weak antibody-binding capacity. Most detection panels can only provide relative quantification of protein concentrations, making it impossible to directly compare different proteins in the same batch, or the same protein across different batches.
Prospects
With the identification of novel proteins and the development of highly specific antibodies, the detection range of PEA can be further expanded. Customized products for specific diseases can be developed, including standards for absolute quantification. As PEA is applied in more and more studies, cross-disease meta-analysis will become important; to explore the proteome more deeply, multi-platform proteomics research combining PEA, Somascan, and MS has been carried out.
Bead-Based Multiplex Proteomics Analysis
Bead-based microarray technology utilizes multiplex antibody-binding analytical methods to achieve high-throughput multiplex protein analysis. Highly specific capture antibodies are immobilized on the surface of fluorescently encoded magnetic beads, which are mixed to form a suspended bead array, allowing simultaneous qualitative and quantitative analysis of multiple proteins in microplates. Among the established analytical methods for cerebrospinal fluid and plasma, 384 target proteins in 384 samples can be analyzed in parallel.
Technical Characteristics
Advantages: High throughput, parallel analysis of multiple target proteins, low sample consumption, simple preparation and workflow, and relatively high sensitivity and specificity. The detection limit range is typically pg/mL to ng/mL. Standard curves can be generated within a single assay, enabling absolute quantification assessment and, over time, the accumulation of a standard curve dataset. Different magnetic beads can be freely combined to a certain extent, allowing customized protein detection panels for specific research fields. The same reagents can also be used to transition from multiplex exploratory research to targeted confirmatory research.
Disadvantages: Not suitable for large-scale studies (cannot include standard curves for all proteins), high cost, and requiring biological and technical reproducibility validation.
Prospects
As the number of antibodies increases, the detection and application range can be further expanded. By developing new detection technologies (such as photo-excited chemiluminescence immunoassay), detection sensitivity and parallel detection performance can be improved.
Enzyme-Linked Immunosorbent Assay
The enzyme-linked immunosorbent assay (ELISA) is an antibody-based technology for detecting trace proteins in liquid matrices.
Technical Characteristics
Advantages: High sensitivity for detecting neuronal trace proteins in cerebrospinal fluid samples.
Disadvantages: Poor sensitivity for blood samples, and optimizing antibodies suitable for ELISA often takes a long time.
Prospects
ELISA dominates clinical diagnosis, but the future of the in vitro diagnostics industry lies in the digitalization and multiplexing of immunoassays.
Chemiluminescence and Electrochemiluminescence
Chemiluminescence (CLIA) and electrochemiluminescence immunoassay (ELC) are both detection methods based on closed antibody systems, capturing immune complexes via bead-coupled biotin-streptavidin, with luminescence as the indicator of the analytical reaction.
Technical Characteristics
Advantages: Compared with colorimetric methods (such as ELISA), it provides absolute quantification of the analytical reaction, with a large dynamic range, and high sensitivity and specificity.
Disadvantages: High cost, limited availability of analyte detection and test panels, and a closed analytical system.
Prospects
CLIA shows great potential for multiplex immunoassays, which are becoming increasingly popular in clinical applications. As these technologies develop, the translation of novel biomarkers into clinical applications will accelerate.
Simoa
Simoa is a bead-based ELISA that can be run on the Quanterix HD-X/HD-1 fully automated analyzer or the SR-X analyzer.
Technical Characteristics
Advantages: High sensitivity, more than 1,000 times higher than ELISA, with a lower detection limit reaching the fg/mL level, enabling effective detection and quantification of ultra-low-abundance proteins. High throughput and multiplex detection capability, with protocols that can be developed and optimized according to experimental objectives.
Disadvantages: High cost of instruments and reagent consumables, and requiring specialized personnel to ensure correct operation and maintenance of the platform.
Prospects
Research shows that Simoa can be further optimized by improving bead reading efficiency; after improvement, it allows detection of proteins at sub-attomolar concentrations, and this sensitivity may open a window of opportunity for biomarker detection in new matrices.
Microfluidic Immunoassay
The ProteinSimple fully automated microfluidic immunoassay instrument provides a microfluidic immunoassay (ELLA) platform, in which specific capture antibodies are coated in microfluidic glass reaction tubes (GNRs), and three GNRs are then embedded in a fully automated microfluidic immunoassay tube. The target analyte binds to the capture antibody, and with the action of the detection antibody, each sample automatically outputs data and the mean of 3 parallel GNRs. Concentrations are generated using a calibrated standard curve, and the system can be used for single and multiplex detection.
Technical Characteristics
Advantages: ELLA is a small benchtop machine that is easy to operate and has high throughput; it offers an open cartridge version that allows customers to set up assays as needed.
Disadvantages: High cost, and sensitivity for NFL or cytokines is inferior to that of Simoa.
Prospects
The small and easy-to-use benchtop ELLA platform can be used for routine detection of low-abundance biomarkers in clinical settings, and can also be used for point-of-care monitoring in emergency or intensive care units.
Combined Application of Proteomics and Transcriptomics
Although proteomic detection results are more direct, transcriptomic analysis also has some important applications in the field of neurodegenerative diseases. Differences in protein abundance between healthy individuals and patients may have multiple causes: 1. Protein abundance directly affects disease, for example, proteins failing to degrade, or becoming phosphorylated or aggregated together; 2. The disease may cause changes in gene regulation that in turn affect protein levels. The latter can be analyzed by RNA-seq, so combined protein and transcriptomic analysis can provide more information and thus reveal the molecular mechanisms of disease occurrence.
[Reference] Teunissen CE, Kimble L, Bayoumy S, et al. Methods to Discover and Validate Biofluid-Based Biomarkers in Neurodegenerative Dementias. Mol Cell Proteomics. 2023 Oct;22(10):100629.
