Table 3.

Advantages and disadvantages of various analytical techniques used for proteins, metabolites, and lipidomics biomarkers.

Techniques	Advantages	Limitations	Biomarker Type
MALDI-TOF-MS [142,143,144,145]	Rapid and straightforward operability Low sample volumes Mostly single-charged registered ions [M-H]+ High throughput High accuracy, resolution, and sensitivity No staining, labelling, anti-body, and hybridisation Suitable for large polypeptides (>30 kDa) detection	Variation in the surface of the MALDI-TOF target Limited dynamic range Sensitive to contaminants Low reproducibility	Proteins
SELDI-TOF-MS [146]	High throughput Low sample volumes High sensitivity Easy operability Suitable for small peptides (∼500 Da) detection Suitable for low MW, modified, truncated, or fragmented proteins detection	Failure of the validation process Low reproducibility Low resolution Biased toward smaller peptides and proteins (<30 kDa) Problems in larger MW proteins and PTM identification Ion suppression Prone to artefacts generation	Proteins
LC-MS [68,147,148,149,150]	High throughput High resolution Suitable for low and high-molecular-weight compounds High sensitivity	Problems in identifying hydrocarbons that produce similar ions Highly manual workflows for sample preparation can benefit from automation The high complexity of the instrumentation’s operation and maintenance when looking at a limited number of analytes	Proteins Metabolites Lipids
GC-MS [147,150]	High-efficiency separations Suitable for nonpolar, volatile, and small molecules High sensitivity High throughput	Limited mass range Limited to thermally stable and volatile compounds Destructive analysis Not suitable for compounds heavier than 1000 Da Time-consuming for sample preparation	Metabolites Lipids
NMR [150,151]	Very high reproducibility High throughput Non-destructive Sample recovery Rapid	Highly skilled operators Low sensitivity Cost is higher than GC-MS and LC-MS Difficult to quantify the noise.	Metabolites
1DGE [68,152,153,154]	Simple workflow Rapid Cost-effective	Limited reproducibility Unsuitable for low-abundance proteins Hydrophobic proteins’ insolubility	Proteins
2DGE [68,152,153,154]	High resolution High throughput Cost-effective	Gel-to-gel variation Lack of sensitivity Poor dynamic range Time-consuming Highly skilled operators Not automated approach	Proteins
2D-DIGE [153,155,156]	Wide dynamic range detection Fewer number of gels Straightforward matching between gels Higher sensitivity and reproducibility over 2DGE	Highly skilled operators Time-consuming Lower throughput Not suitable for extremely acidic, basic, or hydrophobic proteins	Proteins
Immunoassay techniques (ELISA, Western Blot) [152,157,158]	High sensitivity and specificity when looking at a limited number of analytes Cost-effective Simple workflows Highly reproducible Suitable for validation	Resource-intensive efforts Time-consuming Not recognition of posttranslational protein variants Limited multiplexing options Relatively high sample volume Cross-reactivity Stability of reagents affects outcome Limited number of analytes in each analysis	Proteins