Fig. 1 Time development of protein structure analysis technology based on native mass spectrometry (nMS) CID: collision-induced dissociation; ESI: electrospray ionization; SID: surface-induced dissociation; UVPD: ultraviolet photodissociation.

Fig. 2 Native ESI-MS spectra of AtIspF combined with CDP-MEP and Zn2+[41] a. native ESI-MS spectrum of the bare AtIspF; c. AtIspF incubated with CDP-MEP (T∶L=1∶2) in the absence of Zn2+, and the black number pointing to the peak of MS spectrum representing the numbers of CDP-MEPs bound to AtIspF; e. native ESI-MS spectrum of Zn2+-saturated AtIspF; g. AtIspF incubated with CDP-MEP (T∶L=1∶2) in the presence of Zn2+, and the red number pointing to the peak of MS spectrum representing the numbers of CMPs (one of the products in the reaction) bound to AtIspF; b, d, f, h. expanded spectra of the m/z 3800-4000 corresponding to a, c, e, g, respectively. CDP-MEP or L: 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate; CMP: cytidine monophosphate; T: the bare AtIspF trimer.

Fig. 3 (a) Schematic of the modified Orbitrap Eclipse Tribrid mass spectrometer and (b, c) nMS spectra of ammonia channel (AmtB) under different power infrared lasers[52] IR: infrared radiation.

Fig. 4 nMS spectra of CDK12/CDK13-CycK protein complexes with and without inhibitor SR-4835, and schematic diagram of CDK12/CDK13-CycK protein complexes dissociation induced by SR-4835[20] a, b. nMS spectra of CDK12-CycK and CDK13-CycK protein complexes, respectively; c, d. nMS spectra of CDK12-CycK and CDK13-CycK protein complexes in the presence of SR-4835, respectively; e. SR-4835-induced allosteric regulation of CDK12/CDK13-CycK protein complexes dissociation. The concentration ratio of complex to inhibitor was 1∶3.

Fig. 5 MS2 spectra and corresponding dissociation sequence matching results of the native heme/myoglobin (Mb) complex activated by (a) CID, (b) HCD, (c) ETD, (d, e) UVPD; (f) the sequence coverages obtained under different dissociation modes[29] The filled blue circle represents the heme/myoglobin complex (9+) precursor ion and the unfilled blue circle represents apo-myoglobin (no heme) (8+). HCD: high energy collision dissociation; ETD: electron transfer dissociation.

Fig. 6 UVPD analysis of Mb complexed with crown ether (CE)[38] a. the sequence cleavages of Mb8+complexed with different numbers of CEs; b. the heatmap of residue fragmentation yields (FYs) of Mb8+ with different numbers of CE complexation; c, d. the residue-resolved FYs alterations (ΔFYs) between bare Mb and Mb complexed with 1 CE and 2 CEs, respectively; e, f. corresponding to the crystal structures of Mb in c and d, respectively, with red color indicating increased residue FYs and blue color indicating decreased residue FYs.

Fig. 7 UVPD-nMS analysis for the dihydrofolate reductase (DHFR) dynamic structure changes caused by DHFR variant (P21L and W30R)[75] a. the variations in UVPD FYs are shown across the backbone from the N-terminus to the C-terminus for each of the P21L and W30R constructs relative to wild type (WT); b, c. the DHFR crystal structure (1RX3) for P21L and W30R, respectively. The mutated residue is colored by purple.

Fig. 8 UVPD-nMS analysis for the interaction of the C2 domain of protein kinase Cθ (PKCθ) with pY218[63] a, b. nMS and UVPD-nMS spectra of C2 with 14AA-YY and 14AA-YpY, respectively; c. the sequence coverages of apo C2 and holo C2 obtained by UVPD; d. the different fragmentation efficiency between the holo C2 and apo C2; e. predicted possible pocket. 14AA-YY: a peptide consisting of 14 amino acids, which including a tyrosine residue; 14AA-YpY: a peptide containing 14 amino acids, which including a phosphorylated tyrosine residue; apo C2: the C2 of unbound 14AA-YpY; holo C2: the C2 of bound 14AA-YpY.

Fig. 9 nMS and UVPD for the structural characterization of the T4 gp32-ssDNA complex[94] a. the sequence map of gp32 with the backbone cleavage sites leading to the resulting dT12-containing fragment ions. Red, blue and green lines corresponding to the fragment ions containing the C-terminal, N-terminal, and bidirectional dT12-containing holo ions, respectively. b. the gp32 crystal structure with the backbone cleavage sites from which the C-terminal, N-terminal and both C-terminal and N-terminal holo fragment ions originate. The close ssDNA binding cleft is highlighted in a shade of purple.