Modern proteomic research requires both qualitative and quantitative analysis of peptides and proteins to gain insights into biological systems related to normal and disease states. Understanding protein quantities and their post-translational modifications (PTMs) is crucial for identifying significant biological markers for tumors and other diseases, necessitating sensitive and reliable quantitative data. However, quantitative proteomics faces challenges such as sample complexity, the wide dynamic range of protein abundance, and varying physico-chemical properties of proteins, complicating sample handling and analysis. Technological advancements, particularly in separation techniques and mass spectrometry (MS) detection, have led to various approaches for comprehensive proteome assessment. One- and two-dimensional polyacrylamide gel electrophoresis (1D or 2D-PAGE) are powerful separation techniques but struggle with sensitivity, reproducibility, sample loss, and low dynamic range in quantitative aspects. Consequently, 2D-PAGE has largely been supplanted by liquid chromatography (LC) techniques, with LC coupled to molecule-specific MS emerging as a viable alternative for the qualitative characterization of complex mixtures.
