Figure 1.

(A) Overview of analysis by Tandem Mass Spectrometry. The digested peptides are ionized and passed through the first mass analyzer; analyzed in the second analyzer; and detected as MS spectra, survey spectra, or MS1. The high-intensity peaks from the resulting spectra (MS1) are further selected in the first mass analyzer, fragmented in the collision cell, and then analyzed in the second mass analyzer, resulting in the product ions, detected in MS/MS spectra (MS2), which contain the information for peptide sequencing followed by protein identification. (B) Overview of the different modes of data collection in tandem mass spectrometry. In DIA, all precursor ions are analyzed simultaneously and in MS mode (i.e., with low-collision energy) and then fragmented in the collision cell using high-collision energy. Multiple product ions that resulted from the fragmentation of multiple precursor ions are then detected in one spectrum. In DDA, all precursor ions are detected in the survey MS spectrum, and then, the precursor ions that have the highest intensity are selected in the first analyzer (i.e., quadrupole), fragmented in the collision cell, analyzed in the second analyzer (i.e., TOF), and detected as the MS/MS spectrum.