Coupling 193 nm Ultraviolet Photodissociation and Ion Mobility for Sequence Characterization of Conformationally-Selected Peptides

Abstract

Ultraviolet photodissociation (UVPD) has emerged as a useful technique for characterizing peptide, protein, and protein complex primary and secondary structure. 193 nm UVPD, specifically, enables extensive covalent fragmentation of the peptide backbone without the requirement of a specific side chain chromophore and with no precursor charge state dependence. We have modified a commercial quadrupole-ion mobility-time-of-flight (Q-IM-TOF) mass spectrometer to include 193 nm UVPD following ion mobility. Ion mobility (IM) is a gas-phase separation technique that enables separation of ions by their size, shape, and charge, providing an orthogonal dimension of separation to mass analysis. Following instrument modifications, we characterized the performance of, and information that could be generated from, this new setup using the model peptides substance P, melittin, and insulin chain B. These experiments show extensive fragmentation across the peptide backbone and a variety of ion types as expected from 193 nm UVPD. Additionally, y-2 ions (along with complementary a+2 and b+2 ions) N-terminal to proline were observed. Combining the IM separation and mobility gating capabilities with UVPD, we demonstrate the ability to accomplish both mass- and mobility-selection of bradykinin des-Arg9 and des-Arg1 peptides followed by complete sequence characterization by UVPD. The new capabilities of this modified instrument demonstrate the utility of combining IM with UVPD because isobaric species cannot be independently selected with a traditional quadrupole alone.

Figure 1.

Sequence coverage map, (A) full MS/MS, and (B) zoomed-in higher m/z region observed from 193 nm UVPD of substance P 2+. The isotope distribution of each fragment can be clearly resolved with good signal-to-noise as illustrated in (B). 10 laser shots at 1 mJ were used for irradiation within a 1 s trapping time. A wide variety of ion types and extensive sequence coverage were observed. Major fragments are labeled. A break is included in the y-axis so that fragment ions can be more easily observed.

Figure 2.

Sequence coverage map and full MS/MS spectrum observed from 193 nm UVPD of melittin 4+. 10 laser shots at 1 mJ were used for irradiation within a 1 s trapping time. A wide variety of ion types and extensive sequence coverage were observed. Major fragments are labeled. A break is included in the y-axis so that fragment ions can be more easily observed.

Figure 3.

Sequence fragment map and full MS/MS spectrum observed from 193 nm UVPD of insulin chain B 3+. 10 laser shots at 1 mJ were used for irradiation within a 1 s trapping time. A wide variety of ion types and extensive sequence coverage were observed. Major fragments are labeled. A break is included in the y-axis so that fragment ions can be more easily observed.

Figure 4.

IM-UVPD of bradykinin des-Arg1/des-Arg9. (A) arrival time distribution (ATD) of bradykinin des-Arg9 (blue) and des-Arg1 (purple) mixture from separation within the TWIM cell. The m/z of each species is the same so they are unable to be separated from traditional mass analysis. (B) bradykinin des-Arg9 and (C) bradykinin des-Arg1 show the corresponding ATDs of the drift time-selected peptides using mobility gating. Following mobility gating, the peptides were individually trapped within the transfer SRIG and irradiated with the 193 nm laser, resulting in (D) full MS/MS spectrum and (E) zoomed-in spectrum of mobility-selected bradykinin des-Arg9 alongside (F) full MS/MS spectrum and (G) zoomed-in spectrum of mobility-selected bradykinin des-Arg1. (H) and (I) show the corresponding sequence coverage maps for UVPD of bradykinin des-Arg9 and des-Arg1, respectively. A break is included in the y-axis so that fragment ions can be more easily observed. Peaks resulting from laser noise or fragments arising from UVPD the non-mobility-selected peptide due to lack of IM baseline resolution are marked with an asterisk (*).