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. 2020 Jan 10;6(2):eaax8978. doi: 10.1126/sciadv.aax8978

Fig. 1. Inferring the primary structure of protein by MS.

Fig. 1

MS uses two approaches to infer the primary structure of a protein: (A) a bottom-up (BU-MS) approach that is used prevalently and (B) a top-down (TD-MS) approach that analyzes intact protein <70 kDa. According to the process flow in BU-MS, to infer the primary structure, the proteins are first digested by trypsin, and then the resulting peptides 0.8 to 3 kDa in size, on average, are analyzed in the gas phase by MS. First, the mass of the peptides are determined, and then peptide ions are fragmented to inform on the sequence using tandem MS (MS/MS). BU-MS does not inform on the entire sequence but only fragments as represented by the incomplete word “protein.” In contrast, in TD-MS, intact protein ions are introduced in the gas phase and are fragmented (10 kDa in size on average) and analyzed by MS to identify the mass of the protein and protein ion fragments, which are then puzzled out to reveal the primary structure of the protein. Both methods subsequently rely extensively on the searches through databases to identify the protein. The entire sequence can be revealed this way along with PTMs (represented by the capital letter in the word “pRoteins”). CID, collision-induced dissociation; ECD, electron-capture dissociation; ETD, electron-transfer dissociation.