Comparison of Ultraviolet Photodissociation and Collision Induced Dissociation of Adrenocorticotropic Hormone Peptides
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In an effort to better characterize the fragmentation pathways promoted by ultraviolet photoexcitation in comparison to collision induced dissociation (CID), six adrenocorticotropic hormone (ACTH) peptides in a range of charge states were subjected to 266 nm ultraviolet photodissociation (UVPD), 193 nm UVPD, and CID. Similar fragment ions and distributions were observed for 266 nm UVPD and 193 nm UVPD for all peptides investigated. While both UVPD and CID led to preferential cleavage of the Y–S bond for all ACTH peptides [except ACTH (1-39)], UVPD was far less dependent on charge state and location of basic sites for the production of C-terminal and N-terminal ions. For ACTH (1-16), ACTH (1-17), ACTH (1-24), and ACTH (1-39), changes in the distributions of fragment ion types (a, b, c, x, y, z, and collectively N-terminal ions versus C-terminal ions) showed only minor changes upon UVPD for all charge states. In contrast, CID displayed significant changes in the fragment ion type distributions as a function of charge state, an outcome consistent with the dependence on the number and location of mobile protons that is not prominent for UVPD. Sequence coverages obtained by UVPD showed less dependence on charge state than those determined by CID, with the latter showing a consistent decrease in coverage as charge state increased.
KeywordsUltraviolet photodissociation Peptide Sequence coverage Collision induced dissociation
Significant advances in mass spectrometry for proteomics applications have evolved from the development of increasingly powerful informatics methods as well as new ion activation techniques and more sophisticated MS/MS strategies to improve the characterization of peptides in complex mixtures. The latter include deployment of real-time decision tree methods [1, 2], use of targeted ion monitoring methods [3, 4, 5], development of hybrid ion activation methods [6, 7, 8], and strategic ion manipulation based on ion–ion reactions [9, 10] and innovative ion charging concepts [11, 12]. There has also been growing effort to understand and optimize the fragmentation pathways of peptides to improve sensitivity, to enhance recognition of peptides in database searches, and, in some cases, to exploit preferential bond cleavages to provide greater specificity [13, 14, 15]. Although collision- and electron-based methods remain the most universally popular activation methods [16, 17, 18, 19, 20, 21], a number of alternatives (surface induced dissociation [22, 23], photodissociation [24, 25], high energy cation beam activation , and metastable atom activation ) have been developed to afford higher energy deposition and expand the arsenal of ways to energize ions to create meaningful fragmentation patterns.
Photon-based activation methods (termed photodissociation) offer considerable versatility, and this has led to a number of applications for analysis of peptides and proteins. These methods include infrared multiphoton dissociation (IRMPD) [25, 28, 29, 30, 31], ultraviolet photodissociation (UVPD) [25, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59], and visible photodissociation (Vis-PD) [25, 60, 61, 62]. The most common wavelengths used for UVPD of peptides include 157, 193, 266, 351, and 355 nm, each corresponding to ones readily generated by pulsed excimer or YAG lasers. The photodissociation process requires that the peptides of interest possess a suitable chromophore, either via an intrinsic chromophore (e.g., amide bond or side-chain groups of amino acids) or ones added via derivatization of the peptides prior to analysis [63, 64, 65]. For example, peptides do not naturally absorb photons around 350 nm, but they can be tagged with appropriate chromophores to make them undergo photodissociation, ultimately producing conventional b/y-type fragment ions [43, 44, 45]. This strategy has been reported for the identification of antigen binding regions of antibodies , for streamlining bottom-up proteomics , and has also shown to be useful in de novo sequencing by simplifying spectra . The amide bond absorbs 157 and 193 nm photons, thus serving as the chromophore for 157 and 193 nm UVPD of peptides and proteins. The high energy per photon (7.9 eV for 157 nm and 6.4 eV for 193 nm) can elevate ions to excited electronic states, thus accounting for the production of a wide array of a, b, c, x, y, and z ions. Both 193 and 157 nm UVPD have been reported for numerous bottom-up proteomic applications and, more recently, for top down characterization of intact proteins [36, 37]. A detailed investigation of 157 nm UVPD demonstrated that some of the fragmentation pathways were radical directed . Also, a recent study has shown that high energy fragmentation methods where electrons excited to almost ionized states precede C–C backbone cleavages, which lead to formation of a and x ions .
In contrast to 193 and 157 nm photons, 266 nm photons are not absorbed by the peptide backbone but rather by the amino acid side chains of tyrosine, tryptophan, and phenylalanine. Despite the lower energy per photon (4.6 eV), 266 nm UVPD also results in cleavages that lead to formation of a, b, c, x, y, and z ions, similar to the types of ions formed upon absorption of 157 or 193 nm photons . Oh et al. reported the rich array of fragment ions upon 266 nm UVPD of protonated peptides and noted enhanced backbone cleavages adjacent to aromatic amino acids (tryptophan, phenylalanine, and tyrosine) . The Kim group also showed that tryptic peptides derivatized by phenyl isothiocyanate reagents required lower laser powers for efficient photodissociation at 266 nm, thus confirming the enhanced photo-absorption cross-sections attributed to the added aromatic chromophores . Kim and coworkers also analyzed phosphopeptides using 266 nm UVPD, which led to the production of highly characteristic a n – 97 ions affiliated with each phosphorylated residue . Park et al. used 266 nm UVPD to analyze phosphorylated peptides in an FTICR mass spectrometer, finding a consistent loss of 98 Da . The Julian group has shown that absorption of 266 nm photons promotes selective homolytic cleavage of disulfide and C–S bonds, thus providing a means to map cysteine residues in peptides and proteins . Ly and coworkers also utilized 266 nm photons to activate peptides or proteins containing iodinated tyrosines, leading to a process termed radical directed dissociation (RDD) which results in site-localized fragmentation and can be used as a spatially-specific probe of protein structure in the gas phase [53, 54, 56]. In another RDD strategy, Diedrich and Julian showed that phosphorylated sites in peptides could be pinpointed using a Michael addition reaction with naphthalenethiol followed by 266 nm UVPD to selectively cleave the C–S bond installed during the Michael addition reaction . More recently, Tao et al. showed that D- and L-amino acids in peptides could be differentiated using an RDD process initiated by 266 nm UVPD .
This variety of photon-based activation methods has propelled interest in understanding the correlation between the photon wavelength and the outcomes of peptide activation in terms of dissociation efficiency, the types of fragmentation processes, and the potential for selective bond cleavages. Detailed comparisons of the fragmentation pathways and distributions of fragment ions arising from 157, 193, and 266 nm UVPD have not been extensively explored. Recently, Lai et al. compared the fragmentation caused by 193 nm UVPD and 266 nm UVPD for singly protonated peptides, including angiotensin (DRVYIHPF) analogues and bradykinin (RPPGYSPFR) . They noted numerous similarities in the types of fragment ions (both the ones derived from amide and non-amide backbone cleavages and some side-chain cleavages) produced by 193 and 266 nm UVPD along with some variations in relative abundances . Herein, we report the UVPD patterns of six adrenocorticotropic hormone (ACTH) peptides as a function of charge state and amino acid composition. ACTH peptides were chosen as they contain the three amino acids with aromatic side-chains (Phe, Trp, Tyr) that serve as the optimal chromophores for absorption of 266 nm photons (as well as secondary chromophores for 193 nm photons). Our goal is to extend the fundamental understanding of 266 nm UVPD in comparison with 193 nm UVPD and collisional activation.
The six ACTH (human) peptides, ACTH (1-10, sequence SYSMEHFRWG, Mr 1298.4), ACTH (1-14, sequence SYSMEHFRWGKPVG, Mr 1679.9), ACTH (1-16, sequence SYSMEHFRWGKPVGKK, Mr 1936.3), ACTH (1-17, sequence SYSMEHFRWGKPVGKKR, Mr 2092.4), ACTH (1-24, sequence SYSMEHFRWGKPVGKKRRPVKVYP, Mr 2932.5), ACTH (1-39, sequence SYSMEHFRWGKPVGKKRRPVKVYPNGAEDESAEAFPLEF, Mr 4540.1), as well as alpha-bag cell peptide (1-9 sequence APRLRFYSL, Mr 1122.3), [Tyr5] Bradykinin (sequence RPPGYSPFR Mr 1076.2), [Phe2, Nle4] ACTH (1-24 sequence SFS(Nle)EHFRWGKPVGKKRRPVKVYP, Mr 2899.5), and adamtsostatin-16 (sequence SPWSQCATSCGGGVQTR with a disulfide between the two cysteines Mr 1722.9) were purchased from American Peptide Company (Sunnyvale, CA, USA). LC-MS grade acetonitrile and water were obtained from EMD Millipore (Darmstadt, Germany). LC-MS grade formic acid was purchased from Fisher Scientific (Fair Lawn, NJ,USA).
All experiments were carried out using a Thermo Velos Pro dual linear ion trap mass spectrometer (Thermo Scientific, San Jose, CA, USA) or a Thermo Orbitrap Elite mass spectrometer (Thermo Scientific, Bremen, Germany). The dual linear ion trap was used for the MS/MS comparisons based on 266 nm UVPD, 193 nm UVPD, or CID, and the Orbitrap instrument was used primarily to confirm specific fragment ion assignments based on high resolution, high accuracy measurements. The dual linear ion trap was modified to allow UVPD with a Continuum Minilite Nd:YAG laser (Santa Clara, CA, USA) set to 266 nm or a Coherent Excistar excimer laser (Santa Clara, CA, USA) set to 193 nm. The set-up and implementation of UVPD was similar to that described previously . Peptides were diluted in a 50/50 acetonitrile/water solution with 0.1% formic acid to a final concentration of 1 μM and were infused at a flow rate of 3 μL/min. CID was performed on every observed charged state of each of the six ACTH peptides using a normalized collision energy of 35%. 266 nm UVPD was performed on each charge state observed for the six ACTH peptides using 1, 2, 5, or 10 pulses (nominally 6 mJ per pulse) at a 10 Hz laser pulse rate. 193 nm UVPD was also performed for all observed charge states of the six ACTH peptides using 2 pulses and 2 mJ per pulse at 500 Hz laser pulse rate. Only a fraction of the light enters the ion trap because neither focusing nor collimating optics is used, and the laser beam is divergent as it emerges from the laser. Due to the relatively low photon flux through the ion trap, the extent of secondary (or consecutive) fragment ion dissociation is minimal.
Analysis of Spectra
All CID and UVPD spectra were analyzed manually. Lists of fragments were obtained using both Protein Prospector v. 5.12.2 (UCSF) and Proteomics Tool kit (Institute for Systems Biology). Data for the fragment ion distributions was processed using Microsoft Excel. Abundance information was extracted from the raw data and used to calculate the percentage distributions for each type (b and y for CID and a, b, c, x, y, and z for UVPD).
Based on the graphical distributions shown in Supplemental Figures 7 and 9 and the representative series of spectra in Supplemental Figure 8, it is clear that the number of laser pulses has a relatively modest impact on the overall distribution of fragment ions for our UVPD setup. Visual comparison of the spectra in Supplemental Figure 8 confirms that no new abundant ions are produced with an increasing number of laser pulses, suggesting that the majority of fragment ions are produced directly from the precursor ion, not via secondary dissociation of primary fragment ions. (This outcome depends on the laser power and the overlap of the laser beam with the ion cloud, so it would be expected to be instrument-dependent). For all spectral comparisons discussed in the next section, five laser pulses were used for the 266 nm UVPD spectra and two laser pulses were used for the 193 nm UVPD spectra.
The MS/MS spectra for the ACTH peptides are frequently dominated by several highly abundant fragment ions, which are indicative of preferential cleavages. The most predominant fragment ions produced by UVPD and CID are compiled in Supplemental Table 1 for each charge state of each ACTH peptide. For ACTH (1-10), several of the most dominant fragment ions, namely a 2 , y 4 , y 5 , and y 8 , are observed for all three activation methods, with cleavage of the backbone at the Y–S site leading to y 8 and a 2 ions being particularly enhanced. The UVPD spectra exhibit special enhancement of a ions that are not favored in the CID spectra, and these may arise from secondary dissociation of b ions formed with excess internal energy or ones produced directly from the precursor peptide, potentially via dissociation of ions in excited electronic states. The similarities between the 266 nm UVPD and 193 nm UVPD spectra (Figures 1, 3, and 4), both in terms of the identities and distributions of fragment ions, is remarkable. The level of similarities even extends to the formation of a few low abundance z-type fragment ions, which are absent from the corresponding CID mass spectra. Apparently absorption of 266 nm (4.7 eV) or 193 nm (6.4 eV) photons allows access to excited states (whether the same ones or different ones for each of these wavelengths), and the activation process leads to similar fragmentation processes. Absorption of 193 nm photons may occur at the amide backbone as well as the aromatic side chains of W, Y, and F (the sites where 266 nm photons are absorbed), with W having a greater photoabsorption cross-section than Y or F . From previous studies of UVPD, it is believed that dissociation may occur directly from excited electronic states as well as after internal conversion and intramolecular vibrational redistribution [39, 68]. Based on the present results that show a mixture of CID-like fragment ions (b/y) and radical-directed fragment ions (a/x, c/z), it appears that UVPD involves more than one type of process.
The UVPD and CID spectra collected for ACTH (1-14) showed an even more dramatic preferential cleavage of the Y–S bond, leading to the dominant y 12 2+ ion for the 3+ and 4+ charge states (and complementary a 2 and b 2 ions). The lower charge states (1+, 2+) instead exhibited enhanced b 11 and b 13 ions upon 266 nm UVPD and CID, with the former representing a proline-type cleavage that also accounts for the prominent complementary y 3 ion. Interestingly, despite the overlap in the sequences of ACTH (1-10) and ACTH (1-14), the resulting MS/MS spectra had few similarities, an outcome that is largely attributed to the C-terminal ion dominance in the spectra that masks the more subtle variations in the N-terminal ion abundances.
For ACTH (1-16), the Y–S cleavage (formation of y 14 ) remained the most consistently prominent process upon UVPD and CID for the higher charge states (3+, 4+, and 5+) (see Supplemental Figure 4). Cleavage of the backbone between K–K (resulting in b 15 ) was also a significant pathway, which is a hallmark charge-modulated process common for both UVPD and CID. Selected a, c, and z ions were observed in the UVPD (266 and 193 nm) spectra, which again reflected the greater diversity of pathways upon UV photoactivation.
ACTH (1-17) is an interesting example, having just a single additional arginine residue at the C-terminus compared with ACTH (1-16). The resulting UVPD mass spectra (Figure 1) exhibited many of the same preferential cleavage sites as observed for ACTH (1-16), such as the enhanced Y–S cleavage and cleavage adjacent to a basic residue. Similar to ACTH (1-16), the ACTH (1-17) ions in lower charge states (2+ and 3+) underwent a dominant cleavage of the last amino acid in the sequence (K–R bond) resulting in the formation the b 16 ion. UVPD of ACTH (1-17) also show prominent formation of the y 15 ions, which corresponded to the Y–S cleavage for 3+, 4+, 5+, and 6+ charge states, as well as a modest increase in C-terminal ions as the charge state increased.
Four charge states (3+, 4+, 5+, and 6+) were observed for ACTH (1-24), and representative fragmentation patterns generated by UVPD and CID are shown in Supplemental Figure 5. The relative portion of N-terminal ions compared with C-terminal ions produced by UVPD was greater for all charge states compared with the distributions observed for the shorter ACTH peptides. Again, the Y–S bond cleavage was particularly favored, yielding the y 22 (and a 2 ) ions noted previously. The a 22 ion, arising from a V–Y backbone cleavage, was a significant product ion upon UVPD, and it has been observed previously that cleavages C-terminal to valine and N-terminal to tyrosine may be enhanced .
As the largest peptide in the series, ACTH (1-39) generated five charge states (3+, 4+, 5+, 6+ and 7+) upon ESI and some rather unique results for both 266 nm UVPD and CID (Supplemental Figure 6). This peptide sequence is unusual in that there are no basic sites found within the final 18 residues (C-terminus). For 266 nm UVPD the cleavage of the S–Y bond was not observed for any charge state; in fact there was no consistent preferential cleavage for any of the charge states upon UVPD. CID resulted in the dominant formation of a b 35 ion, which arises from cleavage of the F–P bond, a process consistent with the well-known rule of peptide cleavage N-terminal to proline. N-terminal b ions were highly favored upon CID of all charge states, presumably because the most basic charge sites, which were clustered closer to the C-terminus for the shorter peptides, are actually situated in the mid-region of ACTH (1-39). This means that cleavages at backbone sites in the mid-section of the peptide more likely result in fragment ions with protons localized in the first 21 residues, thus enhancing N-terminal ions (such as the dominant b 29 , b 33 , b 35 , b 38 ions, among others, noted in Supplemental Table 1). Interestingly, the distribution of N-terminal versus C-terminal ions for ACTH (1-39) is almost evenly split (50%/50%) upon 266 nm UVPD and 193 nm UVPD. In contrast, N-terminal fragments make up 70%–75% of the fragment ions upon CID. The C-terminal ions that are observed are long ones that contain basic residues from the N-terminal half of the peptide. The notable differences in C-terminal versus N-terminal fragment ion distribution further supports the lower degree of charge-site dependence on the fragmentation pathways observed upon UVPD.
In an effort to elucidate the reason for the apparent preferential cleavage of the Y–S bond observed upon activation of the ACTH peptides, three additional peptides were subjected to CID, 193 nm UVPD, and 266 nm UVPD. The dominant Y–S cleavage could be due to its position in the peptide (the second and third residues), a special lability of the Y–S bond, or a chromophore effect (although it is noted that this bond cleavage is also observed upon CID, not just UVPD). Bradykinin [Tyr5] and alpha-bag cell peptide were chosen because they both have a Y–S bond in the middle of the peptide or near the C-terminus rather than near the N-terminus. The cleavage of the Y–S bond was fairly prominent for both peptides and was only surpassed by proline cleavages (Supplemental Figures 10 and 11), giving evidence that the preferential Y–S cleavage observed for the ACTH peptide is not solely due to location of the Y–S residues near the N-terminus. Another peptide selected was ACTH (1-24) [Phe 2 Nle 4] for which the Tyr in ACTH at position 2 is replaced by Phe. The fragments observed from both UVPD and CID for this peptide are nearly identical to those seen for the original version of the ACTH (1-24) peptide, with the y 22 and a 2 ions produced in great abundance (Supplemental Figure 12). The fact that cleavage between the F–S residues parallels the preferential cleavage observed for the Y–S residues suggests that the preferential cleavage may arise from an aromatic residue/Ser motif. The third peptide analyzed was adamtsostatin-16, which contained a W and S residues at the third and fourth positions, respectively. The resulting UVPD and CID mass spectra are shown in Supplemental Figure 13. In this case, cleavage of the W–S bond is not overly favored. In total, it appears that a new preferential cleavage associated with X–S in which X is Y or F occurs upon UVPD or CID, a feature that might be prevalent in larger scale bottom-up proteomic studies.
Among the series of ACTH peptides, the UVPD spectra of the smaller peptides were more “CID-like” in terms of the portion of a, b, and y ions, particularly for ACTH (1-10) (3+) and ACTH (1-14) (4+). For the longer ACTH peptides and/or ones in lower charge states, the UVPD spectra exhibited the greater diversity of fragment ions that is the established hallmark of UV photoactivation. In terms of sequence coverages (i.e., expressed as a percentage based on the number of backbone cleavages relative to the total number of backbone positions), UVPD and CID typically gave similar coverages for the lower charge states of each peptide (see values in Supplemental Figures 14–18).
Both 266 nm UVPD and 193 nm UVPD generated similar fragment ion distributions for each ACTH peptide, spanning a variety of a, b, c and x, y, z ions. Comparison of the MS/MS results for ACTH (1-10), ACTH (1-14), ACTH (1-16), ACTH (1-17), and ACTH (1-24) revealed that UVPD and CID consistently showed preferential cleavage of the Y–S bond for nearly every charge state. For all of the peptides, the production of C-terminal versus N-terminal ions and overall sequence coverage was far more dependent on the charge state and location of basic sites for CID than for UVPD, an outcome that reflects the reduced prominence of charge-mediated pathways for UVPD. In general, UVPD of the longer peptides [ACTH (1-16), ACTH (1-17), ACTH (1-24), and ACTH (1-39)] showed relatively little change in the distributions of fragment ion types as a function of the charge state of the peptide; there were much greater changes observed for CID. UVPD demonstrated a modest degree of preferential cleavage adjacent to amino acids containing aromatic side chains, suggestive of a chromophore effect, as well as enhanced cleavages adjacent to proline akin to the well-known proline effect documented for CID. This systematic comparative study has demonstrated many similarities in the types and distributions of fragment ions produced by 266 nm UVPD and 193 nm UVPD, with greater overall sequence coverage afforded by 193 nm UVPD. A comparison of the results for 193 nm and 266 nm UVPD shows that both provide better sequence coverage compared with CID for higher charge states of larger peptides; 193 nm UVPD provides better coverage on average than 266 nm UVPD, and the sequence coverages obtained by UVPD did not exhibit the significant dependence on charge state that was observed upon CID.
The authors acknowledge funding from the NSF (CHE-1402753) and the Welch Foundation (F-1155).
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