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Table 5 Overview of proteomic platforms used for biomarker development

From: Clinical proteomics for prostate cancer: understanding prostate cancer pathology and protein biomarkers for improved disease management

Category Platform Multiplex capability Application LOD Advantages Limitations
Antibody-Based ELISA 96 proteins per assay Biomarker evaluation & validation pg/mL Highly sensitive for protein(s) of interest Dependant on antibody availability
Influenced by non-standardised variables e.g. temperature, pH, antibodes used
Large amounts of protein lysate required
Protein microarrays  > 1000 proteins per screen Biomarker evaluation & validation
Biomarker discovery
pg/mL High throughput for multiplexed analysis Reliant on availability of antibodies
Expensive
Biased to pre-selected proteins if used for biomarker discovery
Two antibodies required per protein
Proximity extension assay (PEA) Up to 100 proteins per array Biomarker discovery pg/mL High throughput
Highly sensitive for proteins of interest
Expensive
Biased to pre-selected proteins if used for biomarker discovery
Two antibodies required per protein
Aptamer-based Somascan Up to 2000 proteins per screen Biomarker evaluation & validation
Biomarker discovery
fg/mL Aptamers cheaper to produce
More sensitive than antibody-based techniques
Aptamers available for wider range of molecules
High throughput
Minimal sample required
Biased to pre-selected proteins if used for biomarker discovery
Mass Spectrometry-based DDA e.g. LC–MS/MS 1000′s of proteins Biomarker discovery ng/mL High throughput
Minimal sample required
Unbiased screen of all detectable proteins
No requirement for antibodies or somamers
Complex sample preparation required
Enrichment techniques required to detect very low abundant proteins in clinical samples
DIA e.g. SWATH &
DIA PASEF
1000′s of proteins Biomarker discovery ng/mL High throughput
Minimal sample required
Unbiased screen of all detectable proteins
No requirement for antibodies or somamers
Increased coverage of sample proteome
Has been optimised for analysis of complex sample types such as FFPE tissue
Complex sample preparation required
Enrichment techniques required to detect very low abundant proteins in clinical samples
Requires specialised Mass Spectrometer
Data storage
MRM Up to 100 proteins per run Biomarker evaluation & validation ng/mL Highly selective for proteins of interest
High throughput
No requirement for antibodies/somamers
Wide dynamic range
Complex sample preparation required
Can be affected by interfering signal from complex biological samples
Labour intensive method development
PRM  > 100 proteins per run Biomarker evaluation & validation
Biomarker discovery
ng/mL Greater sensitivity than MRM
Interfering signals filtered out
Method development less labour intensive than MRM
Complex sample preparation required
Measurements not as precise as MRM
Higher LOQ than MRM
  1. LOD: Limits of detection; DDA: Data dependent acquisition; DIA: Data independent acquisition; SWATH:; PASEF:Sequential Window Acquisition of All Theoretical Mass Spectra; Parallel accumulation—serial fragmentation; MRM: multiple reaction monitoring; PRM: parallel reaction monitoring