The accurate quantitation of proteins and peptides in complex biological systems is one of the most challenging areas of proteomics. Mass spectrometry-based approaches have forged significant in-roads allowing accurate and sensitive quantitation and the ability to multiplex vastly complex samples through the application of robust bioinformatic tools. These relative and absolute quantitative measures using label-free, tags, or stable isotope labelling have their own strengths and limitations. The continuous development of these methods is vital for increasing reproducibility in the rapidly expanding application of quantitative proteomics in biomarker discovery and validation. This paper provides a critical overview of the primary mass spectrometry-based quantitative approaches and the current status of quantitative proteomics in biomedical research. 1. Introduction Quantification in a proteomics setting relies on the ability to detect small changes in protein and peptide abundance in response to an altered state [1]. Differential analysis is generated from LC-MS experiments and can be carried out using both label and label-free approaches. For trace amounts of proteins within complex proteomes such as plasma, tears, and urine, no singular technique should be used as a stand-alone guarantee of quantitative precision without hypothesis-driven, targeted approaches. Enrichment and fractionation of specific classes of protein is beneficial during the discovery phase of a project, but because these methods can involve numerous steps, they can become a limiting factor for large scale validation. The variability introduced by multiple methods prior to quantitative mass spectrometry should be assessed, and it is paramount that protein measurements reflect the authentic concentration in the original sample. The development of methods for accurate protein quantitation is one of the most challenging areas of proteomics. Quantitative proteomics comes in two forms: absolute and relative. Relative quantitation compares the levels of a specific protein in different samples with results being expressed as a relative fold change of protein abundance [2]. Absolute quantitation is the determination of the exact amount or mass concentration of a protein, for example, in units of ng/mL of a plasma biomarker. Traditional proteomic quantitation approaches rely on high-resolution protein separation by 2D gels. The use of dyes, fluorophores, or radioactivity to label proteins allows visualization of spots/bands with differential intensities [3, 4]. These methods facilitate
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