Directed-Backbone Dissociation Following Bond-Specific Carbon-Sulfur UVPD at 213 nm
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Ultraviolet photodissociation or UVPD is an increasingly popular option for tandem-mass spectrometry experiments. UVPD can be carried out at many wavelengths, and it is important to understand how the results will be impacted by this choice. Here, we explore the utility of 213 nm photons for initiating bond-selective fragmentation. It is found that bonds previously determined to be labile at 266 nm, including carbon-iodine and sulfur-sulfur bonds, can also be cleaved with high selectivity at 213 nm. In addition, many carbon-sulfur bonds that are not subject to direct dissociation at 266 nm can be selectively fragmented at 213 nm. This capability can be used to site-specifically create alaninyl radicals that direct backbone dissociation at the radical site, creating diagnostic d-ions. Furthermore, the additional carbon-sulfur bond fragmentation capability leads to signature triplets for fragmentation of disulfide bonds. Absorption of amide bonds can enhance dissociation of nearby labile carbon-sulfur bonds and can be used for stochastic backbone fragmentation typical of UVPD experiments at shorter wavelengths. Several potential applications of the bond-selective fragmentation chemistry observed at 213 nm are discussed.
KeywordsLaser Photodissociation Excited state Iodine Disulfide Phosphorylation
Ultraviolet photodissociation (UVPD) is becoming an increasingly popular choice for fragmenting ions in mass spectrometers . UVPD differs from other dissociation techniques such as collision-induced dissociation (CID) [2, 3] or electron-transfer dissociation (ETD)  in several important ways. For example, UVPD causes dissociation through nearly simultaneous operation of two mechanisms, vibrational heating and excited-state dissociation . The relevant timescale for UVPD experiments can also be significantly shorter than CID or ETD, occurring within nanoseconds for experiments utilizing high-powered lasers . Furthermore, the requirement of absorption by a chromophore offers an orthogonal parameter, independent of mass or charge, for controlling which ions will be excited. UVPD experiments have been conducted at many wavelengths including 157, 193, 213, 266, and 355 nm [7, 8, 9, 10, 11, 12, 13]. Wavelength selection significantly impacts fragmentation by modulating the ratio of fragmentation mechanisms available. For example, at 266 nm, very few direct dissociation pathways exist, and the abundance of natural chromophores in analytes is small. At 157 nm, most bonds act as chromophores, and direct dissociation pathways are expected to be more plentiful.
Previous efforts to favor bond-selective direct dissociation, which occurs due to excitation to a dissociative excited state, have primarily utilized 266 nm photons . Early experiments demonstrated that carbon-iodine bonds could be selectively and homolytically cleaved in peptides or proteins, leaving all other bonds intact . Carbon-bromine bonds can also be cleaved in this fashion, though the yield is reduced . Sulfur-sulfur bonds are the only native bonds in peptides or proteins that undergo direct dissociation at 266 nm . Carbon-sulfur bonds can be cleaved with 266 nm photons, but only if the sulfur atom is directly attached to a suitable chromophore, such as naphthalene [18, 19]. Importantly, all of these labile bonds can be cleaved with a high degree of selectivity even in large molecules, meaning that in many cases, a single bond is the only site of dissociation. This degree of control over fragmentation enables a variety of unique applications.
For example, disulfide bond partners from a protein can be easily identified . In this experiment, the protein is digested with disulfide bonds intact and then subjected to chromatographic separation and MS/MS analysis with UVPD at 266 nm. Peptide pairs bound by a disulfide bond cleave into the constituent peptides while other monomeric peptides remain intact. Bond-selective UVPD can also be used to identify UV labile post-translational modifications such as iodination of tyrosine or quinone reactions with cysteine . When coupled with targeted wet-chemistry modifications, bond-selective UVPD can be used to identify sites of phosphorylation by directing backbone fragmentation at the site of modification . All of these previous examples utilized 266 nm photons, which favor direct dissociation but also suffer from weak absorption by many chromophores.
Recently, interest in UVPD at 213 nm has increased because this wavelength is afforded by a solid-state laser, and the photon energy resides at the threshold of absorption by small chromophores such as amide bonds. In theory, this could allow UPVD at 213 nm to favor either direct dissociation or nonspecific dissociation, depending on experimental parameters (such as laser power and excitation time) or molecular composition (most critically, the chromophores available for absorption). Herein, the photochemistry of UVPD at 213 nm is explored for a variety of modified and native peptides. The propensity for bond-selective fragmentation versus undirected UVPD is examined as a function of peptide composition. The capacity for fragmenting both native and synthetically appended C–S bonds with 213 nm photons is investigated. The prospect for using bond-specific C–S bond cleavage to subsequently direct backbone fragmentation at targeted residues is evaluated. Applications of 213 nm light for bond-specific cleavage of S–S bonds, which simultaneously yields C–S bond fragmentation, are explored. Advantages and disadvantages relative to similar experiments conducted at 266 nm are discussed.
Peptides RKRRQTSM, RQSVELHSPQSLPR, RGDC, RPHERNGFTVLCPKN, HCLGKWLGHPDKF, NTWTTCQSIAFPSK, and SHLVEALYLVCGERG were purchased from Anaspec (San Jose, CA). SLRRSSCFGGR and CQDSETRTFY were purchased from Abbiotec (San Diego, CA). CDPGYIGSR was purchased from Apexbio, and CGYGPKKKRKVGG was purchased from American Peptide Company (Sunnyvale, CA). GSNKGAIIGLM (Piscataway, NJ) was purchased from GenScript. DRVYIHPF was purchased from Sigma-Aldrich (St. Louis, MO). Naphthoquinone (NQ), iodoacetamide, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO). Benzoquinone (BQ), benzyl mercaptan (BM), and trifluoroacetic acid (TFA) were purchased from Alfa Aesar (Haverhill, MA). Naphthalenethiol (NT) was purchased from Fluka Analytical (Mexico City, Mexico). Iodomethane was purchased from Arcos Organics (Geel, Belgium). Chloramine-T, sodium metabisulfite, and sodium iodide were purchased from Fisher Chemical (Fairlawn, NJ). Acetonitrile (ACN) and methanol were purchased from Fisher Scientific (Waltham, MA). Water was purified by Millipore Direct-Q (Millipore, Billerica, MA). A Macrotrap holder and Macrotrap consisting of polymeric reversed-phase packing material were purchased from Michrom Bioresources, Inc. (Auburn, CA).
Peptides were iodinated using a previously published method . Briefly, equimolar peptide and sodium iodide were mixed with a two-fold molar excess of chloramine-T for 5 min at room temperature in water. The reaction was then quenched by the addition of 4× molar excess sodium metabisulfite. The products were then purified with a peptide microtrap, rinsed with 0.1% TFA in 90:10 H2O:ACN, and eluted with 0.1%TFA in 2%:98% H2O:ACN.
Modification of peptides was carried out with either benzoquinone or naphthoquinone based on a previous procedure . Stocks of each quinone were prepared fresh prior to each use and stored in the dark to reduce degradation. Quinone stocks were added to peptide solutions in 0.5 to 4× excess of the peptide concentration. The reaction proceeded for 4 h in the dark at room temperature. Following the reaction time, the solution was purified by microtrap rinsing with 0.1% TFA in 90:10 H2O:ACN and eluted in 0.1%TFA in 2%:98% H2O:ACN. The resulting solution was lyophilized, and the powder was redissolved in 50:50 H2O:MeOH in 0.1% formic acid for a final concentration of 4 or 10 μM for MS analysis.
Carbamidomethylation and Methylation of Free Thiols
Carbamidomethylation of cysteine was carried out by preparing a stock solution of 9 mg of iodoacetamide into 1 mL of water. To 25 μL of a 50 mM ammonium bicarbonate buffer, 10 μL of 1 mM peptide was added, followed by 6 μL of the stock iodoacetamide solution. The resulting solution was placed in the dark at room temperature for 20 min. The reaction mixture was lyophilized and the powder dissolved in 50:50 H2O:MeOH in 0.1% formic acid for a final concentration of 4 or 10 μM for MS analysis.
Methylation was carried out by adding 1 μL of iodomethane to 25 μL of ammonium bicarbonate buffer and 10 μL of 1 mM peptide, followed by incubation in the dark at room temperature for 20 min. The modified peptide was purified by peptide trap as described above for MS analysis.
Preparation of Disulfide-Linked Peptides
Disulfide-linked peptides were prepared by adding 10 μL of a 1 mM peptide to 5 μL of DMSO. The reaction was mixed well and placed in a water bath at 37 °C for 18 h. The resulting solution was lyophilized to remove DMSO and dissolved in 50:50 H2O:MeOH in 0.1% formic acid for a final concentration of 4 or 10 μM for MS analysis.
Dephosphorylation and Derivatization of Phosphopeptides
Peptides were dephosphorylated based on previous procedures . Briefly, 10 μL of 1 mM peptide stock was combined with 3 μL of saturated barium hydroxide and incubated at 60 °C for 30 min. The dephosphorylated peptides were subsequently modified through the addition of 5 μL of 50 mM naphthalenethiol (2 mg dissolved in 200 μL dioxane) or 2 μL of benzyl mercaptan at 60 °C over 4 h. Each reagent was 0.5 to 4× excess of the peptide concentration. Purification of the peptides was carried out with a peptide trap. The resulting solution was lyophilized, and the powder was dissolved in 50:50 H2O:MeOH in 0.1% formic acid for a final concentration of 4 or 10 μM for MS analysis. Deamidation of glutamine was observed for only a single residue of the RQSVQLHsAPQSLPR peptide, and it has been previously shown that high temperature can induce deamidation .
Photodissociation of Derivatized and Non-derivatized Peptides
UVPD experiments were performed on an Orbitrap Velos Pro mass spectrometer (Thermo Fisher Scientific, Waltham, MA) with an HESI II electrospray source. The HCD vacuum housing was directly modified with a quartz window to transmit the fifth harmonic (213 nm) of a diode-pumped FQSS 213-Q4 laser (Crylas, Berlin, Germany). The pulse energy was 2.5 μJ @ 1000 Hz. Photodissociation was performed by trapping ions without activation in the HCD cell for either 50, 100, or 200 ms (every millisecond equates to approximately a single pulse from the laser system), followed by mass analysis in the Orbitrap. The resolution was set to 30,000. Peptides were sprayed at 4 μM concentrations at 3 μL/min with electrospray voltages set between 3 and 4 kV and the capillary inlet temperature set to 300 °C.
An LTQ linear ion trap mass spectrometer (Fisher Scientific, Waltham, MA) with a standard ESI source was utilized for fourth harmonic (266 nm) UVPD experiments. A quartz window was installed on the back plate of the LTQ for transmission of laser pulses from a flash lamp-pumped Nd:YAG Minilite laser (Continuum, Santa Clara, CA). A single pulse of the 4-mJ laser was synchronized to occur at activation step of an MS2 experiment which was triggered by the same external delay generator state above. Peptides were sprayed at 10 μM concentrations at 3 μL/min with electrospray voltages set between 3 and 4 kV and the capillary inlet temperature set to 275 °C.
An LTQ linear ion trap mass spectrometer (Fisher Scientific, Waltham, MA) with a standard ESI source with an OPO tunable laser was utilized for 213-nm UVPD experiments of disulfide-linked peptides. A quartz window was installed on the back plate of the LTQ for transmission of laser pulses from a flash lamp-pumped Nd:YAG Minilite laser (Continuum, Santa Clara, CA). Three pulses of the 0.7-mJ laser were synchronized to occur at activation step of an MS2 experiment. Peptides were sprayed at 10 μM concentrations at 3 μL/min with electrospray voltages set between 3 and 4 kV and the capillary inlet temperature set to 275 °C.
Photodissociation of Native C–S Bonds
Modification of Free Thiols
In addition to quinone chemistry, cysteine residues are often capped with acetamide following thiol reduction in proteomics protocols . Photoactivation of acetamide-capped NTWTTCQSIAFPSK is shown in Figure 3c, revealing surprisingly abundant C–S bond cleavage. Amide functional groups are weak chromophores at 213 nm, which may account for the enhanced fragmentation. Again, the characteristic d6 ion is generated and identifies the sequence position of the cysteine residue. Although not a commonly encountered cysteine modification, we also examined methylated cysteine in the peptide HCLGKWLGHPDKF. Methylated cysteine behaves similarly to methionine, with the exception that a modest d-ion is generated. This result is not unexpected because the cysteine side chain is one methylene unit shorter than methionine and will therefore yield an alaninyl radical following C–S bond cleavage. Comparable experiments at 266 nm yield little to no dissociation of the C–S bonds.
C–S Bonds Formed During Identification of Phosphorylation Site Locations
Gas Phase Disulfide Identification
Photoactivation of peptides with 213 nm photons can be used to drive both bond-specific dissociation and traditional nonspecific UVPD. Photons at 213 nm work well for bond-selective photodissociation of labile bonds previously found to undergo direct dissociation at 266 nm. In addition, many native and non-native carbon-sulfur bonds are labile at 213 nm, including several that generate alaninyl radicals that initiate beta dissociation of the peptide backbone at the radical site. The signature d-ions generated by this mechanism can be used to site-specifically identify the location of cysteine or phosphorylated serine/threonine residues. The expanded list of labile carbon-sulfur bonds also allows for more reactive groups to be used in the derivatization of dehydroalanine, enabling quantitative conversion to a photoactive species. For disulfide bond characterization, signature triplets are generated at 213 nm due to dissociation of both the sulfur-sulfur and carbon-sulfur bonds, facilitating identification. Experiments conducted at 213 nm are promising for the further expansion of bond-specific photodissociation and its applications for biomolecular characterization.
The authors gratefully acknowledge assistance from John Syka, Chris Mullen, Chad Weisbrod, Jens Griep-Raming, and Jenny Brodbelt with interfacing the laser with the orbitrap.
The NIH is thanked for financial support (NIGMS grant R01GM107099).
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