Top-Down Characterization of Protein Anions Using Ultraviolet Photodissociation Mass Spectrometry
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Top-Down Characterization of Protein Anions Using Ultraviolet Photodissociation Mass Spectrometry
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Journal of the American Society for Mass Spectrometry

Cite this: J. Am. Soc. Mass Spectrom. 2026, 37, 4, 884–893
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https://doi.org/10.1021/jasms.5c00380
Published February 24, 2026

Copyright © 2026 The Authors. Published by American Chemical Society. This publication is licensed under these Terms of Use.

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Abstract

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Top-down proteomics is primarily performed using electrospray ionization-tandem mass spectrometry (ESI-MS/MS) in the positive mode. Development of methods in the negative mode can potentially facilitate analysis of acidic proteome, but has been hampered by the low ionization efficiency and the lack of effective fragmentation methods for protein anions. Here, we investigate the performance of ultraviolet photodissociation (UVPD) for top-down analysis of protein anions. We employed organic bases as additives in solution to yield highly charged, nonadducted protein anions of high abundance. We compared UVPD with higher energy collisional dissociation (HCD) and activated electron photodetachment (a-EPD) for fragmentation of proteins ranging from 8.6 to 47 kDa. UVPD yielded abundant charge-reduced precursor radicals, in addition to numerous a/x, b/y and c/z fragment ions. UVPD offered 70–95% sequence coverage for proteins below 20 kDa regardless of the presence of disulfide bonds, and 30% coverage for the largest protein studied (47 kDa enolase), higher coverage than HCD and a-EPD. UVPD of deprotonated proteins exhibited several features similar to those of protonated proteins, such as minimal sensitivity to the charge state, production of abundant a/x fragment ions, and fairly uniform backbone cleavages adjacent to each residue (i.e., no prominent preferential cleavage sites).

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Published as part of Journal of the American Society for Mass Spectrometry special issue “Fenn: Photoactivation and Ion Activation”.

Introduction

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Mass spectrometry (MS)-based proteomics has been routinely used to explore protein sequences and identify modifications. (1−4) Both bottom-up and top-down MS proteomics are primarily performed via electrospray ionization (ESI) in positive mode, for which peptides and proteins generally exhibit high ionization efficiencies and produce multiprotonated molecules amenable to characterization by tandem mass spectrometry (MS/MS). (2,3,5) Positive mode is also compatible with liquid chromatography (LC)-MS methods that typically contain an organic acid in the mobile phases to improve chromatographic peak shapes and enhance protonation during ESI. (6) However, the positive ionization mode may impede analysis of some acidic peptides or proteins. Nearly half of the proteins in the human proteome have isoelectric points below 7, (7) and functional proteoforms bearing acidic post-translational modifications (PTMs) such as phosphorylation and sialic acid glycosylation can readily ionize as anions via deprotonation. (8−13) Hence, ESI-MS in negative mode is a compelling alternative to facilitate the characterization of acidic proteins and extend the coverage of proteomics studies.
Analysis in negative mode is primarily hampered by the lack of efficient fragmentation methods for peptide or protein anions. While collision-induced dissociation (CID) for peptide cations produces b and y sequence ions originating from cleavage of backbone amide bonds, (3) it generates less informative spectra dominated by neutral losses of CO2, NH3, and H2O for peptide anions. (14−18) Alternative electron- and photon-based activation methods have been actively developed to enhance the yield of sequence fragments of peptide anions. (19−27) Negative electron transfer dissociation (NETD), (12,22) activated ion NETD (AI-NETD), (23,24) and ultraviolet photodissociation (UVPD) (25−27) were demonstrated to generate high sequence coverages for various peptide anions. For example, using a 193 nm laser, UVPD resulted in high yields of a and x fragments for both high and low charge states of peptide anions. (27) Labile acidic PTMs were retained on the fragment ions in UVPD, allowing their localization, (28−30) as shown for deprotonated phosphopeptides, (13) glycopeptides, (31) and sulfopeptides. (32) UVPD of peptide or protein anions also generated charge-reduced radical ions via electron photodetachment. (33,34) Subsequent collisional activation of these charge-reduced species, a process termed activated electron photodetachment (a-EPD), was explored to obtain sequence information for peptides and small proteins like ubiquitin. (33,34)
Another challenge for negative mode proteomics lies in ionization efficiency. The low abundance of stable anions in solution is one issue, and the occurrence of corona discharge during ESI affects spray stability. (8,35) Proteins in particular may yield a limited range of low charge states and exhibit adduction with counterions and other ligands in negative mode even for denaturing conditions. (36−38) Several promising strategies have been introduced to overcome these obstacles, such as introducing an organic modifier or electron-scavenging solvent to enhance ESI stability and sensitivity. (39−41) Modifying the pKa of the peptide or pH of the solution was also discussed as a means to facilitate deprotonation in ESI. (42) As one example, carbamylation of side-chains of basic amino acids improved the identification of peptides by CID and UVPD in negative mode. (43) Modulating the solution pH by the addition of an organic base, such as piperidine, was advantageous for improving the production of highly charged peptide and protein anions (38,44,45) and was successfully translated to an LC-MS mode for high-throughput proteomics. (8,23) Strategies that combined both positive and negative modes (such as using AI-NETD or UVPD) demonstrated the complementarity of the polarities and broadened the coverage of bottom-up proteomics. (23,46)
Although mass spectrometric identification of proteins is still most commonly implemented on a peptide level via bottom-up methods, analysis of intact proteins via top-down workflows provides insight into true proteoform characterization, facilitating the discovery of sequence variants and mapping combinatorial patterns of PTMs. (2,4,47,48) Most top-down methods have used collision- or electron-based activation techniques for MS/MS in the positive mode. (49−51) Although UVPD has been applied for top-down analysis of proteins in the positive mode, (52−58) UVPD for analysis of intact protein anions remains under-explored. For multiprotonated proteins, UVPD predominantly generates a/x signature ions as well as b/y and c/z ions via nonspecific cleavages along the entire protein backbone, offering high sequence coverages and localization of labile PTMs. (59) Moreover, production of the sequence ions is relatively insensitive to protein charge state or amino acid compositions. (60) Based on these findings, we envisioned the potential of UVPD as a promising alternative MS/MS method for the characterization of protein anions, as explored in this work. In the present study, an organic base is added to the protein solutions to improve the ionization efficiency during negative mode ESI and generate higher charge states, an outcome vital for unfolding the proteins and achieving high sequence coverages. We compare the capabilities of higher energy collisional dissociation (HCD), 193 nm UVPD, and a-EPD for top-down analysis of intact protein anions ranging from 8 to 47 kDa and examine various metrics to assess performance for protein anions.

Experimental Section

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Sample Preparation

Ubiquitin (bovine, Uniprot P0CG53, 8.6 kDa), cytochrome C (equine, Uniprot P00004, 12 kDa), myoglobin (equine, Uniprot P68082, 17 kDa), β-lactoglobulin (bovine, Uniprot P02754, 18 kDa), carbonic anhydrase II (bovine, Uniprot P00921, 29 kDa), and enolase (yeast, Uniprot P00924, 47 kDa) were purchased from Sigma-Aldrich. The sequences are shown in Table S1. LC-MS grade solvents were purchased from Fisher Scientific. Formic acid (FA), ammonia hydroxide, and organic bases including trimethylamine, 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (MTBD) and 1,5-diazabicyclo(4.3.0)non-5-ene (DBN) were purchased from Sigma-Aldrich. 1-(3-Aminopropyl)-2-pyrrolidinone was obtained by hydrolysis of DBN in DBN/H2O (10/90, v/v) solution over a week. (61) Proteins were dissolved at 10 uM in H2O/methanol (50/50, v/v) or H2O/acetonitrile (50/50, v/v) solution. Then 0.01–1% formic acid, ammonium hydroxide, trimethylamine, 1-(3-aminopropyl)-2-pyrrolidinone, MTBD, or DBN were added to the protein solutions to modulate the ionization efficiencies and charge states of proteins in negative mode ESI. β-lactoglobulin with reduced disulfide bonds were prepared by incubating 10 uM protein with 5 mM dithiothreitol (Sigma) in water for 30 min. (3) Then 15 mM iodoacetamide (Sigma) were added and incubated for 30 min to alkylate the free thiols, and alkylation was quenched by additional 5 mM dithiothreitol. The reduced proteins were cleaned up using 10 kDa molecular weight cutoff centrifugal filters (Millipore Amicon).

Top-Down Mass Spectrometry

Solutions were infused via static nanoESI using metal-coated borosilicate glass emitters pulled in-house. The applied ESI voltage was 1000 V for positive mode and −900 V for negative mode. All data were collected on a Thermo Scientific Orbitrap Eclipse Tribrid mass spectrometer modified with a 193 nm laser to perform UVPD in the linear ion trap by addition of a Coherent ArF excimer laser. Spectra were collected using resolving power of 60k for ubiquitin, 120k for cytochrome c and myoglobin, and 240k resolution for β-lactoglobulin, carbonic anhydrase and enolase to resolve isotopes. For tandem MS, individual charge states of proteins were subjected to higher energy collisional dissociation (HCD) using optimized collision energies or UVPD using a single laser pulse of 0.2–2.0 mJ. For a-EPD, the singly charge-reduced precursor ions produced by UVPD were isolated to perform MS3 via CID. Tandem MS spectra were collected using AGC target of 2000% (1e6), S-lens RF of 40, intact protein mode with low pressure setting, full profile spectra mode, and averaging 200 scans. For enolase, in-source CID of 20 V was applied to reduce salt adducts, and 400 scans were averaged to improve the signal-to-noise (S/N) ratio.

Data Analysis

All spectra were deconvoluted using Xtract with an S/N threshold of 10 and a fit factor of 70%. Fragments were identified using deconvoluted spectra and MS-TAFI (62) with a 10 ppm tolerance of mass error. HCD spectra were searched for b/y and c/z ions. (17) For UVPD and a-EPD, ten fragment ion types (a, a+1, b, c, x, x+1, y, y–1, y–2, and z) were searched. Prosight Native (63) was used to check isotopic fits of fragments, for which fragments were searched with a 10 ppm mass error tolerance, an S/N threshold of 10, and a fit score cutoff of 0.65. Ion types, sizes, relative intensities and amino acids adjacent to backbone cleavage sites of all fragment ions were analyzed and plotted based on MS-TAFI, (62) in which a and a+1 ions were grouped, as were x and x+1 ions, and y, y–1, and y–2 ions. For preferential cleavage sites, fragment ions were grouped by the amino acid located N-terminal or C-terminal to the cleavage site, and the intensities were normalized by the count of the amino acid in the protein sequence. Raw mass spectra and identification results can be accessed at https://dataverse.tdl.org/dataverse/Top-down_protein_anion.

Results and Discussion

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Impact of Organic Bases on Ionization of Proteins in the Negative Mode

ESI-MS is commonly practiced in the positive mode to effectively produce multiprotonated proteins without ligands or salt adducts, facilitating isolation and identification of heterogeneous proteoforms by MS/MS. (47) Examination of highly charged proteins, ones which are denatured and presumed to be unfolded, (64) improves sequence coverage obtained by MS/MS, especially for collision- and electron-based methods. (50,65−67) Past studies have reported that negative mode often produced protein anions in low abundance and low charge states, and the charge states exhibited little sensitivity to denaturing solution conditions, change in pH, or introduction of supercharging reagents. (36,37,41) Hence, we first optimized the solution parameters to ensure efficient ionization and production of high charge states in negative mode. As shown in Figure 1, positive mode ESI of denatured myoglobin (17 kDa) containing 1% formic acid (FA) generated a broad array of charge states for the apo protein, with 18+ being the most abundant charge state. For negative mode ESI of a myoglobin solution containing 1% ammonia, the most abundant charge state was 13– and the intensity decreased over 100-fold, despite the same number of acidic (21) and basic (21) residues in the protein sequence. The myoglobin anions also exhibited salt adduction and retention of heme, suggesting refolded or collapsed compact gas-phase structures. The addition of an organic base of high gas-phase basicity, DBN, (45) significantly enhanced the ionization efficiency of myoglobin and eliminated salt adduction. The most abundant charge state shifted to 20–, and the charge state distribution was similar to the one observed in the positive mode under acidic conditions. We tested several bases, including trimethylamine, 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene, 1-(3-aminopropyl)-2-pyrrolidinone, and DBN at varied concentrations, and concluded that 0.05% DBN was optimal (Figure S1). Two of the other organic bases, 1-(3-aminopropyl)-2-pyrrolidinone and 7-methyl-1,5,7-triazabicyclo(4.4.0)dec-5-ene, also yielded highly charged protein anions but were not deemed optimal due to less extensive charging and lower ionization efficiency. Higher concentrations of DBN (such as 0.5%) resulted in production of even higher charge states for carbonic anhydrase II (CA, 29 kDa) (Figure S2a), but caused formation of numerous adducts (+44 or +62 Da), which might originate from base-catalyzed reaction between CO2 in the air and the primary amines of the protein forming carbamic groups (Figure S2d). (68) Lower concentrations of DBN (0.01%) resulted in less extensive charging and less stable electrospray (Figure S2c). Enhanced ionization in the negative mode using 0.05% DBN was demonstrated for proteins ranging from 8.6 to 47 kDa (Figure S3).

Figure 1

Figure 1. MS1 spectra of solutions containing 10 μM myoglobin and (a) 1% formic acid in positive mode, (b) with 1% NH3·H2O in negative mode, and (c) with 0.05% DBN in negative mode. Selected charge states of the protein and the heme are annotated.

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MS/MS Analysis of Myoglobin

The 20– charge state of myoglobin (m/z 846) in 0.05% DBN solution was selected as an initial benchmark for MS/MS of protein anions, and the outcomes of HCD, UVPD, and a-EPD were compared. HCD of myoglobin using optimized activation parameters (i.e., normalized collision energy (NCE) of 15) yielded a spectrum featuring clusters of ion peaks corresponding to neutral losses of CO2 and H2O from both the precursor ions and fragment ions (Figure 2a). A few fragment ions dominated the spectrum and indicated preferential cleavage of specific backbone positions. Both b/y and c/z ions were produced by HCD of myoglobin (as noted previously for peptide anions), (14) but the limited fragmentation provided a sequence coverage of only 18% as shown in the backbone cleavage map (Figures 2b, S4a). Most of the prominent backbone cleavages occurred adjacent to acidic residues (D,E).

Figure 2

Figure 2. MS/MS spectra of myoglobin (20–, m/z 846, indicated by an asterisk in the UVPD spectrum). (a) HCD using NCE of 15. (c) UVPD using 1 laser pulse of 0.5 mJ. (e) a-EPD performed using UVPD (1 laser pulse of 0.3 mJ) followed by isolation of the charge-reduced 19−• radical ions and then CID using NCE of 23 (b), (d), (f) show sequence coverage and backbone cleavage maps based on relative intensities of fragment ions originating from different backbone cleavage sites derived from the corresponding HCD, UVPD, and a-EPD mass spectra.

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For UVPD (1 laser pulse of 0.5 mJ), abundant charge-reduced precursor radicals via electron photodetachment (EPD) were observed, in addition to numerous a/x, b/y and c/z fragment ions that resulted in 86% sequence coverage (Figures 2c,d, S4b). Multiple neutral losses of CO2 also occurred for the charge-reduced precursors. All ten fragment ion types (a, a+1, b, c, x, x+1, y, y–1, y–2, z) were searched and grouped into six categories (a, b, c, x, y, z) that are color-coded. Ion types plotted in Figure 2d were predominantly a/x ions, as well as lower abundance contributions from b/y and c/z ions, consistent with the characteristics of UVPD for protein cations. (27,28,59) Manual examination of the isotopic distribution of all types of fragment ions confirmed that hydrogen shifts that are commonly observed in UVPD spectra of protein cations were observed, resulting in a+1, x+1 and y–1 ions (Figure S5). (59) Replacing the true myoglobin sequence with a false β-lactoglobulin sequence resulted in few matches, confirming the low false positive rate (Figure S6). A few fragments also exhibited neutral loss of CO2, but these were not significant.
Replicates collected on different days demonstrated the consistency of charge reduction and fragmentation for UVPD of protein anions (Figure S7). These results indicated that the energy deposited in the protein anions upon UV photoabsorption induced backbone cleavage or alternatively caused electron detachment, a process reported previously, (34) in conjunction with neutral losses of CO2. Searching ten fragment ion types encompassed the majority of fragment ions and accounted for 21% of the ions in the UVPD spectrum of myoglobin (20–). Although the production of fragments with neutral losses and internal fragments has the potential to increase sequence coverage, these ions also increase both the complexity of the search space and the chances of false discovery and were thus not included in the searches of other proteins in this study. Fragmentation of the 20– and 20+ precursors by UVPD is compared in Figure S8, and the fragmentation efficiencies, calculated by normalizing absolute intensities of fragments to the total ion current (TIC), were similar and the sequence coverages were comparable (86% for 20– and 85% for 20+).
a-EPD is a hybrid MS3 method in which a charge-reduced precursor produced by UV photoactivation is isolated and subjected to collision-induced dissociation (CID). (34) a-EPD has been used previously to increase production of fragment ions for peptide anions and small proteins like ubiquitin. (33,34) The 20– charge state of myoglobin was isolated and subjected to one 0.3 mJ laser pulse, then the charge-reduced 19–• precursors along with their neutral loss species were isolated and subjected to CID (NCE 23) (Figures 2e and S9). a-EPD provided a diverse array of fragment ion types like UVPD. Abundant a/x and c/z ions were produced along with multiple neutral losses similar to HCD, and the sequence coverage of 30% was modest (Figures 2f, S4c, S9d). The backbone cleavage sites from which the c/z ions originated in Figure 2f had substantial overlap with the ones observed in the HCD spectrum. The lower coverage of a-EPD compared to UVPD primarily corresponded to the absence of multiple a/x ions. Isolation and activation of other charge-reduced precursors of myoglobin, such as 18–••, generated a-EPD spectra similar to the one observed for 19–• (Figure S9f,g). However, because of the lower abundance of the 18–•• ion and the prevalence of more uninformative neutral losses, fewer fragments were identified and the resulting sequence coverage was 34%. This initial assessment of a-EPD indicated that collisional activation of charge-reduced precursors was not a compelling MS/MS strategy for protein anions.

MS/MS Analysis of Other Proteins

We then applied HCD, UVPD, and a-EPD for characterization of five other proteins of varied sizes: ubiquitin (8.6 kDa), cytochrome c (12 kDa), β-lactoglobulin (BLG, 18 kDa, with 2 disulfide bonds), carbonic anhydrase II (CA, 29 kDa) and enolase (47 kDa) in addition to more extensive analysis of myoglobin (17 kDa). The MS1 spectra of these proteins in the negative mode using 0.05% DBN to assist deprotonation are shown in Figure S3. Sequence coverages for the three fragmentation methods are summarized in Figure 3. Fragment ion assignments were confirmed by fitting their isotope distribution spectra to theoretical distributions (see examples in Figure S10 for fragment ions produced by UVPD).

Figure 3

Figure 3. Sequence coverage produced by HCD, UVPD and a-EPD, as a function of precursor charge state for (a) ubiquitin, (b) myoglobin, (c) cytochrome c, (d) β-lactoglobulin, (e) carbonic anhydrase and (f) enolase.

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As shown in Figure 3, UVPD provided higher sequence coverage than HCD or a-EPD for all protein anions, and a-EPD always yielded higher coverage than HCD. As the protein size increased, although the sequence coverage consistently declined for all methods due to more complicated spectra (which reduced S/N and confounded assignment of some fragment ions), the relative increase in sequence coverage produced by UVPD compared to that obtained by HCD more than doubled, demonstrating the advantage of using UVPD for top-down analysis in negative mode. UVPD provided over 85% sequence coverage for relatively small proteins such as ubiquitin, cytochrome c and myoglobin, and afforded 25–30% coverage for the largest protein, 47 kDa enolase, where HCD or a-EPD provided on average 5%. Notably, UV photoactivation also caused cleavage of disulfide bonds along with backbone cleavages, yielding high sequence coverage for β-lactoglobulin which has two disulfide bonds. For HCD and a-EPD, sequence coverages decreased with charge state. For example, HCD and a-EPD yielded 27 and 64% sequence coverage, respectively, for the 8– charge state of cytochrome c, but only 17 and 23% for the 16– charge state (Figure 3). Some of the lowest charge states, such as myoglobin (14−) and CA (20−), yielded lower sequence coverage than the higher charge states because of the low abundances of the precursor ions. UVPD exhibited little dependence on charge state, and 83–91% sequence coverage was obtained for cytochrome c for the 8– to 16– charge states, and 36–43% was obtained for CA for the 20– to 36– charge states.
The portions of each type of fragment ion (a/x, b/y, c/z) produced for each of the six proteins by each ion activation methods are displayed in Figure S11, with a collective summary compiled for all proteins and all charge states in Figure 4a. HCD produces nearly equal portions of b/y ions (similar to HCD of protein cations) and c/z ions (unlike HCD of protein cations). UVPD primarily produces a/x ions (70–80% of all ions) via direct cleavage of the C–C bonds of protein backbone, (28) with lower contributions from b/y and c/z ions (all trends similar to UVPD of protein cations). a-EPD generates the most balanced distribution of all ion types (a/x, b/y and c/z), similar to a composite of HCD and UVPD. There are some minor variations as a function of charge state, but these are not consistent trends across the set of proteins.

Figure 4

Figure 4. (a) Fraction of abundance of fragment ion types generated by HCD (HCD−), UVPD (UVPD−), and a-EPD for protein anions and by UVPD for protein cations (UVPD+) with error bars showing standard deviations across six proteins and multiple charge states. (b) Violin plot showing the count of fragment ions as a function of fragment size (expressed as a percentage of the full protein length) for four MS/MS methods.

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The sizes of fragment ions generated by each MS/MS method were compiled for all proteins and charge states to construct the violin plots shown in Figure 4b where the width shows the average count of fragment ions as a function of the fragment size expressed as a percentage of the full protein length. All of the MS/MS methods generated some large fragments constituting nearly the full protein length. The number of fragment ions identified for UVPD, as well as the portion of longer fragment ions, were significantly greater than that produced for a-EPD and HCD, contributing to the high sequence coverage for UVPD. The fragment sizes based on the number of amino acids contained in the fragment ions generated for each protein are mapped in Figure S12. The maps for HCD are more sparse and show considerable gaps across the protein sequences. The UVPD maps are more dense, and the ones for a-EPD show intermediate density and a significant decrease in density as the size of the protein increases.
The maps in Figure S12 also show the impact of laser energy on the sizes of fragment ions produced by UVPD. With increasing laser energy, the count of relatively large fragments declined while the abundance of small fragments increased, an outcome attributed to absorption of multiple photons owing to the greater photon flux for the higher laser energy settings. Multiphoton absorption increases the probability of secondary dissociation.
The impact of the laser energy was also evaluated for UVPD by varying the laser energy from 0.2 mJ to 2.0 mJ per pulse, and the results are summarized in Figure S13. For proteins in lower charge states, a higher laser energy generally resulted in higher sequence coverage. For example, a single 0.5 mJ pulse yielded over 90% sequence coverage for the 6– and 10– charge states of ubiquitin, while 2.0 mJ was needed for the 5– charge state to attain the highest coverage. However, the sequence coverages did not change significantly for many of the proteins across several of the charge states, and thus optimal laser energies were selected to maximize dissociation efficiency and minimize secondary dissociation.

Fragmentation Features of Protein Anions

To analyze whether there are notable preferential backbone cleavage sites for fragmentation of protein anions, we grouped all fragment ions by the amino acid located N-terminal or C-terminal to the backbone cleavage site and reported the outcomes as bar graphs. For example, for the mock sequence AB/CDE, if the dominant backbone cleavage occurs between B and C, then B would be the most prominent residue N-terminal to the cleavage and C would be the most prominent residue C-terminal to the cleavage. To account for the different numbers of amino acids in each protein, the sum of fragment intensities per amino acid was normalized by the count of the corresponding amino acid and then normalized to create the individual bar graphs in Figures S14–S16a,b for each protein and also summed for all six proteins (Figures S14–16c,d). For HCD, backbone cleavage sites with N at the N-terminal or with D, H, N or P at the C-terminal were somewhat preferred for most proteins. However, there were also some variations among proteins. For example, HCD of BLG anions showed preferential cleavage with N-terminal Cys, while HCD of cytochrome c displayed preferential cleavage with N-terminal Pro. The low consistency across proteins might be attributed to several factors: (1) different deprotonation sites, (2) a large amount of fragments that also incorporated neutral losses, (17) (ones not included in this tabulation of fragment ions), and (3) low number of proteins examined (low statistics).
For UVPD, there was a more uniform pattern of backbone cleavages adjacent to each residue, consistent with UVPD of protein cations (Figures 4b, S15). (59) Some of the known preferential cleavage sites for UVPD of protein cations (e.g., N-terminal Pro or C-terminal Tyr) (59) were not observed for UVPD of protein anions. In addition, the frequency of backbone cleavages resulting in product ions with N-terminal Cys was unusually low. Cys was found only in cytochrome c, BLG and enolase, and was covalently connected to heme for cytochrome c or involved in disulfide bonds for BLG, factors which suppressed fragmentation at those sites.
a-EPD produced a somewhat more uniform distribution of backbone cleavage sites compared to HCD (Figure S16), and there were no sites that were strongly preferred. Some of the preferred cleavage sites observed for HCD, such as those adjacent to D, N or P, were also somewhat favored for a-EPD of all proteins. a-EPD also exhibited a moderate preference for cleavage sites adjacent to aromatic amino acids, W and Y at the C-terminal.
The ability of HCD, UVPD, and a-EPD to cleave disulfide bonds for protein anions was examined for β-lactoglobulin (BLG) which has two disulfide bonds (Cys66-Cys160, Cys106-Cys119). HCD of BLG (14−) resulted in few backbone cleavages and sequence coverage of only 2% (Figure 5a). Backbone cleavage adjacent to one of the cysteines was dominant. HCD of the 14+ charge state of BLG resulted in slightly higher sequence coverage (27%), and likewise no cleavage of the disulfide bond (Figure 5d). Selective cleavage of disulfide linkages was reported in a past study using ion trap CID for peptide anions, (18) but this phenomenon was not observed for HCD of BLG anions in the present study. UVPD of BLG (14−) resulted in 74% sequence coverage with many fragment ions that covered the regions spanned by disulfide bonds. Fragmentation of disulfide bonds by UVPD is known to result in either S–S or C–S bond cleavage. (69,70) For UVPD of BLG (14−), the sequence coverage within the region spanned by disulfide bond (Cys66-Cys160) was 62% based on homolytic S–S cleavage, but only 14% based on C–S cleavage at Cys160 or 6% based on Cys66 (Figure S17). These results indicate that conventional homolytic S–S cleavage was the dominant fragmentation pathway for disulfide bond cleavage of protein anions, an outcome also supported by the correlated abundant a65+1 and b160 fragments ions originating from backbone cleavage adjacent to Cys (Figure 5b,f). We only included the conventional homolytic S–S bond cleavage in this study to reduce complexity of the search space and minimize false positives. The sequence coverage obtained for the BLG anion was similar to the 73% coverage generated by UVPD of the 14+ charge state of BLG (Figure 5b,e). However, UVPD produced more abundant fragments within the sequence bracketed by one disulfide bond for the BLG anion compared to the BLG cation. a-EPD also resulted in low sequence coverage of BLG < 15%), and there were only two prominent backbone cleavages (Figure 5c) and both were the same ones noted for UVPD (cleavage between amino acids Glu65/Cys66 and Cys160/His161). Interestingly, for both UVPD and a-EPD, backbone sites adjacent to the disulfide bonds were highly favored for the 14– charge state (Figure 5b,c).

Figure 5

Figure 5. Backbone cleavage maps displaying relative intensities of fragment ions originating from different backbone cleavage sites of β lactoglobulin based on (a) HCD (NCE 14) of 14– charge state, (b) UVPD (1 laser pulse of 1.5 mJ) of 14– charge state, (c) a-EPD of 14– charge state [UVPD (1 laser pulse of 0.5 mJ) followed by isolation of the 13–• radical ions and then CID (NCE 20)], (d) HCD (NCE 14) of the 14+ charge state, and (e) UVPD (1 laser pulse of 1.5 mJ) of 14+ charge state. The backbone cleavage sites adjacent to the C66–C160 disulfide bond that resulted in the most abundant fragment ions are marked with circles and triangles on the backbone cleavage plots for UVPD (5b) and a-EPD (5c). (f) Isotope fitting results of the two of the most abundant fragments originating from cleavage adjacent to C66–C160 disulfide bond produced by UVPD ((a65 + 1)6– and b16012–) and a-EPD (c655– and b16012–) for the 14– charge state (all with score >0.8 in Prosight Native).

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To address the efficiency of UVPD on cleavage of disulfide bonds of protein anions, we compared UVPD spectra of intact nonreduced BLG and BLG after reduction of disulfide bonds (rBLG). For denaturing solution conditions with 0.05% DBN, ESI of rBLG displayed higher charge states than BLG, corresponding to more extended structures (Figure S18a–d). The 14– charge state and BLG and rBLG were subjected to UVPD using one laser pulse of 1.5 mJ (Figure S18e,f). For the proteins produced in the same charge states (i.e., 14– to 22−) for nonreduced BLG and rBLG, UVPD yielded comparable sequence coverages (74% for BLG (14−) and 71% for rBLG (14−)), suggesting UVPD effectively cleaved disulfide bonds for nonreduced BLG (Figure S19a–c). Disulfide bonds exhibited minimal effect on the uniformity of backbone cleavages adjacent to each residue for UVPD (Figure S19d), except that the abundant a65+1 and b160 fragments originating from cleavages adjacent to disulfide bonds upon UVPD of nonreduced BLG were not dominant for UVPD of rBLG, indicating their origins were specifically related to S–S bond cleavage. The region bridged by two disulfide bonds (Cys106-Cys119) in nonreduced BLG yielded few fragment ions and little sequence coverage for any of the MS/MS methods, including UVPD. After reduction of disulfide bonds, UVPD of rBLG accessed this region with a coverage of 77%.

Comparison of UVPD of Protein Anions and Cations

Because UVPD yields the highest sequence coverages of the protein anions relative to HCD or a-EPD, the performance of UVPD for protein anions versus protein cations was evaluated in more detail. Sequence coverages and the portions of fragment ion types (a/x, b/y, c/z) are summarized in Figure S20 for UVPD of each of the six proteins over a range of charge states in negative and positive modes. The consistency of fragment ion type (dominance of a/x ions, Figure 4a) and preferential backbone cleavage sites (Figure S21) between positive and negative modes suggests they share the same major fragmentation pathway. However, the distribution of fragment ion sizes (Figure 4b) was not identical for UVPD of protein anions and cations, with somewhat smaller fragment ions on average generated for UVPD of anions. The number of fragment ions declined gradually with increasing fragment length for UVPD, but the decline was greater for UVPD of protein anions. Moreover, sequence coverages of larger proteins (i.e., 29 kDa carbonic anhydrase and 47 kDa enolase) were significantly greater for UVPD of the cations (Figure S20e,f). These trends mostly affected the analysis of large protein anions, for which sequence coverage was lost more extensively in the middle sequence region. Indeed, UVPD of protonated CA and enolase yielded nearly twice the sequence coverage of deprotonated CA (75% protonated protein vs 40% deprotonated protein) and enolase (50% protonated protein vs 30% deprotonated protein), respectively. The decrease in fragment ions for the larger protein anions could be attributed to the production of the abundant charge-reduced precursors and neutral loss ions that were only observed in negative mode, two features which ineffectively used some of the precursor ion current and masked less abundant fragment ions. For larger proteins such as enolase, more charge-reduced precursors were produced which exacerbated the masking effect (Figure S22).

Conclusions

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While top-down analysis of proteins in the negative mode has been underexplored due to low ionization efficiency and uninformative fragmentation patterns of protein anions, we demonstrate effective top-down analysis by combining organic base additives in solution and UVPD. The organic base, DBN, enabled the production of protein anions of high charge states and high abundance. For top-down fragmentation, UVPD exhibited greater sequence coverage than HCD or a-EPD for proteins ranging from 8.6 to 47 kDa. Specifically, for proteins <20 kDa, UVPD of protein anions resulted in fragmentation as extensive as UVPD of protein cations and offered 70–95% sequence coverages. However, congestion of spectra from the formation of charge-reduced precursors and neutral loss ions caused UVPD to underperform for larger protein anions, although still yielding sequence coverage of 30% for enolase (47 kDa).
This work also compared features of HCD, UVPD and a-EPD in negative mode. The impact of the protein charge state on sequence coverage was significant for HCD and a-EPD, but not notably for UVPD. UVPD produced abundant a/x fragment ions and relatively uniform backbone cleavages adjacent to each residue, distinct from HCD or a-EPD. UVPD also cleaved disulfide bonds, a pathway not prominent for HCD. More detailed analysis of fragments with neutral losses and inclusion of internal fragments would likely allow assignment of many of the fragment ions that are currently unidentified in the MS/MS spectra. Examination of UVPD of proteins carrying acidic PTMs like phosphate and sulfate is underway and may facilitate higher throughput identification of acidic proteome, providing information complementary to the typical top-down workflow (23,26)

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jasms.5c00380.

  • (1) Mass spectra for optimization of ESI-MS in negative mode, (2) sequence maps and isotopic fitting results supporting identification of fragment ions, (3) bar graphs for sequence coverage and feature of fragment ions for varied charge states of proteins using HCD, UVPD (varied laser energy) and a-EPD, (4) bar graphs for preferential cleavage analysis of HCD, UVPD and a-EPD, and (5) mass spectra and bar graphs comparing positive and negative mode UVPD of all proteins (Figure S23) (PDF)

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Author Information

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  • Corresponding Author
  • Author
    • Hanlin Ren - Department of Chemistry, University of Texas at Austin, Austin, Texas 78712, United States
  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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We acknowledge the following funding sources: NSF (Grant CHE-2203602) and the Welch Foundation (Grant F-1155).

References

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This article references 70 other publications.

  1. 1
    Shuken, S. R. An Introduction to Mass Spectrometry-Based Proteomics. J. Proteome Res. 2023, 22 (7), 2151– 2171,  DOI: 10.1021/acs.jproteome.2c00838
    Google Scholar
    There is no corresponding record for this reference.
  2. 2
    Po, A.; Eyers, C. E. Top-Down Proteomics and the Challenges of True Proteoform Characterization. J. Proteome Res. 2023, 22 (12), 3663– 3675,  DOI: 10.1021/acs.jproteome.3c00416
    Google Scholar
    There is no corresponding record for this reference.
  3. 3
    Jiang, Y.; Rex, D. A. B.; Schuster, D.; Neely, B. A.; Rosano, G. L.; Volkmar, N.; Momenzadeh, A.; Peters-Clarke, T. M.; Egbert, S. B.; Kreimer, S. Comprehensive Overview of Bottom-Up Proteomics Using Mass Spectrometry. ACS Meas Sci. Au 2024, 4 (4), 338– 417,  DOI: 10.1021/acsmeasuresciau.3c00068
    Google Scholar
    There is no corresponding record for this reference.
  4. 4
    Chen, B.; Brown, K. A.; Lin, Z.; Ge, Y. Top-Down Proteomics: Ready for Prime Time?. Anal. Chem. 2018, 90 (1), 110– 127,  DOI: 10.1021/acs.analchem.7b04747
    Google Scholar
    There is no corresponding record for this reference.
  5. 5
    Meyer, J. G.; Niemi, N. M.; Pagliarini, D. J.; Coon, J. J. Quantitative shotgun proteome analysis by direct infusion. Nat. Methods 2020, 17 (12), 1222– 1228,  DOI: 10.1038/s41592-020-00999-z
    Google Scholar
    There is no corresponding record for this reference.
  6. 6
    Lenčo, J.; Jadeja, S.; Naplekov, D. K.; Krokhin, O. V.; Khalikova, M. A.; Chocholous, P.; Urban, J.; Broeckhoven, K.; Novakova, L.; Svec, F. Reversed-Phase Liquid Chromatography of Peptides for Bottom-Up Proteomics: A Tutorial. J. Proteome Res. 2022, 21 (12), 2846– 2892,  DOI: 10.1021/acs.jproteome.2c00407
    Google Scholar
    There is no corresponding record for this reference.
  7. 7
    Kurotani, A.; Tokmakov, A. A.; Sato, K. I.; Stefanov, V. E.; Yamada, Y.; Sakurai, T. Localization-specific distributions of protein pI in human proteome are governed by local pH and membrane charge. BMC Mol. Cell Biol. 2019, 20 (1), 36  DOI: 10.1186/s12860-019-0221-4
    Google Scholar
    There is no corresponding record for this reference.
  8. 8
    Penanes, P. A.; Gorshkov, V.; Ivanov, M. V.; Gorshkov, M. V.; Kjeldsen, F. Potential of Negative-Ion-Mode Proteomics: An MS1-Only Approach. J. Proteome Res. 2023, 22 (8), 2734– 2742,  DOI: 10.1021/acs.jproteome.3c00307
    Google Scholar
    There is no corresponding record for this reference.
  9. 9
    Liigand, P.; Kaupmees, K.; Haav, K.; Liigand, J.; Leito, I.; Girod, M.; Antoine, R.; Kruve, A. Think Negative: Finding the Best Electrospray Ionization/MS Mode for Your Analyte. Anal. Chem. 2017, 89 (11), 5665– 5668,  DOI: 10.1021/acs.analchem.7b00096
    Google Scholar
    There is no corresponding record for this reference.
  10. 10
    Leutert, M.; Entwisle, S. W.; Villen, J. Decoding Post-Translational Modification Crosstalk With Proteomics. Mol. Cell. Proteomics 2021, 20, 100129  DOI: 10.1016/j.mcpro.2021.100129
    Google Scholar
    There is no corresponding record for this reference.
  11. 11
    Ruhaak, L. R.; Xu, G.; Li, Q.; Goonatilleke, E.; Lebrilla, C. B. Mass Spectrometry Approaches to Glycomic and Glycoproteomic Analyses. Chem. Rev. 2018, 118 (17), 7886– 7930,  DOI: 10.1021/acs.chemrev.7b00732
    Google Scholar
    There is no corresponding record for this reference.
  12. 12
    McAlister, G. C.; Russell, J. D.; Rumachik, N. G.; Hebert, A. S.; Syka, J. E.; Geer, L. Y.; Westphall, M. S.; Pagliarini, D. J.; Coon, J. J. Analysis of the acidic proteome with negative electron-transfer dissociation mass spectrometry. Anal. Chem. 2012, 84 (6), 2875– 2882,  DOI: 10.1021/ac203430u
    Google Scholar
    There is no corresponding record for this reference.
  13. 13
    Robinson, M. R.; Taliaferro, J. M.; Dalby, K. N.; Brodbelt, J. S. 193 nm Ultraviolet Photodissociation Mass Spectrometry for Phosphopeptide Characterization in the Positive and Negative Ion Modes. J. Proteome Res. 2016, 15 (8), 2739– 2748,  DOI: 10.1021/acs.jproteome.6b00289
    Google Scholar
    There is no corresponding record for this reference.
  14. 14
    Ewing, N. P.; Cassady, C. J. Dissociation of multiply charged negative ions for hirudin (54–65), fibrinopeptide B, and insulin A (oxidized). J. Am. Soc. Mass Spectrom. 2001, 12 (1), 105– 116,  DOI: 10.1016/S1044-0305(00)00195-1
    Google Scholar
    There is no corresponding record for this reference.
  15. 15
    Bowie, J. H.; Brinkworth, C. S.; Dua, S. Collision-induced fragmentations of the (M-H)- parent anions of underivatized peptides: an aid to structure determination and some unusual negative ion cleavages. Mass Spectrom Rev. 2002, 21 (2), 87– 107,  DOI: 10.1002/mas.10022
    Google Scholar
    There is no corresponding record for this reference.
  16. 16
    Brinkworth, C. S.; Dua, S.; McAnoy, A. M.; Bowie, J. H. Negative ion fragmentations of deprotonated peptides: backbone cleavages directed through both Asp and Glu. Rapid Commun. Mass Spectrom. 2001, 15 (20), 1965– 1973,  DOI: 10.1002/rcm.457
    Google Scholar
    There is no corresponding record for this reference.
  17. 17
    Zuo, M.-Q.; Sun, R.-X.; Fang, R.-Q.; He, S.-M.; Dong, M.-Q. Characterization of collision-induced dissociation of deprotonated peptides of 4–16 amino acids using high-resolution mass spectrometry. Int. J. Mass Spectrom. 2019, 445, 116186  DOI: 10.1016/j.ijms.2019.116186
    Google Scholar
    There is no corresponding record for this reference.
  18. 18
    Chrisman, P. A.; McLuckey, S. A. Dissociations of disulfide-linked gaseous polypeptide/protein anions: ion chemistry with implications for protein identification and characterization. J. Proteome Res. 2002, 1 (6), 549– 557,  DOI: 10.1021/pr025561z
    Google Scholar
    There is no corresponding record for this reference.
  19. 19
    Kjeldsen, F.; Horning, O. B.; Jensen, S. S.; Giessing, A. M.; Jensen, O. N. Towards liquid chromatography time-scale peptide sequencing and characterization of post-translational modifications in the negative-ion mode using electron detachment dissociation tandem mass spectrometry. J. Am. Soc. Mass Spectrom. 2008, 19 (8), 1156– 1162,  DOI: 10.1016/j.jasms.2008.04.031
    Google Scholar
    There is no corresponding record for this reference.
  20. 20
    Kalli, A.; Grigorean, G.; Hakansson, K. Electron induced dissociation of singly deprotonated peptides. J. Am. Soc. Mass Spectrom. 2011, 22 (12), 2209– 2221,  DOI: 10.1007/s13361-011-0233-6
    Google Scholar
    There is no corresponding record for this reference.
  21. 21
    Yoo, H. J.; Wang, N.; Zhuang, S.; Song, H.; Hakansson, K. Negative-ion electron capture dissociation: radical-driven fragmentation of charge-increased gaseous peptide anions. J. Am. Chem. Soc. 2011, 133 (42), 16790– 16793,  DOI: 10.1021/ja207736y
    Google Scholar
    There is no corresponding record for this reference.
  22. 22
    Huzarska, M.; Ugalde, I.; Kaplan, D. A.; Hartmer, R.; Easterling, M. L.; Polfer, N. C. Negative electron transfer dissociation of deprotonated phosphopeptide anions: choice of radical cation reagent and competition between electron and proton transfer. Anal. Chem. 2010, 82 (7), 2873– 2878,  DOI: 10.1021/ac9028592
    Google Scholar
    There is no corresponding record for this reference.
  23. 23
    Riley, N. M.; Rush, M. J.; Rose, C. M.; Richards, A. L.; Kwiecien, N. W.; Bailey, D. J.; Hebert, A. S.; Westphall, M. S.; Coon, J. J. The Negative Mode Proteome with Activated Ion Negative Electron Transfer Dissociation (AI-NETD). Mol. Cell. Proteomics 2015, 14 (10), 2644– 2660,  DOI: 10.1074/mcp.M115.049726
    Google Scholar
    There is no corresponding record for this reference.
  24. 24
    Shaw, J. B.; Kaplan, D. A.; Brodbelt, J. S. Activated ion negative electron transfer dissociation of multiply charged peptide anions. Anal. Chem. 2013, 85 (9), 4721– 4728,  DOI: 10.1021/ac4005315
    Google Scholar
    There is no corresponding record for this reference.
  25. 25
    Madsen, J. A.; Kaoud, T. S.; Dalby, K. N.; Brodbelt, J. S. 193-nm photodissociation of singly and multiply charged peptide anions for acidic proteome characterization. Proteomics 2011, 11 (7), 1329– 1334,  DOI: 10.1002/pmic.201000565
    Google Scholar
    There is no corresponding record for this reference.
  26. 26
    Greer, S. M.; Bern, M.; Becker, C.; Brodbelt, J. S. Extending Proteome Coverage by Combining MS/MS Methods and a Modified Bioinformatics Platform Adapted for Database Searching of Positive and Negative Polarity 193 nm Ultraviolet Photodissociation Mass Spectra. J. Proteome Res. 2018, 1340– 1347,  DOI: 10.1021/acs.jproteome.7b00673
    Google Scholar
    There is no corresponding record for this reference.
  27. 27
    Shaw, J. B.; Madsen, J. A.; Xu, H.; Brodbelt, J. S. Systematic comparison of ultraviolet photodissociation and electron transfer dissociation for peptide anion characterization. J. Am. Soc. Mass Spectrom. 2012, 23 (10), 1707– 1715,  DOI: 10.1007/s13361-012-0424-9
    Google Scholar
    There is no corresponding record for this reference.
  28. 28
    Brodbelt, J. S.; Morrison, L. J.; Santos, I. Ultraviolet Photodissociation Mass Spectrometry for Analysis of Biological Molecules. Chem. Rev. 2020, 120 (7), 3328– 3380,  DOI: 10.1021/acs.chemrev.9b00440
    Google Scholar
    There is no corresponding record for this reference.
  29. 29
    LeBlanc, B. M.; Moreno, R. Y.; Escobar, E. E.; Venkat Ramani, M. K.; Brodbelt, J. S.; Zhang, Y. What’s all the phos about? Insights into the phosphorylation state of the RNA polymerase II C-terminal domain via mass spectrometry. RSC Chem. Biol. 2021, 2 (4), 1084– 1095,  DOI: 10.1039/D1CB00083G
    Google Scholar
    There is no corresponding record for this reference.
  30. 30
    Bashyal, A.; Brodbelt, J. S. Uncommon posttranslational modifications in proteomics: ADP-ribosylation, tyrosine nitration, and tyrosine sulfation. Mass Spectrom. Rev. 2024, 43 (2), 289– 326,  DOI: 10.1002/mas.21811
    Google Scholar
    There is no corresponding record for this reference.
  31. 31
    Madsen, J. A.; Ko, B. J.; Xu, H.; Iwashkiw, J. A.; Robotham, S. A.; Shaw, J. B.; Feldman, M. F.; Brodbelt, J. S. Concurrent automated sequencing of the glycan and peptide portions of O-linked glycopeptide anions by ultraviolet photodissociation mass spectrometry. Anal. Chem. 2013, 85 (19), 9253– 9261,  DOI: 10.1021/ac4021177
    Google Scholar
    There is no corresponding record for this reference.
  32. 32
    Robinson, M. R.; Brodbelt, J. S. Integrating Weak Anion Exchange and Ultraviolet Photodissociation Mass Spectrometry with Strategic Modulation of Peptide Basicity for the Enrichment of Sulfopeptides. Anal. Chem. 2016, 88 (22), 11037– 11045,  DOI: 10.1021/acs.analchem.6b02899
    Google Scholar
    There is no corresponding record for this reference.
  33. 33
    Antoine, R.; Joly, L.; Tabarin, T.; Broyer, M.; Dugourd, P.; Lemoine, J. Photo-induced formation of radical anion peptides. Electron photodetachment dissociation experiments. Rapid Commun. Mass Spectrom. 2007, 21 (2), 265– 268,  DOI: 10.1002/rcm.2810
    Google Scholar
    There is no corresponding record for this reference.
  34. 34
    Larraillet, V.; Antoine, R.; Dugourd, P.; Lemoine, J. Activated-electron photodetachment dissociation for the structural characterization of protein polyanions. Anal. Chem. 2009, 81 (20), 8410– 8416,  DOI: 10.1021/ac901304d
    Google Scholar
    There is no corresponding record for this reference.
  35. 35
    Cech, N. B.; Enke, C. G. Practical implications of some recent studies in electrospray ionization fundamentals. Mass Spectrom. Rev. 2001, 20 (6), 362– 387,  DOI: 10.1002/mas.10008
    Google Scholar
    There is no corresponding record for this reference.
  36. 36
    Konermann, L.; Douglas, D. J. Unfolding of proteins monitored by electrospray ionization mass spectrometry: a comparison of positive and negative ion modes. J. Am. Soc. Mass Spectrom. 1998, 9 (12), 1248– 1254,  DOI: 10.1016/S1044-0305(98)00103-2
    Google Scholar
    There is no corresponding record for this reference.
  37. 37
    Sterling, H. J.; Daly, M. P.; Feld, G. K.; Thoren, K. L.; Kintzer, A. F.; Krantz, B. A.; Williams, E. R. Effects of supercharging reagents on noncovalent complex structure in electrospray ionization from aqueous solutions. J. Am. Soc. Mass Spectrom. 2010, 21 (10), 1762– 1774,  DOI: 10.1016/j.jasms.2010.06.012
    Google Scholar
    There is no corresponding record for this reference.
  38. 38
    Kharlamova, A.; McLuckey, S. A. Negative electrospray droplet exposure to gaseous bases for the manipulation of protein charge state distributions. Anal. Chem. 2011, 83 (1), 431– 437,  DOI: 10.1021/ac1027319
    Google Scholar
    There is no corresponding record for this reference.
  39. 39
    Nišavić, M.; Hozic, A.; Hamersak, Z.; Radic, M.; Butorac, A.; Duvnjak, M.; Cindric, M. High-Efficiency Microflow and Nanoflow Negative Electrospray Ionization of Peptides Induced by Gas-Phase Proton Transfer Reactions. Anal. Chem. 2017, 89 (9), 4847– 4854,  DOI: 10.1021/acs.analchem.6b04466
    Google Scholar
    There is no corresponding record for this reference.
  40. 40
    McClory, P. J.; Hakansson, K. Corona Discharge Suppression in Negative Ion Mode Nanoelectrospray Ionization via Trifluoroethanol Addition. Anal. Chem. 2017, 89 (19), 10188– 10193,  DOI: 10.1021/acs.analchem.7b01225
    Google Scholar
    There is no corresponding record for this reference.
  41. 41
    Stevens, A.; Abramsson, M. L.; Agasid, M. T.; Allison, T. M.; Landreh, M. Stabilization of Protein Interactions through Electrospray Additives in Negative Ion Mode Native Mass Spectrometry. Anal. Chem. 2025, 97 (20), 10738– 10744,  DOI: 10.1021/acs.analchem.5c00757
    Google Scholar
    There is no corresponding record for this reference.
  42. 42
    Ogorzalek Loo, R. R.; Lakshmanan, R.; Loo, J. A. What protein charging (and supercharging) reveal about the mechanism of electrospray ionization. J. Am. Soc. Mass Spectrom. 2014, 25 (10), 1675– 1693,  DOI: 10.1007/s13361-014-0965-1
    Google Scholar
    There is no corresponding record for this reference.
  43. 43
    Greer, S. M.; Cannon, J. R.; Brodbelt, J. S. Improvement of shotgun proteomics in the negative mode by carbamylation of peptides and ultraviolet photodissociation mass spectrometry. Anal. Chem. 2014, 86 (24), 12285– 12290,  DOI: 10.1021/ac5035314
    Google Scholar
    There is no corresponding record for this reference.
  44. 44
    Le Blanc, J. C. Y.; Guevremont, R.; Siu, K. W. M. Electrospray mass spectrometry of some proteins and the aqueous solution acid/base equilibrium model in the negative ion detection mode. Int. J. Mass Spectrom. 1993, 125 (2–3), 145– 153,  DOI: 10.1016/0168-1176(93)80037-F
    Google Scholar
    There is no corresponding record for this reference.
  45. 45
    Ganisl, B.; Taucher, M.; Riml, C.; Breuker, K. Charge as you like! Efficient manipulation of negative ion net charge in electrospray ionization of proteins and nucleic acids. Eur. J. Mass Spectrom 2011, 17 (4), 333– 343,  DOI: 10.1255/ejms.1140
    Google Scholar
    There is no corresponding record for this reference.
  46. 46
    Madsen, J. A.; Xu, H.; Robinson, M. R.; Horton, A. P.; Shaw, J. B.; Giles, D. K.; Kaoud, T. S.; Dalby, K. N.; Trent, M. S.; Brodbelt, J. S. High-throughput database search and large-scale negative polarity liquid chromatography-tandem mass spectrometry with ultraviolet photodissociation for complex proteomic samples. Mol. Cell. Proteomics 2013, 12 (9), 2604– 2614,  DOI: 10.1074/mcp.O113.028258
    Google Scholar
    There is no corresponding record for this reference.
  47. 47
    Donnelly, D. P.; Rawlins, C. M.; DeHart, C. J.; Fornelli, L.; Schachner, L. F.; Lin, Z.; Lippens, J. L.; Aluri, K. C.; Sarin, R.; Chen, B. Best practices and benchmarks for intact protein analysis for top-down mass spectrometry. Nat. Methods 2019, 16 (7), 587– 594,  DOI: 10.1038/s41592-019-0457-0
    Google Scholar
    There is no corresponding record for this reference.
  48. 48
    Roberts, D. S.; Loo, J. A.; Tsybin, Y. O.; Liu, X.; Wu, S.; Chamot-Rooke, J.; Agar, J. N.; Pasa-Tolic, L.; Smith, L. M.; Ge, Y. Top-down proteomics. Nat. Rev. Methods Primers 2024, 4 (1), 38  DOI: 10.1038/s43586-024-00318-2
    Google Scholar
    There is no corresponding record for this reference.
  49. 49
    Larson, E. J.; Roberts, D. S.; Melby, J. A.; Buck, K. M.; Zhu, Y.; Zhou, S.; Han, L.; Zhang, Q.; Ge, Y. High-Throughput Multi-attribute Analysis of Antibody-Drug Conjugates Enabled by Trapped Ion Mobility Spectrometry and Top-Down Mass Spectrometry. Anal. Chem. 2021, 93 (29), 10013– 10021,  DOI: 10.1021/acs.analchem.1c00150
    Google Scholar
    There is no corresponding record for this reference.
  50. 50
    Riley, N. M.; Westphall, M. S.; Coon, J. J. Activated Ion-Electron Transfer Dissociation Enables Comprehensive Top-Down Protein Fragmentation. J. Proteome Res. 2017, 16 (7), 2653– 2659,  DOI: 10.1021/acs.jproteome.7b00249
    Google Scholar
    There is no corresponding record for this reference.
  51. 51
    Wei, B.; Zenaidee, M. A.; Lantz, C.; Williams, B. J.; Totten, S.; Ogorzalek Loo, R. R.; Loo, J. A. Top-down mass spectrometry and assigning internal fragments for determining disulfide bond positions in proteins. Analyst 2022, 148 (1), 26– 37,  DOI: 10.1039/D2AN01517J
    Google Scholar
    There is no corresponding record for this reference.
  52. 52
    Dunham, S. D.; Brodbelt, J. S. Enhancing Top-Down Analysis of Proteins by Combining Ultraviolet Photodissociation (UVPD), Proton-Transfer Charge Reduction (PTCR), and Gas-Phase Fractionation to Alleviate the Impact of Nondissociated Precursor Ions. J. Am. Soc. Mass Spectrom. 2024, 35 (2), 255– 265,  DOI: 10.1021/jasms.3c00351
    Google Scholar
    There is no corresponding record for this reference.
  53. 53
    Walker, J. N.; Lam, R.; Brodbelt, J. S. Enhanced Characterization of Histones Using 193 nm Ultraviolet Photodissociation and Proton Transfer Charge Reduction. Anal. Chem. 2023, 95 (14), 5985– 5993,  DOI: 10.1021/acs.analchem.2c05765
    Google Scholar
    There is no corresponding record for this reference.
  54. 54
    Watts, E.; Thyer, R.; Ellington, A. D.; Brodbelt, J. S. Integrated Top-Down and Bottom-Up Mass Spectrometry for Characterization of Diselenide Bridging Patterns of Synthetic Selenoproteins. Anal. Chem. 2022, 94 (32), 11175– 11184,  DOI: 10.1021/acs.analchem.2c01433
    Google Scholar
    There is no corresponding record for this reference.
  55. 55
    Brodbelt, J. S. Deciphering combinatorial post-translational modifications by top-down mass spectrometry. Curr. Opin. Chem. Biol. 2022, 70, 102180  DOI: 10.1016/j.cbpa.2022.102180
    Google Scholar
    There is no corresponding record for this reference.
  56. 56
    Fornelli, L.; Srzentic, K.; Toby, T. K.; Doubleday, P. F.; Huguet, R.; Mullen, C.; Melani, R. D.; Dos Santos Seckler, H.; DeHart, C. J.; Weisbrod, C. R. Thorough Performance Evaluation of 213 nm Ultraviolet Photodissociation for Top-down Proteomics. Mol. Cell. Proteomics 2020, 19 (2), 405– 420,  DOI: 10.1074/mcp.TIR119.001638
    Google Scholar
    There is no corresponding record for this reference.
  57. 57
    Oates, R. N.; Lieu, L. B.; Srzentic, K.; Damoc, E.; Fornelli, L. Characterization of a Monoclonal Antibody by Native and Denaturing Top-Down Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2024, 35 (9), 2197– 2208,  DOI: 10.1021/jasms.4c00224
    Google Scholar
    There is no corresponding record for this reference.
  58. 58
    Cannon, J. R.; Cammarata, M. B.; Robotham, S. A.; Cotham, V. C.; Shaw, J. B.; Fellers, R. T.; Early, B. P.; Thomas, P. M.; Kelleher, N. L.; Brodbelt, J. S. Ultraviolet photodissociation for characterization of whole proteins on a chromatographic time scale. Anal. Chem. 2014, 86 (4), 2185– 2192,  DOI: 10.1021/ac403859a
    Google Scholar
    There is no corresponding record for this reference.
  59. 59
    Lanzillotti, M.; Brodbelt, J. S. Comparison of Top-Down Protein Fragmentation Induced by 213 and 193 nm UVPD. J. Am. Soc. Mass Spectrom. 2023, 34 (2), 279– 285,  DOI: 10.1021/jasms.2c00288
    Google Scholar
    There is no corresponding record for this reference.
  60. 60
    Juetten, K. J.; Brodbelt, J. S. Top-Down Analysis of Supercharged Proteins Using Collision-, Electron-, and Photon-Based Activation Methods. J. Am. Soc. Mass Spectrom. 2023, 34 (7), 1467– 1476,  DOI: 10.1021/jasms.3c00138
    Google Scholar
    There is no corresponding record for this reference.
  61. 61
    Hyde, A. M.; Calabria, R.; Arvary, R.; Wang, X.; Klapars, A. Investigating the Underappreciated Hydrolytic Instability of 1,8-Diazabicyclo[5.4.0]undec-7-ene and Related Unsaturated Nitrogenous Bases. Org. Process Res. Dev. 2019, 23 (9), 1860– 1871,  DOI: 10.1021/acs.oprd.9b00187
    Google Scholar
    There is no corresponding record for this reference.
  62. 62
    Juetten, K. J.; Brodbelt, J. S. MS-TAFI: A Tool for the Analysis of Fragment Ions Generated from Intact Proteins. J. Proteome Res. 2023, 22 (2), 546– 550,  DOI: 10.1021/acs.jproteome.2c00594
    Google Scholar
    There is no corresponding record for this reference.
  63. 63
    Durbin, K. R.; Robey, M. T.; Voong, L. N.; Fellers, R. T.; Lutomski, C. A.; El-Baba, T. J.; Robinson, C. V.; Kelleher, N. L. ProSight Native: Defining Protein Complex Composition from Native Top-Down Mass Spectrometry Data. J. Proteome Res. 2023, 22 (8), 2660– 2668,  DOI: 10.1021/acs.jproteome.3c00171
    Google Scholar
    There is no corresponding record for this reference.
  64. 64
    Li, J.; Santambrogio, C.; Brocca, S.; Rossetti, G.; Carloni, P.; Grandori, R. Conformational effects in protein electrospray-ionization mass spectrometry. Mass Spectrom Rev. 2016, 35 (1), 111– 122,  DOI: 10.1002/mas.21465
    Google Scholar
    There is no corresponding record for this reference.
  65. 65
    Reid, G. E.; Wu, J.; Chrisman, P. A.; Wells, J. M.; McLuckey, S. A. Charge-state-dependent sequence analysis of protonated ubiquitin ions via ion trap tandem mass spectrometry. Anal. Chem. 2001, 73 (14), 3274– 3281,  DOI: 10.1021/ac0101095
    Google Scholar
    There is no corresponding record for this reference.
  66. 66
    Cobb, J. S.; Easterling, M. L.; Agar, J. N. Structural characterization of intact proteins is enhanced by prevalent fragmentation pathways rarely observed for peptides. J. Am. Soc. Mass Spectrom. 2010, 21 (6), 949– 959,  DOI: 10.1016/j.jasms.2010.02.009
    Google Scholar
    There is no corresponding record for this reference.
  67. 67
    Hellinger, J.; Brodbelt, J. S. Impact of Charge State on Characterization of Large Middle-Down Sized Peptides by Tandem Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2024, 35 (8), 1647– 1656,  DOI: 10.1021/jasms.3c00405
    Google Scholar
    There is no corresponding record for this reference.
  68. 68
    Carrera, G. V. S. M.; Jordão, N.; Santos, M. M.; da Ponte, M. N.; Branco, L. C. Reversible systems based on CO2, amino-acids and organic superbases. RSC Adv. 2015, 5 (45), 35564– 35571,  DOI: 10.1039/C5RA03474D
    Google Scholar
    There is no corresponding record for this reference.
  69. 69
    Bonner, J.; Talbert, L. E.; Akkawi, N.; Julian, R. R. Simplified identification of disulfide, trisulfide, and thioether pairs with 213 nm UVPD. Analyst 2018, 143 (21), 5176– 5184,  DOI: 10.1039/C8AN01582A
    Google Scholar
    There is no corresponding record for this reference.
  70. 70
    Macias, L. A.; Brodbelt, J. S. Investigation of Product Ions Generated by 193 nm Ultraviolet Photodissociation of Peptides and Proteins Containing Disulfide Bonds. J. Am. Soc. Mass Spectrom. 2022, 33 (7), 1315– 1324,  DOI: 10.1021/jasms.2c00124
    Google Scholar
    There is no corresponding record for this reference.

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  • Abstract

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    Figure 1

    Figure 1. MS1 spectra of solutions containing 10 μM myoglobin and (a) 1% formic acid in positive mode, (b) with 1% NH3·H2O in negative mode, and (c) with 0.05% DBN in negative mode. Selected charge states of the protein and the heme are annotated.

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    Figure 2

    Figure 2. MS/MS spectra of myoglobin (20–, m/z 846, indicated by an asterisk in the UVPD spectrum). (a) HCD using NCE of 15. (c) UVPD using 1 laser pulse of 0.5 mJ. (e) a-EPD performed using UVPD (1 laser pulse of 0.3 mJ) followed by isolation of the charge-reduced 19−• radical ions and then CID using NCE of 23 (b), (d), (f) show sequence coverage and backbone cleavage maps based on relative intensities of fragment ions originating from different backbone cleavage sites derived from the corresponding HCD, UVPD, and a-EPD mass spectra.

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    Figure 3

    Figure 3. Sequence coverage produced by HCD, UVPD and a-EPD, as a function of precursor charge state for (a) ubiquitin, (b) myoglobin, (c) cytochrome c, (d) β-lactoglobulin, (e) carbonic anhydrase and (f) enolase.

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    Figure 4

    Figure 4. (a) Fraction of abundance of fragment ion types generated by HCD (HCD−), UVPD (UVPD−), and a-EPD for protein anions and by UVPD for protein cations (UVPD+) with error bars showing standard deviations across six proteins and multiple charge states. (b) Violin plot showing the count of fragment ions as a function of fragment size (expressed as a percentage of the full protein length) for four MS/MS methods.

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    Figure 5

    Figure 5. Backbone cleavage maps displaying relative intensities of fragment ions originating from different backbone cleavage sites of β lactoglobulin based on (a) HCD (NCE 14) of 14– charge state, (b) UVPD (1 laser pulse of 1.5 mJ) of 14– charge state, (c) a-EPD of 14– charge state [UVPD (1 laser pulse of 0.5 mJ) followed by isolation of the 13–• radical ions and then CID (NCE 20)], (d) HCD (NCE 14) of the 14+ charge state, and (e) UVPD (1 laser pulse of 1.5 mJ) of 14+ charge state. The backbone cleavage sites adjacent to the C66–C160 disulfide bond that resulted in the most abundant fragment ions are marked with circles and triangles on the backbone cleavage plots for UVPD (5b) and a-EPD (5c). (f) Isotope fitting results of the two of the most abundant fragments originating from cleavage adjacent to C66–C160 disulfide bond produced by UVPD ((a65 + 1)6– and b16012–) and a-EPD (c655– and b16012–) for the 14– charge state (all with score >0.8 in Prosight Native).

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  • References

    This article references 70 other publications.

    1. 1
      Shuken, S. R. An Introduction to Mass Spectrometry-Based Proteomics. J. Proteome Res. 2023, 22 (7), 2151– 2171,  DOI: 10.1021/acs.jproteome.2c00838
      There is no corresponding record for this reference.
    2. 2
      Po, A.; Eyers, C. E. Top-Down Proteomics and the Challenges of True Proteoform Characterization. J. Proteome Res. 2023, 22 (12), 3663– 3675,  DOI: 10.1021/acs.jproteome.3c00416
      There is no corresponding record for this reference.
    3. 3
      Jiang, Y.; Rex, D. A. B.; Schuster, D.; Neely, B. A.; Rosano, G. L.; Volkmar, N.; Momenzadeh, A.; Peters-Clarke, T. M.; Egbert, S. B.; Kreimer, S. Comprehensive Overview of Bottom-Up Proteomics Using Mass Spectrometry. ACS Meas Sci. Au 2024, 4 (4), 338– 417,  DOI: 10.1021/acsmeasuresciau.3c00068
      There is no corresponding record for this reference.
    4. 4
      Chen, B.; Brown, K. A.; Lin, Z.; Ge, Y. Top-Down Proteomics: Ready for Prime Time?. Anal. Chem. 2018, 90 (1), 110– 127,  DOI: 10.1021/acs.analchem.7b04747
      There is no corresponding record for this reference.
    5. 5
      Meyer, J. G.; Niemi, N. M.; Pagliarini, D. J.; Coon, J. J. Quantitative shotgun proteome analysis by direct infusion. Nat. Methods 2020, 17 (12), 1222– 1228,  DOI: 10.1038/s41592-020-00999-z
      There is no corresponding record for this reference.
    6. 6
      Lenčo, J.; Jadeja, S.; Naplekov, D. K.; Krokhin, O. V.; Khalikova, M. A.; Chocholous, P.; Urban, J.; Broeckhoven, K.; Novakova, L.; Svec, F. Reversed-Phase Liquid Chromatography of Peptides for Bottom-Up Proteomics: A Tutorial. J. Proteome Res. 2022, 21 (12), 2846– 2892,  DOI: 10.1021/acs.jproteome.2c00407
      There is no corresponding record for this reference.
    7. 7
      Kurotani, A.; Tokmakov, A. A.; Sato, K. I.; Stefanov, V. E.; Yamada, Y.; Sakurai, T. Localization-specific distributions of protein pI in human proteome are governed by local pH and membrane charge. BMC Mol. Cell Biol. 2019, 20 (1), 36  DOI: 10.1186/s12860-019-0221-4
      There is no corresponding record for this reference.
    8. 8
      Penanes, P. A.; Gorshkov, V.; Ivanov, M. V.; Gorshkov, M. V.; Kjeldsen, F. Potential of Negative-Ion-Mode Proteomics: An MS1-Only Approach. J. Proteome Res. 2023, 22 (8), 2734– 2742,  DOI: 10.1021/acs.jproteome.3c00307
      There is no corresponding record for this reference.
    9. 9
      Liigand, P.; Kaupmees, K.; Haav, K.; Liigand, J.; Leito, I.; Girod, M.; Antoine, R.; Kruve, A. Think Negative: Finding the Best Electrospray Ionization/MS Mode for Your Analyte. Anal. Chem. 2017, 89 (11), 5665– 5668,  DOI: 10.1021/acs.analchem.7b00096
      There is no corresponding record for this reference.
    10. 10
      Leutert, M.; Entwisle, S. W.; Villen, J. Decoding Post-Translational Modification Crosstalk With Proteomics. Mol. Cell. Proteomics 2021, 20, 100129  DOI: 10.1016/j.mcpro.2021.100129
      There is no corresponding record for this reference.
    11. 11
      Ruhaak, L. R.; Xu, G.; Li, Q.; Goonatilleke, E.; Lebrilla, C. B. Mass Spectrometry Approaches to Glycomic and Glycoproteomic Analyses. Chem. Rev. 2018, 118 (17), 7886– 7930,  DOI: 10.1021/acs.chemrev.7b00732
      There is no corresponding record for this reference.
    12. 12
      McAlister, G. C.; Russell, J. D.; Rumachik, N. G.; Hebert, A. S.; Syka, J. E.; Geer, L. Y.; Westphall, M. S.; Pagliarini, D. J.; Coon, J. J. Analysis of the acidic proteome with negative electron-transfer dissociation mass spectrometry. Anal. Chem. 2012, 84 (6), 2875– 2882,  DOI: 10.1021/ac203430u
      There is no corresponding record for this reference.
    13. 13
      Robinson, M. R.; Taliaferro, J. M.; Dalby, K. N.; Brodbelt, J. S. 193 nm Ultraviolet Photodissociation Mass Spectrometry for Phosphopeptide Characterization in the Positive and Negative Ion Modes. J. Proteome Res. 2016, 15 (8), 2739– 2748,  DOI: 10.1021/acs.jproteome.6b00289
      There is no corresponding record for this reference.
    14. 14
      Ewing, N. P.; Cassady, C. J. Dissociation of multiply charged negative ions for hirudin (54–65), fibrinopeptide B, and insulin A (oxidized). J. Am. Soc. Mass Spectrom. 2001, 12 (1), 105– 116,  DOI: 10.1016/S1044-0305(00)00195-1
      There is no corresponding record for this reference.
    15. 15
      Bowie, J. H.; Brinkworth, C. S.; Dua, S. Collision-induced fragmentations of the (M-H)- parent anions of underivatized peptides: an aid to structure determination and some unusual negative ion cleavages. Mass Spectrom Rev. 2002, 21 (2), 87– 107,  DOI: 10.1002/mas.10022
      There is no corresponding record for this reference.
    16. 16
      Brinkworth, C. S.; Dua, S.; McAnoy, A. M.; Bowie, J. H. Negative ion fragmentations of deprotonated peptides: backbone cleavages directed through both Asp and Glu. Rapid Commun. Mass Spectrom. 2001, 15 (20), 1965– 1973,  DOI: 10.1002/rcm.457
      There is no corresponding record for this reference.
    17. 17
      Zuo, M.-Q.; Sun, R.-X.; Fang, R.-Q.; He, S.-M.; Dong, M.-Q. Characterization of collision-induced dissociation of deprotonated peptides of 4–16 amino acids using high-resolution mass spectrometry. Int. J. Mass Spectrom. 2019, 445, 116186  DOI: 10.1016/j.ijms.2019.116186
      There is no corresponding record for this reference.
    18. 18
      Chrisman, P. A.; McLuckey, S. A. Dissociations of disulfide-linked gaseous polypeptide/protein anions: ion chemistry with implications for protein identification and characterization. J. Proteome Res. 2002, 1 (6), 549– 557,  DOI: 10.1021/pr025561z
      There is no corresponding record for this reference.
    19. 19
      Kjeldsen, F.; Horning, O. B.; Jensen, S. S.; Giessing, A. M.; Jensen, O. N. Towards liquid chromatography time-scale peptide sequencing and characterization of post-translational modifications in the negative-ion mode using electron detachment dissociation tandem mass spectrometry. J. Am. Soc. Mass Spectrom. 2008, 19 (8), 1156– 1162,  DOI: 10.1016/j.jasms.2008.04.031
      There is no corresponding record for this reference.
    20. 20
      Kalli, A.; Grigorean, G.; Hakansson, K. Electron induced dissociation of singly deprotonated peptides. J. Am. Soc. Mass Spectrom. 2011, 22 (12), 2209– 2221,  DOI: 10.1007/s13361-011-0233-6
      There is no corresponding record for this reference.
    21. 21
      Yoo, H. J.; Wang, N.; Zhuang, S.; Song, H.; Hakansson, K. Negative-ion electron capture dissociation: radical-driven fragmentation of charge-increased gaseous peptide anions. J. Am. Chem. Soc. 2011, 133 (42), 16790– 16793,  DOI: 10.1021/ja207736y
      There is no corresponding record for this reference.
    22. 22
      Huzarska, M.; Ugalde, I.; Kaplan, D. A.; Hartmer, R.; Easterling, M. L.; Polfer, N. C. Negative electron transfer dissociation of deprotonated phosphopeptide anions: choice of radical cation reagent and competition between electron and proton transfer. Anal. Chem. 2010, 82 (7), 2873– 2878,  DOI: 10.1021/ac9028592
      There is no corresponding record for this reference.
    23. 23
      Riley, N. M.; Rush, M. J.; Rose, C. M.; Richards, A. L.; Kwiecien, N. W.; Bailey, D. J.; Hebert, A. S.; Westphall, M. S.; Coon, J. J. The Negative Mode Proteome with Activated Ion Negative Electron Transfer Dissociation (AI-NETD). Mol. Cell. Proteomics 2015, 14 (10), 2644– 2660,  DOI: 10.1074/mcp.M115.049726
      There is no corresponding record for this reference.
    24. 24
      Shaw, J. B.; Kaplan, D. A.; Brodbelt, J. S. Activated ion negative electron transfer dissociation of multiply charged peptide anions. Anal. Chem. 2013, 85 (9), 4721– 4728,  DOI: 10.1021/ac4005315
      There is no corresponding record for this reference.
    25. 25
      Madsen, J. A.; Kaoud, T. S.; Dalby, K. N.; Brodbelt, J. S. 193-nm photodissociation of singly and multiply charged peptide anions for acidic proteome characterization. Proteomics 2011, 11 (7), 1329– 1334,  DOI: 10.1002/pmic.201000565
      There is no corresponding record for this reference.
    26. 26
      Greer, S. M.; Bern, M.; Becker, C.; Brodbelt, J. S. Extending Proteome Coverage by Combining MS/MS Methods and a Modified Bioinformatics Platform Adapted for Database Searching of Positive and Negative Polarity 193 nm Ultraviolet Photodissociation Mass Spectra. J. Proteome Res. 2018, 1340– 1347,  DOI: 10.1021/acs.jproteome.7b00673
      There is no corresponding record for this reference.
    27. 27
      Shaw, J. B.; Madsen, J. A.; Xu, H.; Brodbelt, J. S. Systematic comparison of ultraviolet photodissociation and electron transfer dissociation for peptide anion characterization. J. Am. Soc. Mass Spectrom. 2012, 23 (10), 1707– 1715,  DOI: 10.1007/s13361-012-0424-9
      There is no corresponding record for this reference.
    28. 28
      Brodbelt, J. S.; Morrison, L. J.; Santos, I. Ultraviolet Photodissociation Mass Spectrometry for Analysis of Biological Molecules. Chem. Rev. 2020, 120 (7), 3328– 3380,  DOI: 10.1021/acs.chemrev.9b00440
      There is no corresponding record for this reference.
    29. 29
      LeBlanc, B. M.; Moreno, R. Y.; Escobar, E. E.; Venkat Ramani, M. K.; Brodbelt, J. S.; Zhang, Y. What’s all the phos about? Insights into the phosphorylation state of the RNA polymerase II C-terminal domain via mass spectrometry. RSC Chem. Biol. 2021, 2 (4), 1084– 1095,  DOI: 10.1039/D1CB00083G
      There is no corresponding record for this reference.
    30. 30
      Bashyal, A.; Brodbelt, J. S. Uncommon posttranslational modifications in proteomics: ADP-ribosylation, tyrosine nitration, and tyrosine sulfation. Mass Spectrom. Rev. 2024, 43 (2), 289– 326,  DOI: 10.1002/mas.21811
      There is no corresponding record for this reference.
    31. 31
      Madsen, J. A.; Ko, B. J.; Xu, H.; Iwashkiw, J. A.; Robotham, S. A.; Shaw, J. B.; Feldman, M. F.; Brodbelt, J. S. Concurrent automated sequencing of the glycan and peptide portions of O-linked glycopeptide anions by ultraviolet photodissociation mass spectrometry. Anal. Chem. 2013, 85 (19), 9253– 9261,  DOI: 10.1021/ac4021177
      There is no corresponding record for this reference.
    32. 32
      Robinson, M. R.; Brodbelt, J. S. Integrating Weak Anion Exchange and Ultraviolet Photodissociation Mass Spectrometry with Strategic Modulation of Peptide Basicity for the Enrichment of Sulfopeptides. Anal. Chem. 2016, 88 (22), 11037– 11045,  DOI: 10.1021/acs.analchem.6b02899
      There is no corresponding record for this reference.
    33. 33
      Antoine, R.; Joly, L.; Tabarin, T.; Broyer, M.; Dugourd, P.; Lemoine, J. Photo-induced formation of radical anion peptides. Electron photodetachment dissociation experiments. Rapid Commun. Mass Spectrom. 2007, 21 (2), 265– 268,  DOI: 10.1002/rcm.2810
      There is no corresponding record for this reference.
    34. 34
      Larraillet, V.; Antoine, R.; Dugourd, P.; Lemoine, J. Activated-electron photodetachment dissociation for the structural characterization of protein polyanions. Anal. Chem. 2009, 81 (20), 8410– 8416,  DOI: 10.1021/ac901304d
      There is no corresponding record for this reference.
    35. 35
      Cech, N. B.; Enke, C. G. Practical implications of some recent studies in electrospray ionization fundamentals. Mass Spectrom. Rev. 2001, 20 (6), 362– 387,  DOI: 10.1002/mas.10008
      There is no corresponding record for this reference.
    36. 36
      Konermann, L.; Douglas, D. J. Unfolding of proteins monitored by electrospray ionization mass spectrometry: a comparison of positive and negative ion modes. J. Am. Soc. Mass Spectrom. 1998, 9 (12), 1248– 1254,  DOI: 10.1016/S1044-0305(98)00103-2
      There is no corresponding record for this reference.
    37. 37
      Sterling, H. J.; Daly, M. P.; Feld, G. K.; Thoren, K. L.; Kintzer, A. F.; Krantz, B. A.; Williams, E. R. Effects of supercharging reagents on noncovalent complex structure in electrospray ionization from aqueous solutions. J. Am. Soc. Mass Spectrom. 2010, 21 (10), 1762– 1774,  DOI: 10.1016/j.jasms.2010.06.012
      There is no corresponding record for this reference.
    38. 38
      Kharlamova, A.; McLuckey, S. A. Negative electrospray droplet exposure to gaseous bases for the manipulation of protein charge state distributions. Anal. Chem. 2011, 83 (1), 431– 437,  DOI: 10.1021/ac1027319
      There is no corresponding record for this reference.
    39. 39
      Nišavić, M.; Hozic, A.; Hamersak, Z.; Radic, M.; Butorac, A.; Duvnjak, M.; Cindric, M. High-Efficiency Microflow and Nanoflow Negative Electrospray Ionization of Peptides Induced by Gas-Phase Proton Transfer Reactions. Anal. Chem. 2017, 89 (9), 4847– 4854,  DOI: 10.1021/acs.analchem.6b04466
      There is no corresponding record for this reference.
    40. 40
      McClory, P. J.; Hakansson, K. Corona Discharge Suppression in Negative Ion Mode Nanoelectrospray Ionization via Trifluoroethanol Addition. Anal. Chem. 2017, 89 (19), 10188– 10193,  DOI: 10.1021/acs.analchem.7b01225
      There is no corresponding record for this reference.
    41. 41
      Stevens, A.; Abramsson, M. L.; Agasid, M. T.; Allison, T. M.; Landreh, M. Stabilization of Protein Interactions through Electrospray Additives in Negative Ion Mode Native Mass Spectrometry. Anal. Chem. 2025, 97 (20), 10738– 10744,  DOI: 10.1021/acs.analchem.5c00757
      There is no corresponding record for this reference.
    42. 42
      Ogorzalek Loo, R. R.; Lakshmanan, R.; Loo, J. A. What protein charging (and supercharging) reveal about the mechanism of electrospray ionization. J. Am. Soc. Mass Spectrom. 2014, 25 (10), 1675– 1693,  DOI: 10.1007/s13361-014-0965-1
      There is no corresponding record for this reference.
    43. 43
      Greer, S. M.; Cannon, J. R.; Brodbelt, J. S. Improvement of shotgun proteomics in the negative mode by carbamylation of peptides and ultraviolet photodissociation mass spectrometry. Anal. Chem. 2014, 86 (24), 12285– 12290,  DOI: 10.1021/ac5035314
      There is no corresponding record for this reference.
    44. 44
      Le Blanc, J. C. Y.; Guevremont, R.; Siu, K. W. M. Electrospray mass spectrometry of some proteins and the aqueous solution acid/base equilibrium model in the negative ion detection mode. Int. J. Mass Spectrom. 1993, 125 (2–3), 145– 153,  DOI: 10.1016/0168-1176(93)80037-F
      There is no corresponding record for this reference.
    45. 45
      Ganisl, B.; Taucher, M.; Riml, C.; Breuker, K. Charge as you like! Efficient manipulation of negative ion net charge in electrospray ionization of proteins and nucleic acids. Eur. J. Mass Spectrom 2011, 17 (4), 333– 343,  DOI: 10.1255/ejms.1140
      There is no corresponding record for this reference.
    46. 46
      Madsen, J. A.; Xu, H.; Robinson, M. R.; Horton, A. P.; Shaw, J. B.; Giles, D. K.; Kaoud, T. S.; Dalby, K. N.; Trent, M. S.; Brodbelt, J. S. High-throughput database search and large-scale negative polarity liquid chromatography-tandem mass spectrometry with ultraviolet photodissociation for complex proteomic samples. Mol. Cell. Proteomics 2013, 12 (9), 2604– 2614,  DOI: 10.1074/mcp.O113.028258
      There is no corresponding record for this reference.
    47. 47
      Donnelly, D. P.; Rawlins, C. M.; DeHart, C. J.; Fornelli, L.; Schachner, L. F.; Lin, Z.; Lippens, J. L.; Aluri, K. C.; Sarin, R.; Chen, B. Best practices and benchmarks for intact protein analysis for top-down mass spectrometry. Nat. Methods 2019, 16 (7), 587– 594,  DOI: 10.1038/s41592-019-0457-0
      There is no corresponding record for this reference.
    48. 48
      Roberts, D. S.; Loo, J. A.; Tsybin, Y. O.; Liu, X.; Wu, S.; Chamot-Rooke, J.; Agar, J. N.; Pasa-Tolic, L.; Smith, L. M.; Ge, Y. Top-down proteomics. Nat. Rev. Methods Primers 2024, 4 (1), 38  DOI: 10.1038/s43586-024-00318-2
      There is no corresponding record for this reference.
    49. 49
      Larson, E. J.; Roberts, D. S.; Melby, J. A.; Buck, K. M.; Zhu, Y.; Zhou, S.; Han, L.; Zhang, Q.; Ge, Y. High-Throughput Multi-attribute Analysis of Antibody-Drug Conjugates Enabled by Trapped Ion Mobility Spectrometry and Top-Down Mass Spectrometry. Anal. Chem. 2021, 93 (29), 10013– 10021,  DOI: 10.1021/acs.analchem.1c00150
      There is no corresponding record for this reference.
    50. 50
      Riley, N. M.; Westphall, M. S.; Coon, J. J. Activated Ion-Electron Transfer Dissociation Enables Comprehensive Top-Down Protein Fragmentation. J. Proteome Res. 2017, 16 (7), 2653– 2659,  DOI: 10.1021/acs.jproteome.7b00249
      There is no corresponding record for this reference.
    51. 51
      Wei, B.; Zenaidee, M. A.; Lantz, C.; Williams, B. J.; Totten, S.; Ogorzalek Loo, R. R.; Loo, J. A. Top-down mass spectrometry and assigning internal fragments for determining disulfide bond positions in proteins. Analyst 2022, 148 (1), 26– 37,  DOI: 10.1039/D2AN01517J
      There is no corresponding record for this reference.
    52. 52
      Dunham, S. D.; Brodbelt, J. S. Enhancing Top-Down Analysis of Proteins by Combining Ultraviolet Photodissociation (UVPD), Proton-Transfer Charge Reduction (PTCR), and Gas-Phase Fractionation to Alleviate the Impact of Nondissociated Precursor Ions. J. Am. Soc. Mass Spectrom. 2024, 35 (2), 255– 265,  DOI: 10.1021/jasms.3c00351
      There is no corresponding record for this reference.
    53. 53
      Walker, J. N.; Lam, R.; Brodbelt, J. S. Enhanced Characterization of Histones Using 193 nm Ultraviolet Photodissociation and Proton Transfer Charge Reduction. Anal. Chem. 2023, 95 (14), 5985– 5993,  DOI: 10.1021/acs.analchem.2c05765
      There is no corresponding record for this reference.
    54. 54
      Watts, E.; Thyer, R.; Ellington, A. D.; Brodbelt, J. S. Integrated Top-Down and Bottom-Up Mass Spectrometry for Characterization of Diselenide Bridging Patterns of Synthetic Selenoproteins. Anal. Chem. 2022, 94 (32), 11175– 11184,  DOI: 10.1021/acs.analchem.2c01433
      There is no corresponding record for this reference.
    55. 55
      Brodbelt, J. S. Deciphering combinatorial post-translational modifications by top-down mass spectrometry. Curr. Opin. Chem. Biol. 2022, 70, 102180  DOI: 10.1016/j.cbpa.2022.102180
      There is no corresponding record for this reference.
    56. 56
      Fornelli, L.; Srzentic, K.; Toby, T. K.; Doubleday, P. F.; Huguet, R.; Mullen, C.; Melani, R. D.; Dos Santos Seckler, H.; DeHart, C. J.; Weisbrod, C. R. Thorough Performance Evaluation of 213 nm Ultraviolet Photodissociation for Top-down Proteomics. Mol. Cell. Proteomics 2020, 19 (2), 405– 420,  DOI: 10.1074/mcp.TIR119.001638
      There is no corresponding record for this reference.
    57. 57
      Oates, R. N.; Lieu, L. B.; Srzentic, K.; Damoc, E.; Fornelli, L. Characterization of a Monoclonal Antibody by Native and Denaturing Top-Down Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2024, 35 (9), 2197– 2208,  DOI: 10.1021/jasms.4c00224
      There is no corresponding record for this reference.
    58. 58
      Cannon, J. R.; Cammarata, M. B.; Robotham, S. A.; Cotham, V. C.; Shaw, J. B.; Fellers, R. T.; Early, B. P.; Thomas, P. M.; Kelleher, N. L.; Brodbelt, J. S. Ultraviolet photodissociation for characterization of whole proteins on a chromatographic time scale. Anal. Chem. 2014, 86 (4), 2185– 2192,  DOI: 10.1021/ac403859a
      There is no corresponding record for this reference.
    59. 59
      Lanzillotti, M.; Brodbelt, J. S. Comparison of Top-Down Protein Fragmentation Induced by 213 and 193 nm UVPD. J. Am. Soc. Mass Spectrom. 2023, 34 (2), 279– 285,  DOI: 10.1021/jasms.2c00288
      There is no corresponding record for this reference.
    60. 60
      Juetten, K. J.; Brodbelt, J. S. Top-Down Analysis of Supercharged Proteins Using Collision-, Electron-, and Photon-Based Activation Methods. J. Am. Soc. Mass Spectrom. 2023, 34 (7), 1467– 1476,  DOI: 10.1021/jasms.3c00138
      There is no corresponding record for this reference.
    61. 61
      Hyde, A. M.; Calabria, R.; Arvary, R.; Wang, X.; Klapars, A. Investigating the Underappreciated Hydrolytic Instability of 1,8-Diazabicyclo[5.4.0]undec-7-ene and Related Unsaturated Nitrogenous Bases. Org. Process Res. Dev. 2019, 23 (9), 1860– 1871,  DOI: 10.1021/acs.oprd.9b00187
      There is no corresponding record for this reference.
    62. 62
      Juetten, K. J.; Brodbelt, J. S. MS-TAFI: A Tool for the Analysis of Fragment Ions Generated from Intact Proteins. J. Proteome Res. 2023, 22 (2), 546– 550,  DOI: 10.1021/acs.jproteome.2c00594
      There is no corresponding record for this reference.
    63. 63
      Durbin, K. R.; Robey, M. T.; Voong, L. N.; Fellers, R. T.; Lutomski, C. A.; El-Baba, T. J.; Robinson, C. V.; Kelleher, N. L. ProSight Native: Defining Protein Complex Composition from Native Top-Down Mass Spectrometry Data. J. Proteome Res. 2023, 22 (8), 2660– 2668,  DOI: 10.1021/acs.jproteome.3c00171
      There is no corresponding record for this reference.
    64. 64
      Li, J.; Santambrogio, C.; Brocca, S.; Rossetti, G.; Carloni, P.; Grandori, R. Conformational effects in protein electrospray-ionization mass spectrometry. Mass Spectrom Rev. 2016, 35 (1), 111– 122,  DOI: 10.1002/mas.21465
      There is no corresponding record for this reference.
    65. 65
      Reid, G. E.; Wu, J.; Chrisman, P. A.; Wells, J. M.; McLuckey, S. A. Charge-state-dependent sequence analysis of protonated ubiquitin ions via ion trap tandem mass spectrometry. Anal. Chem. 2001, 73 (14), 3274– 3281,  DOI: 10.1021/ac0101095
      There is no corresponding record for this reference.
    66. 66
      Cobb, J. S.; Easterling, M. L.; Agar, J. N. Structural characterization of intact proteins is enhanced by prevalent fragmentation pathways rarely observed for peptides. J. Am. Soc. Mass Spectrom. 2010, 21 (6), 949– 959,  DOI: 10.1016/j.jasms.2010.02.009
      There is no corresponding record for this reference.
    67. 67
      Hellinger, J.; Brodbelt, J. S. Impact of Charge State on Characterization of Large Middle-Down Sized Peptides by Tandem Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2024, 35 (8), 1647– 1656,  DOI: 10.1021/jasms.3c00405
      There is no corresponding record for this reference.
    68. 68
      Carrera, G. V. S. M.; Jordão, N.; Santos, M. M.; da Ponte, M. N.; Branco, L. C. Reversible systems based on CO2, amino-acids and organic superbases. RSC Adv. 2015, 5 (45), 35564– 35571,  DOI: 10.1039/C5RA03474D
      There is no corresponding record for this reference.
    69. 69
      Bonner, J.; Talbert, L. E.; Akkawi, N.; Julian, R. R. Simplified identification of disulfide, trisulfide, and thioether pairs with 213 nm UVPD. Analyst 2018, 143 (21), 5176– 5184,  DOI: 10.1039/C8AN01582A
      There is no corresponding record for this reference.
    70. 70
      Macias, L. A.; Brodbelt, J. S. Investigation of Product Ions Generated by 193 nm Ultraviolet Photodissociation of Peptides and Proteins Containing Disulfide Bonds. J. Am. Soc. Mass Spectrom. 2022, 33 (7), 1315– 1324,  DOI: 10.1021/jasms.2c00124
      There is no corresponding record for this reference.

  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jasms.5c00380.

    • (1) Mass spectra for optimization of ESI-MS in negative mode, (2) sequence maps and isotopic fitting results supporting identification of fragment ions, (3) bar graphs for sequence coverage and feature of fragment ions for varied charge states of proteins using HCD, UVPD (varied laser energy) and a-EPD, (4) bar graphs for preferential cleavage analysis of HCD, UVPD and a-EPD, and (5) mass spectra and bar graphs comparing positive and negative mode UVPD of all proteins (Figure S23) (PDF)


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