The largest protein complex fragmented here to test the SID performance was 14-mer 801 kDa GroEL, normal charge in 200 mM ammonium acetate and charge-reduced species using TEAA (Figure ). We isolated 68+ and 47+ GroEL at 11,850 and 16,950 m/z, respectively, with an isolation window of 200 m/z using quadrupole, and fragmented them using SID. The SID energies were adjusted to 7,520 eV and 7,480 eV, respectively, to compare their results under an approximately equal SID energy level. Figure shows the SID results from both selected ions. The SID products cover the entire m/z range selected for analysis. The peaks at high m/z are well resolved and less broad than on other platforms we have modified. Fragments corresponding 10-to 13-mers at 30,000 to 40,000 m/z were not reported previously for other instrument platforms. After deconvolving the spectrum using UniDec , the results show that, after SID, 47+ GroEL (Figure ) has fewer monomers and 13-mers formed, and it is more favorable to produce 7-mers, which reflects a more native-like structure compared to the SID result of 68+ GroEL (Figure ). Analysis of a complex mixture using the modified TIMS-Q-TOF. When the sample is a complex mixture, sometimes the protein complex of interest can overlap with others under the same m/z. Using TIMS to separate the mixture by ion mobility (IM) and isolating a narrow m/z range of interest before SID makes the identification of SID fragments easier. GTPase, Ras, is a key component of the mitogen-activated protein kinase signaling pathway, cycling between guanosine diphosphate-bound inactive and guanosine triphosphate (GTP)-bound active states. It forms a complex with a specific guanine nucleotide exchange factor, Son of Sevenless (SOS). Ras*GTP binds at the distal site, which modulates the activity of SOS, while Ras binds at the active site, facilitating nucleotide exchange. To investigate the HRas-SOS complex structure, HRas*GTP and SOS were mixed and ionized and 4 different proteins and protein complexes, HRas*GTP, SOS, HRas*GTP-SOS, and HRas*GTP-SOS-HRas, were observed in the MS1 spectrum (Figure ). We were interested in studying the structure of the full HRas*GTP-SOS-HRas complex. However, from the mobiligram shown in Figure , there were always other protein complexes overlapped with HRas*GTP-SOS-HRas in m/z. Those overlapped protein complexes, which could not be separated without IM or by narrowing the m/z range using quadrupole, were also fragmented when performing MS/MS on HRas*GTP-SOS-HRas, convoluting interpretation of results. Therefore, TIMS was used to separate overlapped species prior to quadrupole isolation and MS/MS to improve the structural analysis of the HRas*GTP-SOS-HRas complex.