2  Introduction

2.1 Mass Spectrometry Fundamentals

A mass spectrometer consists of three main components: an ionization source, a mass analyzer, and a detector (Dass 2007). A wide range of mass spectrometers, built around different mass analysis techniques, have been developed for proteomics. These include Orbitrap-based instruments, such as the Orbitrap Exploris 480 (Bekker-Jensen et al. 2020; Denisov et al. 2021) and the Orbitrap Astral (Heil et al. 2023; Guzman et al. 2024), as well as time-of-flight (TOF) instruments, such as the Bruker timsTOF, which couples trapped ion mobility spectrometry (TIMS) with TOF mass analysis to add an additional dimension of ion separation (Meier et al. 2018). During injection into the instrument, peptides are ionized using electrospray ionization (ESI), which imparts one or more electrical charges to the peptide molecules (Fenn et al. 1989). T hese charged peptide ions, referred to as precursor ions, are transferred to the mass analyzer, where electromagnetic fields separate them according to their mass-to-charge (m/z) ratios (McDonald and Yates 2002). The instrument then records a full mass spectrum (MS1), which measures the m/z values and relative abundances of the precursor ions.

The four principal types of mass analyzers used in proteomics are quadrupole, ion trap, time-of-flight (TOF), and Fourier transform (FT)-based analyzers, such as the Orbitrap (Peters-Clarke et al. 2024). Following the MS1 scan, selected precursor ions are fragmented in a process known as tandem mass spectrometry (MS/MS or MS2). This fragmentation step is essential for determining the amino acid sequence of a peptide by generating characteristic fragment ions (Steen and Mann 2004).

The most widely used fragmentation method is collision-induced dissociation (CID), in which selected precursor ions collide with an inert gas, causing cleavage of the peptide backbone (Peters-Clarke et al. 2024). The resulting fragment ions are commonly classified as b-ions and y-ions (Roepstorff and Fohlman 1984). Modern mass spectrometers frequently employ higher-energy collisional dissociation (HCD), a refined form of CID in which ions are accelerated to higher collision energies, producing more efficient and reproducible fragmentation (Olsen et al. 2007).

2.2 Data Acquisition Strategies

There are several data acquisition method to select peptides from the MS1 for further fragmentation into MS2. There are three main acquisition types in proteomics, in which the precursors are selected from the MS1. The three main data collection strategies are: targeted analysis, data-dependent acquisition (DDA) (Stahl et al. 1996), and data-independent acquisition (DIA). Targeted analysis includes techniques such as selected/multiple reaction monitoring (SRM/MRM) (Lange et al. 2008) and parallel reaction monitoring (PRM) (Peterson et al. 2012), in which a predefined set of precursor ions, and in the case of SRM/MRM their corresponding fragment ions, are monitored throughout the run rather than selected dynamically.

  • See Section 4.1 for details on the database search approach used with DDA.
  • See Section 4.2 for details on the DIA search approach.