Table 5.

Overview of proteomic platforms used for biomarker development

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

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