Protein S-sulfinylation (R–SO2–) and S-sulfonylation (R–SO3–) are irreversible oxidative post-translational modifications of cysteine residues. Greater than 5% of cysteines are reported to occupy these higher oxidation states, which effectively inactivate the corresponding thiols and alter the electronic and physical properties of modified proteins. Such higher oxidation states are reached after excessive exposure to cellular oxidants, and accumulate across different disease states. Despite widespread and functionally relevant cysteine oxidation across the proteome, there are currently no robust methods to profile higher order cysteine oxidation. Traditional data-dependent liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods generally miss low-occupancy modifications in complex analyses. Here, we present a data-independent acquisition (DIA) LC/MS-based approach, leveraging the high IR absorbance of sulfoxides at 10.6 μm, for selective dissociation and discovery of S-sulfonated peptides. Across peptide standards and protein digests, we demonstrate selective infrared multiphoton dissociation (IRMPD) of S-sulfonated peptides in the background of unmodified peptides. This selective DIA IRMPD LC/MS-based approach allows identification and annotation of S-sulfonated peptides across complex mixtures while providing sufficient sequence information to localize the modification site.

Sulfated N-glycans released from bovine thyroid stimulating hormone (bTSH) were ionized with the divalent metal cations Ca2+, Mg2+, and Co by electrospray ionization (ESI). These metal-adducted species were subjected to infrared multiphoton dissociation (IRMPD) and electron capture dissociation (ECD) and the corresponding fragmentation patterns were compared. IRMPD generated extensive glycosidic and cross-ring cleavages, but most product ions suffered from sulfonate loss. Internal fragments were also observed, which complicated the spectra. ECD provided complementary structural information compared with IRMPD, and all observed product ions retained the sulfonate group, allowing sulfonate localization. To our knowledge, this work represents the first application of ECD towards metal-adducted sulfated N-glycans released from a glycoprotein. Due to the ability of IRMPD and ECD to provide complementary structural information, the combination of the two strategies is a promising and valuable tool for glycan structural characterization. The influence of different metal ions was also examined. Calcium adducts appeared to be the most promising species because of high sensitivity and ability to provide extensive structural information.

In top-down proteomics, intact gaseous proteins are fragmented in a mass spectrometer by, e.g., electron capture dissociation (ECD) to obtain structural information. By far, most top-down approaches involve dissociation of protein cations. However, in electrospray ionization of phosphoproteins, the high acidity of phosphate may contribute to the formation of intramolecular hydrogen bonds or salt bridges, which influence subsequent fragmentation behavior. Other acidic proteins or proteins with regions containing multiple acidic residues may also be affected similarly. Negative ion mode, on the other hand, may enhance deprotonation and unfolding of multiply phosphorylated or highly acidic protein regions. Here, activated ion electron detachment dissociation (AI-EDD) and negative ion infrared multiphoton dissociation (IRMPD) were employed to investigate the fragmentation of intact proteins, including multiply phosphorylated β-casein, calmodulin, and glycosylated ribonuclease B. Compared to AI-ECD and positive ion IRMPD, AI-EDD and negative ion IRMPD provide complementary protein sequence information, particularly in regions with high acidity, including the multiply phosphorylated region of β-casein.

Positive ion mode collision-activated dissociation tandem mass spectrometry (CAD MS/MS) of O-sulfopeptides precludes determination of sulfonated sites due to facile proton-driven loss of the highly labile sulfonate groups. A previously proposed method for localizing peptide and protein O-sulfonation involves derivatization of nonsulfonated tyrosines followed by positive ion CAD MS/MS of the corresponding modified sulfopeptides for diagnostic sulfonate loss. This indirect method relies upon specific and complete derivatization of nonsulfonated tyrosines. Alternative MS/MS activation methods, including positive ion metastable atom-activated dissociation (MAD) and metal-assisted electron transfer dissociation (ETD) or electron capture dissociation (ECD) provide varying degrees of sulfonate retention. Sulfonate retention has also been reported following negative ion MAD and electron detachment dissociation (EDD), which also operates in negative ion mode in which sulfonate groups are less labile than in positive ion mode. However, an MS/MS activation technique that can effectively preserve sulfonate groups while providing extensive backbone fragmentation (translating to sequence information, including sulfonated sites) with little to no noninformative small molecule neutral loss has not previously been realized. Here, we report that negative ion CAD, EDD, and negative ETD (NETD) result in sulfonate retention mainly at higher charge states with varying degrees of fragmentation efficiency and sequence coverage. Similar to previous observations from CAD of sulfonated glycosaminoglycan anions, higher charge states translate to a higher probability of deprotonation at the sulfonate groups thus yielding charge-localized fragmentation without loss of the sulfonate groups. However, consequently, higher sulfonate retention comes at the price of lower sequence coverage in negative ion CAD. Fragmentation efficiency/sequence coverage averaged 19/6% and 33/20% in EDD and NETD, respectively, both of which are only applicable to multiply-charged anions. In contrast, the recently introduced negative ion ECD showed an average fragmentation efficiency of 69% and an average sequence coverage of 82% with complete sulfonate retention from singly- and doubly-deprotonated sulfopeptide anions.

The generation of gaseous polyanions with a Coulomb barrier has attracted attention as exemplified by previous studies of fullerene dianions. However, this phenomenon has not been reported for biological anions. By contrast, electron attachment to multiply charged peptide and protein cations has seen a surge of interest due to the high utility for tandem mass spectrometry (MS/MS). Electron capture dissociation (ECD) and electron transfer dissociation (ETD) involve radical-driven fragmentation of charge-reduced peptide/protein cations to yield N–Cα backbone bond cleavage, resulting in predictable c′/z•-type product ions without loss of labile post-translational modifications (PTMs). However, acidic peptides, e.g., with biologically important PTMs such as phosphorylation and sulfonation, are difficult to multiply charge in positive ion mode and show improved ionization in negative-ion mode. We found that peptide anions ([M – nH]n−, n ≥ 1) can capture electrons within a rather narrow energy range (∼3.5–6.5 eV), resulting in charge-increased radical intermediates that undergo dissociation analogous to that in ECD/ETD. Gas-phase zwitterionic structures appear to play an important role in this novel MS/MS technique, negative-ion electron capture dissociation (niECD).

Collision-activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD) of Ag-adducted phospholipids were investigated as structural tools. Previously, determination of the acyl chains at the two phospholipid esterification sites has been performed based on the R1COO−/R2COO− ratio in negative ion mode CAD tandem mass spectrometry. However, the observed product ion ratio is dependent on the extent of unsaturation of the fatty acyl group at sn-2 as well as on the total chain length. Similarly, in positive ion mode CAD with/without alkaline or alkaline earth metal adduction, the ratio of product ions resulting from either R1COOH or R2COOH neutral losses is dependent on the nature of the phospholipid polar headgroup. Ag+ ion chromatography, in which silver ions are part of the stationary phase, can provide information on double bond number/distribution as well as double bond configuration (cis/trans) because of interaction between Ag+ ions and olefinic π electrons of fatty acids and lipids. We hypothesized that interactions between double bonds and Ag+ may be utilized to also reveal phospholipid esterification site information in tandem mass spectrometry. CAD and IRMPD of Ag-adducted phospholipids with unsaturated fatty acids (RxCOOH, x = 1 or 2) provided characteristic product ions, [RxCOOH + Ag]+, and their neutral losses. The characteristic product ions and their abundances do not depend on the type of polar headgroup or the number of double bonds of unsaturated acyl chains. Tandem mass spectrometry of Cu-adducted phospholipids was also performed for comparison based on the Lewis acid and base properties of Cu+ and phospholipid double bonds, respectively.

κ-Carrageenan sulfated oligosaccharides containing one, two, and four sulfate groups were complexed with divalent metal cations and electrosprayed in positive ion mode. The resulting metal complexes were subjected to sustained off-resonance irradiation collision-activated dissociation (SORI-CAD) and electron capture dissociation (ECD). The presence of divalent metal cations in the electrospray solution facilitates formation of doubly positively charged precursor ions in the gas phase. Unsurprisingly, abundant sulfate group (SO3) loss is observed in SORI CAD of sulfated oligosaccharides complexed with divalent metal cations. By contrast, sulfation is retained in ECD of metal-sulfated oligosaccharide complexes, providing information on the site of sulfation. Most product ions resulting from ECD are C/Z- or B/Y-type ions, corresponding to cleavage of glycosidic bonds linking two sugar units. However, several A/X-type product ions corresponding to sugar cross-ring cleavage were also observed in ECD. Such cross-ring fragments are important in carbohydrate analysis because they provide linkage information.Graphical abstractResearch highlights▶ Sulfated oligosaccharide dications from divalent metal adduction. ▶ ECD yields extensive fragmentation with sulfate retention. ▶ CAD mainly results in sulfate loss, precluding sulfate localization.

Dissociation of singly charged species is more challenging compared with that of multiply charged precursor ions because singly charged ions are generally more stable. In collision activated dissociation (CAD), singly charged ions also gain less kinetic energy in a fixed electric field compared with multiply charged species. Furthermore, ion–electron and ion–ion reactions that frequently provide complementary and more extensive fragmentation compared with CAD typically require multiply charged precursor ions. Here, we investigate electron induced dissociation (EID) of singly deprotonated peptides and compare the EID fragmentation patterns with those observed in negative ion mode CAD. Fragmentation induced upon electron irradiation and collisional activation is not specific and results in the formation of a wide range of product ions, including b-, y-, a-, x-, c-, and z-type ions. Characteristic amino acid side chain losses are detected in both techniques. However, differences are also observed between EID and CAD spectra of the same species, including formation of odd-electron species not seen in CAD, in EID. Furthermore, EID frequently results in more extensive fragmentation compared with CAD. For modified peptides, EID resulted in retention of sulfonation and phosphorylation, allowing localization of the modification site. The observed differences are likely due to both vibrational and electronic excitation in EID, whereas only the former process occurs in CAD.

Double bond locations in fatty acids can be determined from characteristic charge-remote fragmentation patterns of alkali metal-adducted fatty acids following high energy collision activated dissociation (CAD). With low energy CAD, several chemical derivatization methods, including ozonization, epoxidation, and hydroxylation, have been used to generate characteristic fragments. However, high energy CAD is not universally available and involves a high degree of scattering, causing product ion loss. Further, derivatization reactions involve side reactions and sample loss. Here, we analyzed metal-adducted fatty acids to investigate the utility of electron induced dissociation (EID) for determining double bond location. EID has been proposed to involve both electronic excitation, similar to high energy CAD, and vibrational exciation. Various metals (Li, Zn, Co, Ni, Mg, Ca, Fe, and Mn) were investigated to fix one charge at the carboxylate end of fatty acids to promote charge-remote fragmentation. EID of Mn(II)-adducted fatty acids allowed determination of all double bond locations of arachidonic acid, linolenic acid, oleic acid, and stearic acid. For Mn(II)-adducted fatty acids, reduced characteristic charge-remote product ion abundances at the double bond positions are indicative of double bond locations. However, other metal adducts did not generally provide characteristic product ion abundances at all double bond locations.

Electron capture dissociation (ECD), which provides more extensive sequence coverage compared to “slow heating” tandem mass spectrometric techniques such as collision-activated dissociation, constitutes a promising method for de novo sequencing of peptides and proteins. We have previously examined and characterized the ECD fragmentation behavior of small to medium size doubly and triply protonated peptides from trypsin, chymotrypsin and Glu C digestion. Here, we extend that work to longer, more highly charged proteolytic peptides from Lys N, Lys C and Glu C digestion to compare the extent of fragmentation obtained from each type of proteolytically derived peptide and also to examine the effectiveness of each enzyme in terms of peptide sequence coverage in ECD. Our findings demonstrate that medium size peptides (i.e., 1600–4800 Da) at high charge states (+3 to +6) exhibit very similar ECD fragmentation behavior independent of which proteolytic enzyme was used for digestion. The average peptide sequence coverage we obtained for Lys N and Glu C generated peptides was 81%, and for Lys C proteolytic peptides we obtained an average sequence coverage of 80%. Most of the peptides we examined, 82%, showed high ECD sequence coverage ranging from 70–100%, whereas 13% of the peptides showed a moderate sequence coverage ranging from 50–60%. Only 5% of the peptides showed an average sequence coverage below 50%. Furthermore, the extent of fragmentation, measured by the total number of backbone c- and z-type product ions, was very similar for Lys N, Lys C and Glu C derived peptides.

Gas-phase ion-electron reactions, including electron capture dissociation (ECD) and electron detachment dissociation (EDD), are advantageous for characterization of protein posttranslational modifications (PTMs), because labile modifications are not lost during the fragmentation process. However, at least two positive charges and relatively abundant precursor ions are required for ECD due to charge reduction and lower fragmentation efficiency compared to conventional gas-phase fragmentation techniques. Both these criteria are difficult to fulfill for phosphopeptides due to their acidic character. The negative ion mode operation of EDD is more compatible with phosphopeptide ionization, but EDD suffers from a fragmentation efficiency even lower than that of ECD. Recently, metal oxides such as ZrO 2 and TiO 2 have been shown to provide selective enrichment of phosphopeptides from proteolytic digests. Here, we utilize this enrichment strategy to improve ECD and EDD of phosphopeptides. This approach allowed determination of the locations of phosphorylation sites in highly acidic, multiply phosphorylated peptides from complex peptide mixtures by ECD. For singly phosphorylated peptides, EDD provided complementary sequence information compared to ECD.

In bottom-up proteomics, proteolytically derived peptides from proteins of interest are analyzed to provide sequence information for protein identification and characterization. Electron capture dissociation (ECD), which provides more random cleavages compared to “slow heating” techniques such as collisional activation, can result in greater sequence coverage for peptides and proteins. Most bottom-up proteomics approaches rely on tryptic doubly protonated peptides for generating sequence information. However, the effectiveness, in terms of peptide sequence coverage, of tryptic doubly protonated peptides in ECD remains to be characterized. Herein, we examine the ECD fragmentation behavior of 64 doubly- and 64 triply protonated peptides (i.e., a total of 128 peptide ions) from trypsin, Glu-C, and chymotrypsin digestion in a Fourier transform ion cyclotron resonance mass spectrometer. Our findings indicate that when triply protonated peptides are fragmented in ECD, independent of which proteolytic enzyme was used for protein digestion, more c- and z-type product ions are observed, and the number of complementary fragment pairs increases dramatically (44%). In addition, triply protonated peptides provide an increase (26%) in peptide sequence coverage. ECD of tryptic peptides, in both charge states, resulted in higher sequence coverage compared to chymotryptic and Glu-C digest peptides. The peptide sequence coverage we obtained in ECD of tryptic doubly protonated peptides (64%) is very similar to that reported for electron transfer dissociation of the same peptide type (63%).

Several phosphate-containing metabolites, including nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), adenosine 5′-diphosphate ribose (ADP-r), adenosine 5′-triphosphate (ATP), and guanosine 5′-triphosphate (GTP), have been characterized with electron capture dissociation (ECD) and sustained off-resonance irradiation collision-activated dissociation (SORI-CAD) tandem mass spectrometry (MS/MS) in positive-ion mode. Calcium complexation was used to successfully produce abundant doubly charged cationic precursor ions with or without hydration. This approach enabled application of ECD to acidic metabolites for the first time. Fragmentation pathways observed in ECD and SORI-CAD of calcium-adducted phosphate-containing metabolites were complementary. Unique fragmentation was observed in ECD compared to SORI-CAD MS/MS, including ribose cross-ring cleavage for NAD and NADP, and generation of hydrated product ions, including cross-ring fragments, for hydrated ATP and GTP. A combination of ECD and CAD appears promising for maximizing structural information about metabolites.

We have investigated electron capture dissociation (ECD) of doubly protonated peptides with few or no basic amino acid residues (BAARs). For peptides containing one His, abundant b-type ions were only found when His was located adjacent to the N-terminus. Interestingly, b-type ions, particularly b5+, were found to be the dominant product ions in ECD of peptides without BAARs. Fragmentation patterns of luteinizing hormone releasing hormone (LHRH) and vasopressin (VP), containing one Arg and one His, respectively, were compared to those of Q8-LHRH and oxytocin (OT) in which the BAAR is replaced with a non-BAAR. More b-type ions were found for Q8-LHRH and OT than for LHRH and VP. We also performed ECD of melittin and found no b-type ions from ECD of the 4+ charge state; however, many low abundance b-type ions were produced in ECD of the 5+ charge state. Possible mechanisms for the formation of b-type ions are discussed and we propose that such ions are formed as a consequence of protons being located at backbone amide nitrogens.

Siderophores are high-affinity iron-chelating ligands produced by microorganisms to scavenge vital Fe3+ from the environment. Thus, siderophores constitute potential therapeutic targets and their structural determination is important for exploiting their therapeutic value. Here, the virulence-associated siderophore petrobactin from Bacillus anthracis was characterized with electron capture dissociation (ECD). Fragmentation of doubly protonated petrobactin was investigated and compared to sustained off-resonance irradiation collision-activated dissociation (SORI CAD) and infrared multiphoton dissociation (IRMPD) of both the singly and doubly protonated species. These experiments demonstrate that ECD provides additional information (complementary bond cleavages) on the structure of petrobactin compared to both SORI CAD and IRMPD. Furthermore, complexes of petrobactin with divalent (Ca2+, Fe2+, and Co2+) and trivalent (Fe3+ and Ga3+) metal cations were also subjected to SORI CAD and ECD. Again, most structural information was obtained from the ECD spectra. However, significant differences were found in both SORI CAD and ECD of metal complexes, dependent on the nature of the metal ion. Intriguingly, unique behavior, consistent with a recently proposed solution-phase structure, was observed for the highly preferred Fe3+—petrobactin complex.

Electron detachment dissociation (EDD) has recently been shown by Amster and coworkers to constitute a valuable analytical approach for structural characterization of glycosaminoglycans. Here, we extend the application of EDD to neutral and sialylated oligosaccharides. Both branched and linear structures are examined, to determine whether branching has an effect on EDD fragmentation behavior. EDD spectra are compared to collisional activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD) spectra of the doubly and singly deprotonated species. Our results demonstrate that EDD of both neutral and sialylated oligosaccharides provides structural information that is complementary to that obtained from both CAD and IRMPD. In all cases, EDD resulted in additional cross-ring cleavages. In most cases, cross-ring fragmentation obtained by EDD is more extensive than that obtained from IRMPD or CAD. Our results also indicate that branching does not affect EDD fragmentation, contrary to what has been observed for electron capture dissociation (ECD).

Electron capture dissociation (ECD) has been proposed to be a non-ergodic process, i.e. to provide backbone dissociation of gas-phase peptides faster than randomization of the imparted energy. One potential consequence could be that ECD can fragment deuterated peptides without causing hydrogen scrambling and thereby provide amino acid residue-specific amide hydrogen exchange rates. Such a feature would improve the resolution of approaches involving solution-phase amide hydrogen exchange combined with mass spectrometry for protein structural characterization. Here, we explore this hypothesis using melittin, a haemolytic polypeptide from bee venom, as our model system. Exchange rates in methanol calculated from consecutive c-type ion pairs show some correlation with previous NMR data: the amide hydrogens of leucine 13 and alanine 15, located at the unstructured kink surrounding proline 14 in the melittin structure adopted in methanol, appear as fast exchangers and the amide hydrogens of leucine 16 and lysine 23, buried within the helical regions of melittin, appear as slow exchangers. However, calculations based on c-type ions for other amide hydrogens do not correlate well with NMR data, and evidence for deuterium scrambling in ECD was obtained from z˙-type ions.

We have recently demonstrated that both electron capture dissociation (ECD) and electron detachment dissociation (EDD) can provide complementary sequence-specific cleavage of DNA compared with collision activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD). However, EDD is preferred because of more extensive fragmentation at higher sensitivity (due to its negative ion mode operation). Here, we extend the radical ion chemistry of these two gas-phase ion-electron reaction techniques to the characterization of RNA. Compared with DNA, rather limited information is currently available on the gas-phase fragmentation of RNA. We found that the ECD fragmentation patterns of the oligoribonucleotides A6, C6, and CGGGGC are nucleobase dependent, suggesting that cleavage proceeds following electron capture at the nucleobases. Only limited backbone cleavage was observed in ECD. EDD, on the other hand, provided complete sequence coverage for the RNAs A6, C6, G6, U6, CGGGGC, and GCAUAC. The EDD fragmentation patterns were different from those observed with CAD and IRMPD in that the dominant product ions correspond to d- and w-type ions rather than c- and y-type ions. The minimum differences between oligoribonucleotides suggest that EDD proceeds following direct electron detachment from the phosphate backbone.

We have investigated electron capture dissociation (ECD) of doubly protonated peptides with few or no basic amino acid residues (BAARs). For peptides containing one His, abundant b-type ions were only found when His was located adjacent to the N-terminus. Interestingly, b-type ions, particularly b5+, were found to be the dominant product ions in ECD of peptides without BAARs. Fragmentation patterns of luteinizing hormone releasing hormone (LHRH) and vasopressin (VP), containing one Arg and one His, respectively, were compared to those of Q8-LHRH and oxytocin (OT) in which the BAAR is replaced with a non-BAAR. More b-type ions were found for Q8-LHRH and OT than for LHRH and VP. We also performed ECD of melittin and found no b-type ions from ECD of the 4+ charge state; however, many low abundance b-type ions were produced in ECD of the 5+ charge state. Possible mechanisms for the formation of b-type ions are discussed and we propose that such ions are formed as a consequence of protons being located at backbone amide nitrogens.

Siderophores are high-affinity iron-chelating ligands produced by microorganisms to scavenge vital Fe3+ from the environment. Thus, siderophores constitute potential therapeutic targets and their structural determination is important for exploiting their therapeutic value. Here, the virulence-associated siderophore petrobactin from Bacillus anthracis was characterized with electron capture dissociation (ECD). Fragmentation of doubly protonated petrobactin was investigated and compared to sustained off-resonance irradiation collision-activated dissociation (SORI CAD) and infrared multiphoton dissociation (IRMPD) of both the singly and doubly protonated species. These experiments demonstrate that ECD provides additional information (complementary bond cleavages) on the structure of petrobactin compared to both SORI CAD and IRMPD. Furthermore, complexes of petrobactin with divalent (Ca2+, Fe2+, and Co2+) and trivalent (Fe3+ and Ga3+) metal cations were also subjected to SORI CAD and ECD. Again, most structural information was obtained from the ECD spectra. However, significant differences were found in both SORI CAD and ECD of metal complexes, dependent on the nature of the metal ion. Intriguingly, unique behavior, consistent with a recently proposed solution-phase structure, was observed for the highly preferred Fe3+–petrobactin complex.