Decoding the Challenges: navigating Intact Peptide Mass Spectrometry-Based Analysis for Biological Applications.

Quantitative analysis of peptides in biological fluids offers a high diagnostic and prognostic tool to reflect the pathophysiological condition of the patient. Recently, methods based on liquid chromatography coupled with mass spectrometry (LC-MS) for the quantitative determination of intact peptides have been replacing traditionally used ligand-binding assays, which suffer from cross-reactivity issues. The use of "top-down" analysis of peptides is rapidly increasing since it does not undergo incomplete or non-reproducible digestion like "bottom-up" approaches. However, the low abundance of peptides and their peculiar characteristics, as well as the complexity of biological fluids, make their quantification challenging. Herein, the analytical pitfalls that may be encountered during the development of an LC-MS method for the analysis of intact peptides in biological fluids are discussed. Challenges in the pre-analytical phase, stability after sampling and sample processing, significantly impact the accuracy of peptide quantification. Emerging techniques, such as microextractions, are becoming crucial for improved sample cleanup and enrichment of target analytes. A comparison between the roles of high-resolution and low-resolution mass spectrometry in the quantification of intact peptides, as well as the introduction of supercharging reagents to enhance ionization, will be discussed.