Mass spectrometry (MS) is an effective method for the high-throughput analysis of biomolecules, such as genes, proteins, transcripts, and metabolites. This article reviews the principles, components, and applications of mass spectrometry, with an emphasis on its role in the study of peptide drug conjugates. Peptide drug conjugates are complex molecules that combine a peptide with a drug to enhance its delivery and efficacy. Mass spectrometry can measure and characterize these molecules by detecting and identifying the ions within a sample and determining their mass-to-charge ratio (m/z). This review discusses the main components of a mass spectrometer, including the ion source, mass analyser, and ion transducer, and their functions in generating mass spectra. It further describes the key characteristics and performance parameters of different types of mass analysers, such as sensitivity, mass accuracy, mass resolving power, speed, and linear dynamic range. This review highlights the applications of mass spectrometry in the high-throughput analysis of complex protein or peptide mixtures, sequencing, and quantifying proteins or peptides, detecting post-translational modifications of proteins, and predicting their ۳D folding and topology. Moreover, this review compares the two primary approaches used to study proteins using mass spectrometry, the top-down, and bottom-up approaches, and the three main proteomics approaches: shotgun, targeted, and data-independent acquisition. Furthermore, the paper provides an in-depth discussion of ionization sources, focusing on
electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), and their advantages and disadvantages. ESI, the most widely used ionization technique for liquid samples, can generate multi-charged ions, which are essential for analysing large biomolecules with limited mass to charge m/z ranges. This review demonstrates the potential of mass spectrometry for advancing research in the field of peptide drug conjugates, by providing high sensitivity, precision, and accuracy, and enabling the characterization and identification of these complex molecules.
