Infrared multiphoton dissociation for enhanced de novo sequence interpretation of N-terminal sulfonated peptides in a quadrupole ion trap

Infrared multiphoton dissociation (IRMPD) of N-terminal sulfonated peptides improves de novo sequencing capabilities in a quadrupole ion trap mass spectrometer. Not only does IRMPD promote highly efficient dissociation of the N-terminal sulfonated peptides but also the entire series of y ions down to the y(1) fragment may be detected due to alleviation of the low-mass cutoff problem associated with conventional collisional activated dissociation (CAD) methods in a quadrupole ion trap. Commercial de novo sequencing software was applied for the interpretation of CAD and IRMPD MS/MS spectra collected for seven unmodified peptides and the corresponding N-terminal sulfonated species. In most cases, the additional information obtained by N-terminal sulfonation in combination with IRMPD provided significant improvements in sequence identification. The software sequence tag results were combined with a commercial database searching algorithm to interpret sequence information of a tryptic digest on alpha-casein s1. Energy-variable CAD studies confirmed a 30-40% reduction in the critical energies of the N-terminal sulfonated peptides relative to unmodified peptides. This reduction in dissociation energy facilitates IRMPD in a quadrupole ion trap.     

Probing ligand binding to duplex DNA using KMnO4 reactions and electrospray ionization tandem mass spectrometry

An electrospray ionization tandem mass spectrometry (ESI-MS/MS) strategy employing the thymine-selective KMnO4 oxidation reaction to detect conformational changes and ligand binding sites in noncovalent DNA/drug complexes is reported. ESI-MS/MS is used to detect specific mass shifts of the DNA ions that are associated with the oxidation of thymines. This KMnO4 oxidation/ESI-MS/MS approach is an alternative to conventional gel-based oxidation methods and affords excellent sensitivity while eliminating the reliance on radiolabeled DNA. Comparison of single-strand versus duplex DNA indicates that the duplexes exhibit a significant resistance to the reaction, thus confirming that the oxidation process is favored for unwound or single-strand regions of DNA. DNA complexes containing different drugs including echinomycin, actinomycin-D, ethidium bromide, Hoechst 33342, and cis-C1 were subjected to the oxidation reaction. Echinomycin, a ligand with a bisintercalative binding mode, was found to induce the greatest KMnO4 reactivity, while Hoechst 33342, a minor groove binder, caused no increase in the oxidation of DNA. The oxidation of echinomycin/DNA complexes containing duplexes with different sequences and lengths was also assessed. Duplexes with thymines closer to the terminal ends of the duplex demonstrated a greater increase in the degree of oxidation than those with thymines in the middle of the sequence. Collisional activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD) experiments were used to determine the site of oxidation based on oligonucleotide fragmentation patterns.     

Infrared multiphoton dissociation and electron-induced dissociation as alternative MS/MS strategies for metabolite identification

A major challenge encountered in mass spectrometric metabolite analysis is the identification and structural characterization of metabolites. Fourier transform ion cyclotron resonance mass spectrometry is a valuable technique for metabolite structural determination because it provides accurate masses and allows for multiple MS/MS fragmentation strategies, including infrared multiphoton dissociation (IRMPD) and electron-induced dissociation (EID). Collision activated dissociation (CAD) is currently the most commonly used MS/MS technique for metabolite structural characterization. In contrast, IRMPD and EID have had very limited, if any, application for metabolite characterization. Here, we explore IRMPD and EID of phosphate-containing metabolites and compare the resulting fragmentation patterns to those of CAD. Our results show that CAD, IRMPD, and EID provide complementary structural information for phosphate-containing metabolites. Overall, CAD provided the most extensive fragmentation for smaller (<600 Da) phosphate-containing metabolites; however, IRMPD generated more extensive fragmentation for larger (>600 Da) phosphate-containing metabolites, particularly for species containing increased numbers of phosphate groups. EID generally provided complementary fragmentation to CAD and showed extensive fragmentation with relatively evenly abundant product ions, regardless of metabolite size. However, EID fragmentation efficiency is lower than those of CAD and IRMPD.     

Infrared multiphoton dissociation for quantitative shotgun proteomics

We modified a dual-cell linear ion trap mass spectrometer to perform infrared multiphoton dissociation (IRMPD) in the low-pressure trap of a dual-cell quadrupole linear ion trap (dual-cell QLT) and perform large-scale IRMPD analyses of complex peptide mixtures. Upon optimization of activation parameters (precursor q-value, irradiation time, and photon flux), IRMPD subtly, but significantly, outperforms resonant-excitation collisional-activated dissociation (CAD) for peptides identified at a 1% false-discovery rate (FDR) from a yeast tryptic digest (95% confidence, p = 0.019). We further demonstrate that IRMPD is compatible with the analysis of isobaric-tagged peptides. Using fixed QLT rf amplitude allows for the consistent retention of reporter ions, but necessitates the use of variable IRMPD irradiation times, dependent upon precursor mass to charge (m/z). We show that IRMPD activation parameters can be tuned to allow for effective peptide identification and quantitation simultaneously. We thus conclude that IRMPD performed in a dual-cell ion trap is an effective option for the large-scale analysis of both unmodified and isobaric-tagged peptides.     

Consecutive ion activation for top down mass spectrometry: improved protein sequencing by nozzle-skimmer dissociation

Mass spectra produced by nozzle-skimmer dissociation (NSD) have been little used in the past for structural characterization. NSD cannot be used on mass-separated ions (MS/MS), and for electrosprayed protein ions, previous NSD spectra showed backbone cleavages similar to those from energetic methods such as collisionally activated dissociation (CAD) or infrared multiphoton dissociation (IRMPD). However, our experimental configuration with Fourier transform (FT) MS makes possible three consecutive steps of NSD ion activation: thermal in the entrance capillary and collisional in both the nozzle-skimmer (N-S) region and the region after the skimmer before the quadrupole entrance lens (S-Q). In the high-pressure N-S region of adjustable path length, ions undergo high-frequency, low-energy collisions to rupture weak noncovalent or covalent bonds, with these "denatured" products then subjected to high-energy collisions in the low-pressure S-Q region to cleave strong backbone bonds. These NSD spectra, plus those from variable capillary thermal activation, of 8+ to 11+ ubiquitin ions electrosprayed from denatured solution show backbone cleavages between 74 of 75 amino acid pairs, vs 66 for CAD and 50 for IRMPD in the FTMS cell. Thermal activation by the inlet capillary of the newly desolvated 6+, 7+ ubiquitin ions from electrospraying the native conformer increases the NSD yield from 8% at 56 degrees C to 96% at 76 degrees C, but with little change in product branching ratios; this capillary heating has no effect on CAD or IRMPD of these ions collected in the FTMS cell. Ion desolvation with its concomitant H-bond strengthening appears to produce a transiently stable conformer whose formation can be prevented by capillary heating. The far more complex and stable noncovalent tertiary structures of large protein ions in the gas phase have made MS/MS difficult; initial inhibition of tertiary structure formation with immediate NSD ("prefolding dissociation") appears promising for the top down characterization of a 200-kDa protein.     

Infrared multiphoton dissociation of O-linked mucin-type oligosaccharides

Oligosaccharides are known to play important roles in many biological processes. In the study of oligosaccharides, collision-induced dissociation (CID) is the most common dissociation method to elucidate the sequence and connectivity. However, a disadvantage of CID is the decrease in both the degree and efficiency of dissociation with increasing mass. In the present study, we have successfully performed infrared multiphoton dissociation (IRMPD) on 39 O-linked mucin-type oligosaccharide alditols (both neutral and anionic). CID and IRMPD spectra of several oligosaccharides were also compared. They yielded nearly identical fragment ions corresponding to the lowest energy fragmentation pathways. The characteristic fragmentations of structural motifs, which can provide the linkage information, were similarly presented in both CID and IRMPD spectra. Multistage of CID (MS(3) or MS(4)) is commonly needed to completely sequence the oligosaccharides, while IRMPD of the same compounds yielded the fragment ions corresponding to the loss of the first residue to the last residue during a single-stage tandem MS (MS(2)). Finally, it is shown that the fragmentation efficiency of IRMPD increases with the increasing size of oligosaccharides.     

Fragmentation of singly protonated peptides via a combination of infrared and collisional activation

The coupling of matrix-assisted laser desorption/ionization (MALDI) to Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) provides an exceptionally capable platform for peptide analysis, but an important limitation of this approach is the difficulty in obtaining informative tandem mass spectra (MS/MS) of singly protonated peptides. This difficulty is especially pronounced with peptide ions containing basic amino acid residues (for example, tryptic peptides). While such ions can be fragmented in some instrument configurations, most FTICR instruments have comparatively little facility for high-energy fragmentation. Here, a novel MS/MS approach implemented with MALDI-FTICR-MS and specifically intended for enhanced fragmentation of singly protonated peptides is described. The method involves infrared irradiation in concert with the simultaneous application of sustained off-resonance irradiation collision-induced dissociation (SORI-CID). This form of MS/MS, described as a combination of infrared and collisional activation (CIRCA), is shown to provide a greater capacity for dissociation of singly charged model peptide ions as compared to infrared multiphoton dissociation (IRMPD) or SORI-CID alone. Overall, the CIRCA approach is demonstrated to be a feasible technique for accessing useful fragmentation pathways of singly charged peptides, including those harboring basic amino acid residues--a crucial feature in the context of proteomics.     

High-throughput peptide identification from protein digests using data-dependent multiplexed tandem FTICR mass spectrometry coupled with capillary liquid chromatography

Tandem mass spectrometry (MS/MS) plays an important role in the unambiguous identification and structural elucidation of biomolecules. In contrast to conventional MS/MS approaches for protein identification where an individual polypeptide is sequentially selected and dissociated, a multiplexed-MS/MS approach increases throughput by selecting several peptides for simultaneous dissociation using either infrared multiphoton dissociation (IRMPD) or multiple frequency sustained off-resonance irradiation (SORI) collisionally induced dissociation (CID). The high mass measurement accuracy and resolution of FTICR combined with knowledge of peptide dissociation pathways allows the fragments arising from several different parent ions to be assigned. Herein we report the application of multiplexed-MS/MS coupled with on-line separations for the identification of peptides present in complex mixtures (i.e., whole cell lysate digests). Software was developed to enable "on-the-fly" data-dependent peak selection of a subset of polypeptides from each FTICR MS acquisition. In the subsequent MS/MS acquisitions, several coeluting peptides were fragmented simultaneously using either IRMPD or SORI-CID techniques. The utility of this approach has been demonstrated using a bovine serum albumin tryptic digest separated by capillary LC where multiple peptides were readily identified in single MS/MS acquisitions. We also present initial results from multiplexed-MS/MS analysis of a D. radiodurans whole cell digest to illustrate the utility of this approach for high-throughput analysis of a bacterial proteome.     

Combined electron capture and infrared multiphoton dissociation for multistage MS/MS in a Fourier transform ion cyclotron resonance mass spectrometer

We have mounted a permanent on-axis dispenser cathode electron source inside the magnet bore of a 9.4-T Fourier transform ion cyclotron resonance mass spectrometer. This configuration allows electron capture dissociation (ECD) to be performed reliably on a millisecond time scale. We have also implemented an off-axis laser geometry that enables simultaneous access to ECD and infrared multiphoton dissociation (IRMPD). Optimum performance of both fragmentation techniques is maintained. The analytical utility of performing either ECD or IRMPD on a given precursor ion population is demonstrated by structural characterization of several posttranslationally modified peptides: IRMPD of phosphorylated peptides results in few backbone (b- and y-type) cleavages, and product ion spectra are dominated by neutral loss of H3PO4. In contrast, ECD provides significantly more backbone (c- and z*-type) cleavages without loss of H3PO4. For N-glycosylated tryptic peptides, IRMPD causes extensive cleavage of the glycosidic bonds, providing structural information about the glycans. ECD cleaves all backbone bonds (except the N-terminal side of proline) in a 3-kDa glycopeptide with no saccharide loss. However, only a charge-reduced radical species and some side chain losses are observed following ECD of a 5-kDa glycopeptide from the same protein. An MS3 experiment involving IR laser irradiation of the charge-reduced species formed by electron capture results in extensive dissociation into c- and z-type fragment ions. Mass-selective external ion accumulation is essential for the structural characterization of these low-abundance (modified) peptides.     

Amplification of infrared multiphoton dissociation efficiency in a quadruple ion trap using IR-active ligands

A strategy for increasing the efficiency of infrared multiphoton dissociation (IRMPD) in a quadrupole ion trap (QIT) is described. IR-active ligands (IRALs) are incorporated into noncovalent complexes of the type [M2+(analyte) IRAL]+, where M is a transition metal such as copper or cobalt and IRAL is an auxiliary ligand with an IR-active phosphonate functional group. The complexes are formed via self-assembly in solution directly prior to ESI-MS analysis. We demonstrate this new IRMPD approach for the structural characterization of flavonoids. The fragment ions obtained by IRMPD are similar to those obtained by CAD and allow facile isomer differentiation of flavonoids. Fourier transform infrared absorption attenuated total reflectance (FTIR-ATR) and energy-variable CAD experiments indicate that the high IRMPD efficiencies stem from the very large IR absorptivities of the IR-active ligands.     

Hybrid activation methods for elucidating nucleic acid modifications

Hybrid tandem mass spectrometry (MS/MS) techniques combining electron transfer (ET) and collision activated dissociation (CAD), infrared multiphoton dissociation (IRMPD), or ultraviolet photodissociation (UVPD) were implemented and evaluated for the characterization of a series of oligonucleotides and oligoribonucleotides, including both native single strands and single strands containing platinated, phosphorothioated, and 2'-O-methylated modification sites. ET-IRMPD and ET-UVPD of oligodeoxynucleotides and oligoribonucleotides resulted in rich fragmentation with respect to production of w, a, z, and d ions for DNA, and c, y, w, a-B, d, and z ions for RNA, with many product ions retaining the modification and thus allowing site specific identification. ET-IRMPD caused more extensive secondary dissociation of the ions, in addition to a broader distribution of detectable sequence ions attributed to using a lower mass cutoff. ET-UVPD promoted higher energy fragmentation pathways and created the most diverse MS/MS spectra. The numerous products generated by the hybrid MS/MS techniques resulted in specific and extensive backbone cleavages which allowed the modification sites of multiply modified oligonucleotides to be elucidated.     

Determination of phospholipid regiochemistry by Ag(I) adduction and tandem mass spectrometry

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 R(1)COO(-)/R(2)COO(-) 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 R(1)COOH or R(2)COOH 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 (R(x)COOH, x = 1 or 2) provided characteristic product ions, [R(x)COOH + 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.     

Infrared multiple photon dissociation spectroscopy and computational studies of O-glycosylated peptides

The infrared multiple photon dissociation (IRMPD) spectra of O-glycosylated peptides in the gas phase were studied in the IR scanning range of 5.7-9.5 μm. Fragmentation of protonated and sodiated O-glycopeptides was investigated using electrospray ionization (ESI) Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry (MS) with a free electron laser (FEL). FEL is used in the IRMPD technique as a tunable IR light source. In the IRMPD spectroscopic analysis of the protonated O-glycopeptide, fragment ions of the b/y and B/Y types were observed in the range of 5.7-9.5 μm, corresponding to the cleavage of the backbone in the parent amino acid sequence and glycosyl bonds, whereas the spectra of the sodiated glycopeptide showed major peaks of photoproducts of the B/Y type in the range of 8.4-9.5 μm. The IRMPD spectra of the O-glycopeptides were compared with simulated IR spectra for the structures obtained from the molecular dynamics.     

Comparative study of photodissociation and surface-induced dissociation by laser desorption Fourier transform mass spectrometry.

Photodissociation (PD) and surface-induced dissociation (SID) are compared for structural analysis of several nonvolatile compounds analyzed by laser desorption Fourier transform mass spectrometry (LD/FTMS). SID and PD of a porphyrin and two metalloporphyrins were investigated using a variety of experimental conditions. Optimum structural information is obtained from PD when parent ions are irradiated for relatively long times (10-30 s) using 575-nm radiation and short times (0.5-1 s) using 308- or 388-nm radiation. Shorter irradiation times in the visible region resulted in less efficient production of structurally significant product ions, while longer times in the ultraviolet region produced more nonspecific fragment ions, apparently at the expense of more structurally significant fragment ions. SID conversion efficiencies for the porphyrins are estimated for collision energies from 25 to 360 eV, with maximum conversion efficiency found using 62- and 115-eV collision energies for the two porphyrins studied. Results from a concurrent study on the combined use of PD and SID for MS/MS/MS are discussed in the context of these results. The MS3 ion spectra generated by the two dissociation techniques differ more significantly than MS2 product ion spectra. These data suggest some general guidelines for MSn studies of nonvolatile compounds analyzed by LD/FTMS, employing PD and SID for ion activation.     

Infrared multiphoton dissociation in an external ion reservoir.

In this work we present a novel scheme for performing infrared multiphoton dissociation (IRMPD) external to the mass analyzer in an external ion reservoir consisting of an rf-only multipole and a pair of electrostatic lens elements. Ions generated by electrospray ionization (ESI) are accumulated in an rf-only hexapole and dissociated by irradiation at 10.6 microns from a CW CO2 laser in the source region of the mass spectrometer. This scheme is unique from other IRMPD schemes as dissociation occurs in a spatially distinct region of the spectrometer and is independent of the mass spectrometry platform used to analyze the fragment ions. The effectiveness of the technique is demonstrated with ESI IRMPD FTICR mass spectrometry of a 20-mer phosphorothioate oligonucleotide. A comparison of the external IRMPD scheme with nozzle-skimmer dissociation and conventional in-cell IRMPD reveals a significant improvement in signal-to-noise ratio and fragment yield, particularly for larger, more highly charged fragment ions.     

Enabling MALDI-FTICR-MS/MS for high-performance proteomics through combination of infrared and collisional activation

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is a central tool for proteomic analysis, yet the singly protonated tryptic peptide ions produced by MALDI are significantly more difficult to dissociate for tandem mass spectrometry (MS/MS) than the corresponding multiply protonated ions. In order to overcome this limitation, current proteomic approaches using MALDI-MS/MS involve high-energy collision-induced dissociation (CID). Unfortunately, the use of high-energy CID complicates product ion spectra with a significant proportion of irrelevant fragments while also reducing mass accuracy and mass resolution. In order to address the lack of a high-resolution, high mass accuracy MALDI-MS/MS platform for proteomics, Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) and a recently developed MS/MS technique termed CIRCA (for combination of infrared and collisional activation) have been applied to proteomic analysis. Here, CIRCA is shown to be suitable for dissociating singly protonated tryptic peptides, providing greater sequence coverage than either CID or infrared multiphoton dissociation (IRMPD) alone. Furthermore, the CIRCA fragmentation spectra are of sufficient quality to allow protein identification based on the MS/MS spectra alone or in concert with the peptide mass fingerprint (PMF). This is accomplished without compromising mass accuracy or mass resolution. As a result, CIRCA serves to enable MALDI-FTICR-MS/MS for high-performance proteomics experiments.     

Collision-activated infrared multiphoton dissociation in a quadrupole ion trap mass spectrometer

We propose and demonstrate a new method for multiple-stage mass spectrometry (MSn), collision-activated infrared multiphoton dissociation (CA-IRMPD), which is very effective for the quadrupole ion trap mass spectrometer (QITMS). CA-IRMPD uses a combination of focused laser irradiation (beam radius, approximately 0.4 mm) and collisional activation by a supplemental AC voltage between endcap electrodes. This combination enables IRMPD, which has conventionaLly been ineffective above 10(-4) Torr, to be used under a standard bath gas pressure of 2-8 mTorr. CA-IRMPD can produce richer spectra of product ions than CID or IRMPD while maintaining high sensitivity and mass resolution; thus, it will contribute to an accurate determination of peptide sequences.     

Electron detachment dissociation and negative ion infrared multiphoton dissociation of electrosprayed intact proteins

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.     

Thermally assisted infrared multiphoton photodissociation in a quadrupole ion trap

Thermally assisted infrared multiphoton photodissociation (TA-IRMPD) provides an effective means to dissociate ions in the quadrupole ion trap mass spectrometer (QITMS) without detrimentally affecting the performance of the instrument. IRMPD can offer advantages over collision-induced dissociation (CID). However, collisions with the QITMS bath gas at the standard pressure and ambient temperature cause IR-irradiated ions to lose energy faster than photons can be absorbed to induce dissociation. The low pressure required for IRMPD (< or = 10(-5) Torr) is not that required for optimal performance of the QITMS (10(-3) Torr), and sensitivity and resolution suffer. TA-IRMPD is performed with the bath gas at an elevated temperature. The higher temperature of the bath gas results in less energy lost in collisions of the IR-excited ions with the bath gas. Thermal assistance allows IRMPD to be used at or near optimal pressures, which results in an approximately 1 order of magnitude increase in signal intensity. Unlike CID, IRMPD allows small product ions, those less than about one-third the m/z of the parent ion, to be observed. IRMPD should also be more easily paired with fluctuating ion sources, as the corresponding fluctuations in resonant frequencies do not affect IRMPD. Finally, while IR irradiation nonselectively causes dissociation of all ions, TA-IRMPD can be made selective by using axial expansion to move ions away from the path of the laser beam.     

Characterization of o-sulfopeptides by negative ion mode tandem mass spectrometry: superior performance of negative ion electron capture dissociation

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.