Peptidomics in the Spotlight: Advanced Sample Treatment Techniques and Analytical Insights

In recent years, the analysis of intact peptides (peptidomics) has remained a relatively under-explored field, where its potential as a bridge between proteomics and metabolomics has only recently been recognized. Currently, immunoassay techniques are the primary method for quantifying endogenous peptides in biological samples, mainly due to their high-throughput and minimal sample manipulation. However, antibody-based strategies face important limitations, particularly in terms of specificity.Liquid chromatography-tandem mass spectrometry has emerged as a powerful and reliable alternative for the analysis of native peptides in biological fluids. While conventional sample preparation approaches, such as protein precipitation, enable rapid and broad peptide extraction, they are often inadequate for quantifying low-abundance peptides, leading to matrix effects and ion suppression.This chapter explores the critical aspects of antibody-free sample preparation in peptidomics, emphasizing advanced and innovative approaches. We also discuss methods for peptide isolation and purification, spanning from traditional approaches such as protein precipitation and solid-phase extraction to more recent microextraction techniques. Special attention is given to microextraction by packed sorbent, ultrafiltration, and electromembrane extraction, which offer significant advantages in efficiency, sensitivity, and scalability. By minimizing both solvent and sample requirements, these approaches provide new opportunities for improving analytical performance in peptidomics.