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. Author manuscript; available in PMC: 2021 Nov 5.
Published in final edited form as: J Invest Dermatol. 2018 Jun;138(6):1236–1242. doi: 10.1016/j.jid.2018.01.001

Figure 1. General steps of a typical LC-MS/MS experiment.

Figure 1.

A) After isolation during the experiment of interest, proteins are treated with proteolytic enzymes (e.g. trypsin), then subjected to liquid chromatography (LC; explained in B)). Separated peptides are then ionized (i.e. by exposing drops of peptide-containing eluate from LC to a strong electric field, an atomic gas is formed) and separated by their mass (m)-to-charge (z) ratios in the first mass spectrometer (MS1). Precursor ions of a given m/z are then further fragmented by collision-induced dissociation (CID) and the ion fragments separated again (MS2). Resulting fragment ion spectra are recorded and analyzed as detailed in the text. The basic principle of reverse-phase LC is illustrated in B); the most hydrophobic peptides interact best with the non-polar stationary phase, whereas the least hydrophobic components elute first. Complete elution off the column, including the most non-polar peptides, is ensured by gradually increasing the concentration of non-polar solvents in the mobile phase.