Combined Infrared Multiphoton Dissociation with Ultraviolet Photodissociation for Ubiquitin Characterization
Herein we report the successful implementation of the consecutive and simultaneous photodissociation with high (213 nm) and low (10.6 μm) energy photons (HiLoPD, high-low photodissociation) on ubiquitin in a quadrupole-Orbitrap mass spectrometer. Absorption of high-energy UV photon is dispersed over the whole protein and stimulates extensive C–Cα backbone fragmentation, whereas low-energy IR photon gradually increases the internal energy and thus preferentially dissociates the most labile amide (C–N) bonds. We noticed that simultaneous irradiation of UV and IR lasers on intact ubiquitin in a single MS/MS experiment provides a rich and well-balanced fragmentation array of a/x, b/y, and z ions. Moreover, secondary fragmentation from a/x and z ions leads to the formation of satellite side-chain ions (d, v, and w) and can help to distinguish isomeric residues in a protein. Implementation of high-low photodissociation in a high-resolution mass spectrometer may offer considerable benefits to promote a comprehensive portrait of protein characterization.
KeywordsPhotodissociation UVPD IRMPD Ubiquitin Top-down proteomics
Photon-based activation methods, including ultraviolet photodissociation (UVPD) [1, 2, 3] and infrared multiphoton dissociation (IRMPD) [4, 5, 6], have received great attention as alternative to electron-driven methods [7, 8, 9, 10, 11]. In recent years, UVPD has been implemented in high resolution mass spectrometry and employed for peptide and whole protein characterizations [12, 13, 14, 15, 16, 17, 18, 19]. High energy UV photons preferentially cleave Cα–C bond in peptides and proteins producing abundant a/x ions. Other fragment ions, such as c/z and y ions, are also detected in UVPD providing nearly complete sequence coverage [12, 20].
Contrasting to UVPD and electron transfer dissociation (ETD), multiple low energy IR photon excitation selectively breaks the most labile amide (C–N) bonds and generates b and y ions similar to the traditional slow-heating collision activation dissociation (CAD) method . IRMPD has been implemented in different instruments, including quadrupole ion traps  and dual pressure linear ion traps [4, 6, 23]. Vasicek et al. reported the execution of IRMPD in the high collision dissociation (HCD) cell of a modified hybrid linear ion trap-Orbitrap mass spectrometer .
The dissociation mechanisms involved after high and low energy photon excitations are quite different. Absorption of a single high energy photon (in the UV) is sufficient to induce dissociation of a peptide and protein in the gas phase. On the other hand, multiple absorption of low energy photons (in the IR) are required before fragmentation. Excitation is followed by fast internal vibrational redistribution (IVR) and causes a slow and steady rise of the internal energy until it exceeds the dissociation threshold and thus induces cleavage of the labile bonds .
Despite some analytical challenges, coupling of high and low energy activation pathways in a single MS/MS event is expected to offer diverse fragmentation arrays and thus deliver improved, efficient, and well-balanced fragmentation for whole protein characterization. Tsybin et al. reported the implementation of IRMPD with electron capture dissociation (ECD) in a FT-ICR mass spectrometer . Electron and photon irradiation significantly improved the formation of sequence ions for peptides and proteins. Simultaneous IR photoactivation with ETD, known as activated ion electron transfer dissociation (AI-ETD), is also implemented in an ion trap-Orbitrap Elite system . Moreover, tandem ETD spectra exhibited abundant peaks related to unreacted and charge reduced precursors. Hybrid AI-ETD showed better performance for lower charge states and production of specific fragment ions. The combination of UVPD with ETD (known as ETUVPD) in an ion trap-Orbitrap has also been reported . The combined ETUVPD method showed balanced fragment ions with increased number of c and z ions. The fragmentation efficiency of ETD can also be enhanced by other means, such as additional activation with CID and HCD, known as ETciD and EThcd [28, 29]. These hybrid methods showed rich fragmentation spectra compared with CID, HCD, and ETD alone.
Although a few studies report on coupling electron and photon based methods, integrating electron-driven technique with low or high collision activation approaches, so far there is no study reporting the combination of high and low energy photons for characterizing protein within a single MS/MS framework. Here, we report the implementation of a method combining solid-state fifth harmonic 213 nm laser excitation with 10.6 μm CO2 laser excitation in hybrid quadrupole-Orbitrap mass spectrometer using different excitation schemes (consecutive IR + UV, UV + IR, and simultaneous UV/IR) for top-down characterization of ubiquitin. This high-low energy photon-based method (HiLoPD) improves the fragmentation pattern, providing well-proportioned a/x, b/y, and z-ions with richness of secondary fragment ions, including d, v, and w.
Materials and Methods
Laser Setup and Experiments
In order to irradiate ions only when they are in the HCD cell, the voltage on test-point 18 (TP18), located on Q-Exactive electronic board, was monitored. In our experimental conditions, the falling edge (–10 V → –350 V) on the TP18 is used to determine the moment when ions are ejected from the C-trap to the HCD-cell (see Supplementary Figure S1). Two independent TTL pulses are then generated, with width and delay adjustable with regards to the TP18 trigger. The TTL pulses are used to lift the gate on the CO2 laser and open the shutter on the UV beam path.
Three different coupling schemes between IR and UV were implemented (Supplementary Figure S1). In Scheme I, CO2 laser was ON for 1 s and then followed by 4 UV pulses (0.2 s). In Scheme II, four pulses of UV were admitted in the HCD cell first, and followed by 1 s of CO2 laser. In the first two schemes, IR and UV were used consecutively: when CO2 laser was ON, the UV laser was OFF and vice versa. In Scheme III, the CO2 laser was turned ON and the UV shutter was open concomitantly. As in previous schemes, IR was left ON for 1 s while the UV shutter was left open for 0.2 s (four pulses). In each scheme, the coupled IR/UV irradiation takes place during single HCD events in MS2 sequences.
All experiments were performed on a hybrid quadrupole-Orbitrap Q-Exactive mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) equipped with a HESI ion source. Ubiquitin (76 residues, 8.6 kDa) from bovine erythrocytes was obtained from Sigma-Aldrich and used without any further purification. Ubiquitin samples were prepared at 10 μM concentration in 50/49/1 (v/v/v) methanol/water/acetic acid and directly infused to MS at a flow rate of 5 μL/min. All mass spectra were acquired using a mass range of 200–2000 m/z and resolving power of 140,000 at m/z 400. The automatic gain control (AGC) target was set to 5 × 106 and the maximum injection time was set at 250 ms. The isolation width was 8–10 Th. To avoid collisions and CID contamination, HCD collision energy was set to the minimum 2 eV. All experiments were performed for three microscans and averaging for 50 scans.
Raw files were deconvoluted and de-isotoped to the neutral monoisotopic masses using Xtract algorithm provided by Thermo Scientific Inc. Manual analysis of IRMPD, UVPD, and combined UVPD and IRMPD data was performed with the aid of ProSight Light software  and Protein Prospector v5.14.4. (http://prospector.ucsf.edu/prospector/mshome.htm). All major ion types (a, a + 1, a + 2, b – 1, b, b + 1, b + 2, c – 1, c, c + 1, x – 1, x, x + 1, x + 2, y, y – 1, y – 2, z – 1, z, z + 1) were considered. We observed a substantial number of secondary fragment ions, including d, v, and w, which were analyzed by Protein Prospector. H2O and NH3 losses from the fragment ions were also considered. Single protein mode with a fragment mass tolerance set to 15 ppm was used for all methods.
Results and Discussion
Optimization of IRMPD on Intact Protein
The IRMPD on the +12 charge state ion of ubiquitin induces a total of 141 fragment ions of which 41 are b-type and 98 are y-type ions. Exact masses and assignments of the ions detected in the IRMPD of the 12+ precursor ion (m/z = 714.7279) of ubiquitin are summarized in Supplementary Table S1. For this charge state, twice as many y-type ions as b-type ions are identified. The sequence coverage for the +12 ion is 59% (44 bonds break), which is significantly higher than the coverage of 24% (18 bonds break) reported earlier when IRMPD was first implemented in high resolution Orbitrap mass spectrometer (Supplementary Figure S5) . We found that a combination of lower pressure (HV ~9.3 × 10–6 mbar) and longer irradiation time (~1 s) are optimal for characterization of intact protein by IRMPD in a quadrupole-Orbitrap system.
UVPD, IRMPD, and HiLoPD on Ubiquitin
The IRMPD experiment on the +13 ion of ubiquitin allows detecting a total of 121 fragment ions (Figure 4a). Exact masses and assignments of ions detected in the IRMPD of the +13 ion (m/z = 659.8249) of ubiquitin are summarized in Supplementary Table S3. Among them, 49 ions are b-type and 67 ions are y-type fragments. The formation of only b- and y-type ions is expected from cleavages of C–N bonds proceeding via vibrationally-excited ground state dissociation. H2O and NH3 losses from the b and y ions are also observed, with H2O losses being more widespread than NH3 loss. The loss of water is energetically favorable from the protonated acidic group . Ubiquitin has seven threonine (T), six glutamic acid (E), five aspartic acid (D), and three serine (S) residues, which may promote the extensive water loss. Low (z = +1) to high charge states (z = +12) of the b and y ions are detected. The same fragment ion is often observed in many different charge states. For example, +2, +3, and +4 charge states b17 ions are detected at m/z 952.5491, 635.3695, and 476.7778, respectively. The IRMPD sequence coverage of this charge state precursor ion is 44%.
In the consecutive Scheme II, when irradiation with UV laser pulses is followed by IR irradiation, the overall number of detected fragment ions is considerably higher compared with Scheme I (Figure 4a). The number of b- and y-type ions increased sharply compared with both UVPD and Scheme I. The relaxation following electronic excitation either by light emission, internal conversion through a conical intersection or via fragmentation is expected to be fast (typically ranging from fs to ns timescales). In Scheme II, IR excitation occurs after electronic excitation and relaxation has occurred. The UV laser promotes excited states dissociation, whereas IR laser subsequently leads to the ground state dissociation (Figure 5b). The combination of the two dissociation mechanisms explains the large amount of detected fragment ions in Scheme II. The sequence coverage for this charge state precursor ion is 71% and is comparable to the one observed in UVPD.
For a lower charge state (z = +8), the simultaneous irradiation in Scheme III (HiLoPD) also showed a balanced fragmentation pattern. The total number of detected fragment ions for this charge state is higher than both the ones observed with IRMPD and UVPD (Figure 4b) alone. Large numbers of b and y ions are observed for this lower charge state, and c-type ions are also detected. Only z-type ions remain essentially the same as in UVPD. The sequence coverage of the +8 ion of ubiquitin obtained with HiLoPD (Scheme III) is 85%. Formation of d, v, and w ions is also observed for this charge state similar to +13 precursor ion.
Overall, IRMPD selectively produces b/y and b-H2O/y-H2O ions, whereas UVPD preferentially yields to a + 1/x + 1, a/x, y – 1, y – NH3, z, v, and w ions (Supplementary Figure S7a). The hybrid HiLoPD (Scheme III) method generates b/y, b – H2O/y – H2O, x, x + 1, y – 1, y – 2, y – NH3, z, v, and w ions. Bond breaking and sequence coverage of high (z = +13) and low (z = +8) charge state ions of ubiquitin obtained by IRMPD, UVPD, and HiLoPD (Scheme III) are shown in Supplementary Figure S7b and c. HiLoPD allows to improve the efficiency of structural characterization of ubiquitin compared with IRMPD and UVPD. Moreover, sequence coverages obtained with HiLoPD are similar to those theoretically expected by combining UVPD and IRMPD (calculated IR + UV, see Supplementary Figure S7c).
We report IRMPD, 213 nm UVPD, and HiLoPD patterns of ubiquitin in a hybrid quadrupole-Orbitrap mass spectrometer. Improved performance of IRMPD is observed when a combination of very low pressure and longer irradiation time in the HCD cell are used. Significant numbers of b/y ions and neutral losses of NH3 and H2O are detected by IRMPD. Similar to excimer 193 nm UVPD, solid-state 213 nm UVPD can promote Cα–C cleavage generating abundant a/x, y, and z fragment ions for ubiquitin.
The coupling of low-energy IRMPD and high-energy UVPD was implemented using three different irradiation schemes. In Scheme I, where IR irradiation is followed by UV, the number of detected fragment ions is decreased compared with the one obtained by UVPD only, which is mainly due to intense IR fragmentation prior to UV excitation. When UV irradiation was followed by IR (Scheme II), the total number of detected fragment ions is slightly increased. In Scheme III, while UV and IR lasers irradiation are simultaneous, the total number of detected fragment ions is maximal. Excited and ground state dissociation channels promote widespread fragmentation of ubiquitin precursor ions. Compared with UVPD, b/y-type ions are increased. We observed that while a/x fragment ions are decreasing, nearly equal number of d, v, and w ions emerge, which can lead to identifying the isomeric residues in a protein.
The research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Program (FP7/2007-2013 Grant agreement No. 320659). The authors thank Christian Clavier for his invaluable technical assistance. They also thank Dr. Steven Daly (ILM, CNRS et Université Lyon 1, France) for improving the English of this manuscript.
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