Ultraviolet Photodissociation of ESI- and MALDI-Generated Protein

Technical Note pubs.acs.org/jpr

Cite This: J. Proteome Res. XXXX, XXX, XXX−XXX

Ultraviolet Photodissociation of ESI- and MALDI-Generated Protein Ions on a Q‑Exactive Mass Spectrometer Marialaura Dilillo,† Erik L. de Graaf,†,⊥ Avinash Yadav,†,‡ Mikhail E. Belov,§ and Liam A. McDonnell*,†,∥ †

Fondazione Pisana per la Scienza ONLUS, 56107 San Giuliano Terme, Pisa, Italy Scuola Normale Superiore di Pisa, 56126 Pisa, Italy § Spectroglyph LLC, Kennewick, Washington 99338, United States ∥ Center for Proteomics and Metabolomics, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands

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‡

S Supporting Information *

ABSTRACT: The identification of molecular ions produced by MALDI or ESI strongly relies on their fragmentation to structurally informative fragments. The widely diffused fragmentation techniques for ESI multiply charged ions are either incompatible (ECD and ETD) or show lower efficiency (CID, HCD), with the predominantly singly charged peptide and protein ions formed by MALDI. In-source decay has been successfully adopted to sequence MALDI-generated ions, but it further increases spectral complexity, and it is not compatible with mass-spectrometry imaging. Excellent UVPD performances, in terms of number of fragment ions and sequence coverage, has been demonstrated for electrospray ionization for multiple proteomics applications. UVPD showed a much lower charge-state dependence, and so protein ions produced by MALDI may exhibit equal propensity to fragment. Here we report UVPD implementation on an Orbitrap Q-Exactive Plus mass spectrometer equipped with an ESI/EPMALDI. UVPD of MALDI-generated ions was benchmarked against MALDI-ISD, MALDI-HCD, and ESI-UVPD. MALDIUVPD outperformed MALDI-HCD and ISD, efficiently sequencing small proteins ions. Moreover, the singly charged nature of MALDI-UVPD avoids the bioinformatics challenges associated with highly congested ESI-UVPD mass spectra. Our results demonstrate the ability of UVPD to further improve tandem mass spectrometry capabilities for MALDI-generated protein ions. Data are available via ProteomeXchange with identifier PXD011526. KEYWORDS: Q-Exactive, MALDI, ultraviolet photodissociation, MS/MS

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INTRODUCTION The ability to characterize proteoforms and post-translational modifications is dependent on the information content of the tandem mass spectra; the number of structurally informative fragments that contribute sequence information is, in turn, dependent on

analysis have established themselves as the method of choice, particularly for top-down analysis of proteins.1−3 Specific fragmentation techniques include collision-induced dissociation (CID),4 higher energy collisional dissociation (HCD),5 electron capture dissociation (ECD),6 electron-transfer dissociation (ETD),7 and photodissociation using multiple lowenergy (IRMPD) 8 or single higher energy photons (UVPD).9−11 More extensive sequence information can be obtained by combining tandem mass spectrometry methods from sequential tandem mass spectrometry scans but more so by simultaneous application of collisional/photoactivation with ETD.3,12−15 The vast majority of top-down protein characterization and protein identification experiments are performed using electrospray ionization because the multiple charging is either essential to the fragmentation technique (ECD, ETD) or

(i) the fragmentation technique(s), as it determines the types of fragment that may be observed, the extent of protein fragmentation, as well as whether labile posttranslational modifications are retained (ii) mass accuracy, as it strongly influences the ability to assign fragment identities to the tandem mass spectral peaks (iii) mass resolution, as it defines the degree to which unique fragment ions may be resolved in the often congested tandem mass spectra For these reasons hybrid instruments that combine multiple fragmentation techniques with accurate-mass, high-resolution © XXXX American Chemical Society

Received: November 13, 2018 Published: November 28, 2018 A

DOI: 10.1021/acs.jproteome.8b00896 J. Proteome Res. XXXX, XXX, XXX−XXX

Journal of Proteome Research

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aids the fragmentation, for instance, by increasing the effective collision energy in CID, which is directly proportional to the precursor charge. Furthermore, multiple charging enables the detection of both C-terminal and N-terminal fragments from the same fragmentation event, thereby increasing the sequence coverage. Nevertheless MALDI-based analyses remain popular because of their ability to sample complementary parts of the proteome and to rapidly measure protein signatures.16−18 MALDI-based analyses are widely used to identify microorganisms, identify biomarkers, and record protein distributions directly from tissue samples.19−22 Recent reports have demonstrated how the same accurate-mass, high-resolution analysis characteristics of hybrid mass spectrometers that benefit protein characterization are also beneficial for MALDI profiling and MALDI mass spectrometry imaging, specifically enabling more components to be resolved and provisional identities to be assigned.22−24 Despite these improvements, the key challenge for MALDI profiling and MALDI mass spectrometry imaging of proteins remains the definitive identification of many of the detected molecular ions because of the incompatibility or poor performance of CID and ETD tandem mass spectrometry methods with the large singly charged protein ions generated by MALDI. Whereas in-source decay (ISD) can be effective for isolated proteins and also benefits from higher mass resolution and accurate mass (more fragments resolved and assigned),25 the absence of a precursor ion isolation step makes it difficult to use for the identification of proteins from complex mixtures. (Note: Exceptions in which fragments can be attributed to specific proteins have been reported.26,27) The recently reinvigorated UVPD method has a much lower charge-state dependence than ETD and CID;11,28−34 the direct excitement of amide bonds by the high-energy photons is not a charge-driven process, and so protein ions produced by MALDI (singly charged) or ESI (multiply charged) may exhibit near-equal propensity to fragment. It should be noted, however, that although UVPD of MALDI-generated ions would still generate N- and C-terminal fragments from the same fragmentation event, the predominantly singly charged nature of MALDI ions means only one of the complementary fragments could be detected.35−37 On the contrary, the singly charged nature of MALDI-MS/MS spectra means they lack the high mass spectral congestion characteristic of ESI-UVPD experiments, in which the multiple charge states (z = 8−30) combined with the large number of fragment types lead to many overlapping isotopic peak envelopes, severely complicating the assignment of peaks in the mass spectra to specific fragments. Here we report the implementation of UVPD on a QExactive Plus mass spectrometer equipped with a combi-ion source that includes both elevated pressure MALDI and ESI interfaces,38 thus enabling MALDI-UVPD to be benchmarked against MALDI-ISD and ESI-UVPD performed on the same mass spectrometer. ESI-UVPD fragmentation implemented on a Q-Exactive mass spectrometer was recently reported,39,40 but this is the first example of MALDI-UVPD on an Orbitrap system.

Technical Note

EXPERIMENTAL SECTION

Chemicals and Reagents

α-Cyano-4-hydroxycinnamic acid (α-CHCA), bradykinin, thymosin β4 (SRP3324 Human recombinant with an Nterminal R), myoglobin from equine skeletal muscle, formic acid, trifluoroacetic acid, methanol, acetonitrile, and water were purchased from Sigma-Aldrich (St. Louis, MO). Indium-tinoxide (ITO)-coated glass slides were purchased from Bruker Daltonics (Bremen, Germany). All laser optics, mounts, alignment tools, and safety equipment were purchased from Thorlabs (Newton, NJ). MALDI Sample Preparation

Bradykinin peptide and thymosin β4 were dissolved in Milli-Q water to a final concentration of 1 mg/mL and then stored at −20 °C until use. CHCA stock solution was prepared at 10 mg/mL in 70/30 (v/v) ACN/H2O 0.2% TFA. One μL of analyte solution was mixed with 2 μL of matrix stock solution before spotting on ITO-coated microscope slides (Bruker Daltonics). ESI Sample Preparation

Standard protein solutions were prepared at 10 μM in 50/50 (v/v) ACN/H2O 1% formic acid, aliquoted, and stored at −20 °C until use. Direct infusion experiments were performed at 5 μL/min flow rate with a 4 kV spray voltage. The AGC target was set at 1e6 with 10 ms maximum injection time. Data Acquisition

All experiments except for those involving ETD were performed using an ESI/EP-MALDI combi-ion source (Spectroglyph, Kennewick, WA) coupled to a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific, Bremen, Germany).38 MALDI experiments were performed using a frequency-tripled Nd:YAG laser (Laser Export, Moscow, Russia), wavelength 355 nm, focused to ∼30 μm on the MALDI target. Each pixel was analyzed with 30 laser shots operated at 2 kHz. MALDI experiments were performed at 6.5 Torr, and the ions were axially extracted (40 V extraction voltage) into a series of concentric ion funnels. All MALDI experiments were performed with a fixed injection time of 400 ms. MS/MS precursor ions were selected with the quadrupole and fragmented with HCD or UVPD. To enable UVPD, the charge collector at the rear of the HCD cell was removed, and the rear flange replaced with a BaF2 window aligned coaxially with the HCD cell. A 193 nm ArF laser (ExciStar XS 500, Santa Clara, CA) was then aligned to pass through the HCD cell. A schematic of the instrumental setup is shown in Figure S1a. The transmission efficiency through the HCD cell, relative to that set in the Excistar software, was measured at 70%. The laser energy referred to throughout the text refers to the energy set in the software. ESI-UVPD of peptide and protein standards was used for thee optimization of laser timing, pulse energy, and number of laser pulses. The instrument trigger for the injection of ions from the c-trap into the Orbitrap was used as a reference to trigger a pulse generator (Berkeley Nucleonics, model 577), which generated a TTL pulse train at 500 Hz with a userdefined delay and number of pulses to trigger the UVPD laser. The optimum fragmentation conditions were obtained using a single laser pulse, delayed for 247 and 502 ms, for 70k and 140k mass resolution, respectively (Figures S2 and S3). Further details regarding UVPD implementation and optiB


Technical Note


Figure 1. MALDI-UVPD of thymosin β4. The singly charged nature of the fragment ions results in a ladder of fragments that is readily interpretable. The inserts at the bottom of the figure show close-ups of two regions of the MS/MS spectrum, demonstrating that most ions could be assigned via known UVPD fragmentation channels. Internal fragment (INT); neutral loss (NL). The spectrum was acquired using 1.6 mJ pulse energy and 16 scans (1 microscan each).

x/x+1/y/z fragments, and Protein Prospector was used for the assignment of d/w/z fragments and internal fragments. The mass-spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via PRIDE41 partner repository with the data set identifier PXD011526.

mization of the irradiation conditions can be found in the Supporting Information. The laser pulse energy was independently controlled using the Excistar laser software. In this manner, the laser energy, delay, and number of laser shots could be independently varied to optimize UVPD fragmentation. MALDI-ISD experiments were performed on the EPMALDI equipped Q-Exactive by increasing the on-target laser fluence and source gas pressure. ETD experiments were performed using an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) with 120k resolution at m/z 400, AGC 5e5, and maximum injection time of 200 ms. ETD experiments were performed with 10 ms reaction time (reagent target 2e5). The raw tandem mass spectra were visualized and processed with the Xcalibur 3.0 package and deisotoping using the Xtract algorithm. ProsightLite was used for assignment of a/a+1/b/c/

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RESULTS CID and ETD are routinely used to obtain sequence information from ESI-generated peptide and protein ions but are less/not effective for MALDI-generated protein ions because of their very large m/z and predominantly singly charged nature. UVPD has recently been demonstrated to provide extensive sequence information from ESI-generated protein ions and exhibits a low charge-state dependence11 but to date has only been investigated with MALDI-generated peptide and small protein ions on TOF/TOF-based mass spectrometers characterized by much shorter reaction times, lower mass resolution, and lower mass accuracy than the C


Technical Note

Journal of Proteome Research hybrid Orbitrap instruments used for the recent protein characterization experiments.9,30,31 Here we report a comparison of MALDI-UVPD, with ESIUVPD and MALDI-ISD using the same Orbitrap system. UVPD was implemented on a Q-Exactive Plus mass spectrometer, previously equipped with a MALDI/ESI combisource. To benchmark the performance of the UVPD implementation, we compared the sequence coverage of myoglobin obtained with ESI-UVPD with that obtained using ESI-HCD and ESI-ETD (Figure S4). Overall, ESI-UVPD using the QExactive performed similarly to recent using hybrid instruments11,39 outperforming other fragmentation techniques. The same parameters were then used for MALDI-UVPD and compared with MALDI-HCD and MALDI-ISD. For the comparison a polypeptide, bradykinin and a small protein, thymosin β4, were used with monoisotopic masses 1059.57 and 5205.63, respectively. Experiments with larger proteins were not satisfactory because the Q-Exactive Plus instrument has an upper limit of 6000 m/z. In the case of small peptides, such as bradykinin, both MALDI-HCD and MALDI-UVPD provided complete sequence coverage, but MALDI-UVPD produced a larger number of fragments (38 ions assigned for MALDI-UVPD at 2.5 mJ vs 12 for MALDI-HCD at 30 NCE, Figure S5). The difference between the two fragmentation techniques became more evident for the small protein thymosin β4. UVPD (2.5 mJ pulse energy) of the singly charged ions generated by MALDI provided extensive and informative fragmentation, displaying both N-terminal and C-terminal fragments (Figure 1). The fragment ion annotations reported in the insets of Figure 1 demonstrate the rich and but noncongested spectra, thus making fragment ion assignment more straightforward. On the contrary, MALDI-HCD was characterized by low fragmentation efficiency; sequence coverage remained below 60% even with NCE values as high as 90 (Figure 2a). In agreement with previous reports, the charge state of the precursor ions was much more critical for HCD than UVPD. HCD of the multiply charged ions generated by ESI gave high sequence coverage at low NCE. For example, the [M+8H+]8+ ion of thymosin β4 gave 66% sequence coverage with 30 NCE, and the [M+7H+]7+ ion gave 98% sequence coverage at 25 NCE (Figure 2b). This reflects the increased effective collision energy of multiply charged ions (collision energy is proportional to charge state) as well as the detection of complementary fragments from each multiply charged precursor ion. In contrast, UVPD provided consistently high sequence coverage for the multiply charged ions generated by ESI and the singly charged ions generated by MALDI. ESIUVPD provided >90% sequence coverage for all pulse energies investigated (Figure 2b); for MALDI-UVPD, increasing the pulse energy from 1.5 to 3.5 mJ increased the sequence coverage from 50 to 77%. The ions generated by ESI and MALDI have been effectively thermalized prior to fragmentation, and so the differences in their fragmentation behavior can be attributed solely to their charge-state dependence. MALDI-UVPD was also compared with MALDI-ISD. MALDI-ISD was performed using 2,5-dihydroxybenzoic acid (DHB) as the matrix by increasing the source pressure to 9.5 Torr and increasing the laser fluence. Sixteen ISD fragments of thymosin β4 could be assigned, corresponding to 30% sequence coverage. The MALDI-ISD spectrum was dominated

Figure 2. Sequence coverage obtained for UVPD and HCD fragmentation of the singly charged thymosin β4 ion generated by MALDI (a) and the 7+ and 8+ ions generated by ESI (b). The spectrum was acquired using 16 scans (1 microscan each).

by c ions, up to c15 (Figure S6). In contrast, MALDI-UVPD provided high sequence coverage with fragment ions spanning the entire protein sequence (Figure 1). In previous work,42 we demonstrated 68% sequence coverage for MALDI-ISD of thymosin β4 using the long-life liquid matrix DHB/glycerol. In this case, the extended sequence coverage was obtained at the expense of experimental time: The long-life matrix enabled the results from a large number of laser shots and microscans to be accumulated, thereby increasing sensitivity, but the experiments were long (ca. 40 min for each spectrum). MALDIUVPD provided more informative spectra and higher sequence coverage in seconds (e.g. Figure 2). The nature of the fragment ions produced by UVPD has been investigated for the multiply charged protein ions generated by ESI11,13,39,43 and previously for singly charged peptide ions produced by MALDI.9,31 Importantly the previous peptide MALDI-UVPD reports were based on MALDI-TOF experiments, which are characterized by a much shorter reaction time scale than the high-mass-resolution Fourier transform mass analyzers now used for top-down UVPD (10−6 vs 10−1 s respectively). Figure 3a shows a summary of the UVPD fragmentation channels for MALDIand ESI-generated thymosin β4 ions, in which fragments have been grouped into prompt fragmentation (a+1, x+1), Nterminal fragments (a, b, and c), C-terminal fragments (x, y, and z), satellite fragments due to side-chain cleavages (d, w, v), and internal fragments. Thymosin β4 has an N-terminal arginine, and so the fragments obtained from the singly charged ions generated by MALDI were dominated by Nterminal fragments, whereas the multiply charged ions generated by ESI led to the production of N-terminal and CD


Technical Note


Figure 3. (a) Fragment ion channels detected for thymosin β4 UVPD and their dependence on UVPD laser pulse energy for the singly charged [M + H]+ ions generated by MALDI and the multiply charged [M+7H]7+ and [M+8H]8+ ions generated by ESI. (b) Distribution of the fragment ion channels and their dependence on laser pulse energy. Spectra acquired using 16 scans (1 microscan each).

matched by a concomitant increase in the number of b fragment ions (Figure 3b). The high sequence coverage seen with UVPD is due to simultaneous activity of multiple fragmentation channels, namely, prompt dissociation to form the a+1 and x+1 radical fragment ions, which can then eliminate a hydrogen atom to become a and x ions, as well as internal conversion of the absorbed photons’ energy with a concomitant increase in vibrational energy leading to fragment ions characteristic of vibrational excitation (b/y fragments).43 It was previously observed with MALDI-UVPD of tryptic-like peptides that when the peptides had an N-terminal lysine (lower gas-phase basicity than arginine) the c/z and b/y fragmentation channels began to compete because the proton was more mobile.10,37,44 The MALDI-UVPD results reported here for the small protein thymosin β4 (containing the N-terminal arginine) are consistent, namely, the near-parallel increases in a+1, b, and c fragments with increasing laser pulse energies; for the multiply charged ions generated by ESI, the increased propensity for b/y fragments at higher laser pulse energies reflects the greater mobility of the additional protons. The singly charged precursor ions generated by MALDI limit the simultaneous detection of the complementary fragments (e.g., b and y fragments) from the same fragmentation event but are offset by the lower mass spectral complexity. The difference in mass spectral congestion is clearly shown in Figures S7 and S8. MALDI-UVPD of thymosin β4 led to the exclusive generation of singly charged

terminal fragments as well as abundant internal fragments. This is in agreement with previous peptide MALDI-UVPD studies, which focused on peptides with N- or C-terminal arginine to mimic proteolytic peptides. It is noteworthy that at higher pulse energies the number of N-terminal fragments from MALDI-UVPD is comparable to that obtained from ESIUVPD. These results confirm that the charge state has little effect on the efficiency of UVPD but is necessary for the detection of the MALDI-UVPD fragments.10,37,44 A close examination of the fragment ions in Figure 3a revealed that increasing the laser pulse energy increased the number of internal ions (from 4 to 20), satellite ions due to side-chain cleavage (d, v, and w ions; from 5 to 9), and neutral losses (from 4 to 12) for MALDI-UVPD; for ESI-UVPD, similar increases were seen, but the overall number of fragment ions was greater, reflecting the greater likelihood of their being detected. The number of N-terminal fragments (a, b, and c) from MALDI-UVPD was comparable to that from ESI-UVPD (Figure 3a), but close examination of the N- and C-terminal fragments revealed subtle differences. Figure 3b shows that for MALDI-UVPD the major fragment ion series were the a fragment ions, closely followed by b, c, and a+1 fragment ions, and with the prompt-dissociation a+1 fragment ions increasing at higher pulse energies. For ESI-UVPD the major fragment series were the a+1 and b fragment ions, but the a+1 fragments decreased in number at higher pulse energies, which was E


Technical Note

Journal of Proteome Research fragments (Figure S7), whereas ESI-UVPD of the [M+7H]7+ ion of thymosin β4 generated fragment ions spanning the charge states 1+ to 7+ as well as a much larger number of Cterminal and internal fragments, resulting in a large number of overlapping isotopic envelopes (Figure S8). The degree of mass spectral congestion in ESI-UVPD increases with increasing size of the precursor ion, owing to the larger number of fragments spanning the same m/z space. The singly charged nature of MALDI-UVPD avoids the bioinformatics challenge associated with highly congested ESI-UVPD mass spectra, and it was found here that raising the MALDI-UVPD pulse energy also enabled the sequence coverage to be increased (Figure 2). The availability of MALDI matrix preparations that generate multiply charged ions provides future opportunities to tune the charge state of the protein ions generated by MALDI to increase the detection of complementary fragments while maintaining low mass spectral complexity.45,46

ORCID

Mikhail E. Belov: 0000-0002-7353-4789 Present Address ⊥

E.L.d.G.: Sanquin Research, 1066 CX Amsterdam, The Netherlands. Notes

The authors declare no competing financial interest. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via PRIDE41 partner repository with the data set identifier PXD011526.

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REFERENCES

(1) Zhou, M.; Pasǎ-Tolić, L.; Stenoien, D. L. Profiling of Histone Post-Translational Modi fi cations in Mouse Brain with HighResolution Top-Down Mass Spectrometry. J. Proteome Res. 2017, 16, 599−608. (2) Cleland, T. P.; Dehart, C. J.; Fellers, R. T.; Vannispen, A. J.; Greer, J. B.; LeDuc, R. D.; Parker, W. R.; Thomas, P. M.; Kelleher, N. L.; Brodbelt, J. S. High-Throughput Analysis of Intact Human Proteins Using UVPD and HCD on an Orbitrap Mass Spectrometer. J. Proteome Res. 2017, 16 (5), 2072−2079. (3) Zheng, Y.; Fornelli, L.; Compton, P. D.; Sharma, S.; Canterbury, J.; Mullen, C.; Zabrouskov, V.; Fellers, R. T.; Thomas, P. M.; Licht, J. D.; et al. Unabridged Analysis of Human Histone H3 by Differential Top-Down Mass Spectrometry Reveals Hypermethylated Proteoforms from MMSET/NSD2 Overexpression. Mol. Cell. Proteomics 2016, 15 (3), 776−790. (4) Mitchell Wells, J.; McLuckey, S. A. Collision-Induced Dissociation (CID) of Peptides and Proteins. Methods Enzymol. 2005, 402, 148−185. (5) Olsen, J. V.; Macek, B.; Lange, O.; Makarov, A.; Horning, S.; Mann, M. Higher-energy C-trap dissociation for peptide modification analysis. Nat. Methods 2007, 4 (9), 709−712. (6) Zubarev, R. a; Horn, D. M.; Fridriksson, E. K.; Kelleher, N. L.; Kruger, N. a; Lewis, M. a; Carpenter, B. K.; McLafferty, F. W. Electron capture dissociation for structural characterization of multiply charged protein cations. Anal. Chem. 2000, 72 (3), 563−573. (7) Zhurov, K. O.; Fornelli, L.; Wodrich, M. D.; Laskay, Ü . a; Tsybin, Y. O. Principles of electron capture and transfer dissociation mass spectrometry applied to peptide and protein structure analysis. Chem. Soc. Rev. 2013, 42 (12), 5014−5030. (8) Vasicek, L. A.; Ledvina, A. R.; Shaw, J.; Griep-Raming, J.; Westphall, M. S.; Coon, J. J.; Brodbelt, J. S. Implementing photodissociation in an orbitrap mass spectrometer. J. Am. Soc. Mass Spectrom. 2011, 22 (6), 1105−1108. (9) Hettick, J. M.; McCurdy, D. L.; Barbacci, D. C.; Russell, D. H. Optimization of sample preparation for peptide sequencing by MALDI-TOF photofragment mass spectrometry. Anal. Chem. 2001, 73 (22), 5378−5386. (10) Kim, T.-Y.; Thompson, M. S.; Reilly, J. P. Peptide photodissociation at 157 nm in a linear ion trap mass spectrometer. Rapid Commun. Mass Spectrom. 2005, 19 (12), 1657−1665. (11) Shaw, J. B.; Li, W.; Holden, D. D.; Zhang, Y.; Griep-Raming, J.; Fellers, R. T.; Early, B. P.; Thomas, P. M.; Kelleher, N. L.; Brodbelt, J. S. Complete Protein Characterization Using Top-Down Mass Spectrometry and Ultraviolet Photodissociation. J. Am. Chem. Soc. 2013, 135 (34), 12646−12651. (12) Zhao, Y.; Riley, N. M.; Sun, L.; Hebert, A. S.; Yan, X.; Westphall, M. S.; Rush, M. J. P.; Zhu, G.; Champion, M. M.; Medie, F. M.; et al. Coupling Capillary Zone Electrophoresis with Electron Transfer Dissociation and Activated Ion Electron Transfer Dissoci-

CONCLUSIONS The Q-Exactive Plus mass spectrometer equipped with an ESI/ EP-MALDI combisource and UVPD enabled the comparison of MALDI-UVPD with ESI-UVPD, MALDI-ISD, and MALDIHCD. UVPD outperformed HCD fragmentation for the singly charged ions generated by MALDI, efficiently sequencing small proteins. It was demonstrated that at higher molecular weight, MALDI-UVPD outperforms MALDI-HCD and MALDI-ISD in terms of sequence coverage and speed and is expected to increase further with increasing precursor ion molecular weight. MALDI MS of proteins up to ∼20 kDa has been performed using Fourier transform ion cyclotron resonance mass spectrometry. MALDI-UVPD on a Q-Exactive instrument modified for the efficient isolation, trapping, and detection of small- to medium-sized protein ions (the commercial instrument is only equipped to detect masses up to m/z 6000) would enable MALDI-UVPD to be utilized for routine characterization of proteins. ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.8b00896.

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ACKNOWLEDGMENTS

L.A.M. and M.D. acknowledge the European Union (ERANET: TRANSCAN 2) for their financial support.

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UVPD implementation and optimization. Figure S1. Schematic and timing of the UVPD-enabled Q-Exactive Plus. Figure S2. Comparison of different irradiation strategies for MALDI-UVPD. Figure S3. Laser trigger delay optimization. Figure S4. Dependence of ETD, HCD, and UVPD sequence coverage on charge state. Figure S5. Comparison of MALDI-HCD and MALDIUVPD of bradykinin. Figure S6. Example MALDI-ISD spectrum of thymosin β4. Figure S7. MALDI-UVPD of thymosin β4. Figure S8. ESI-UVPD of thymosin β4. Figure S9. Dependence of ESI-UVPD sequence coverage on laser pulse energy. Figure S10. ESI-UVPD of myoglobin (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. F


Technical Note

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