The ability to understand the function of a protein often relies on knowledge about its detailed structure. Sometimes, seemingly insignificant changes in the primary structure of a protein, like an amino acid substitution, can completely disrupt a protein's function. Long-lived proteins (LLPs), which can be found in critical areas of the human body, like the brain and eye, are especially susceptible to primary sequence alterations in the form of isomerization and epimerization. Because long-lived proteins do not have the corrective regeneration capabilities of most other proteins, points of isomerism and epimerization that accumulate within the proteins can severely hamper their functions and can lead to serious diseases like Alzheimer's disease, cancer and cataracts. Whereas tandem mass spectrometry (MS/MS) in the form of collision-induced dissociation (CID) generally excels at peptide characterization, MS/MS often struggles to pinpoint modifications within LLPs, especially when the differences are only isomeric or epimeric in nature. One of the most prevalent and difficult-to-identify modifications is that of aspartic acid between its four isomeric forms: l -Asp, l -isoAsp, d -Asp, and d -isoAsp. In this study, peptides containing isomers of Asp were analyzed by charge transfer dissociation (CTD) mass spectrometry to identify spectral features that could discriminate between the different isomers. For the four isomers of Asp in three model peptides, CTD produced diagnostic ions of the form c n +57 on the N-terminal side of iso-Asp residues, but not on the N-terminal side of Asp residues. Using CTD, the l - and d forms of Asp and isoAsp could also be differentiated based on the relative abundance of y - and z ions on the C-terminal side of Asp residues. Differentiation was accomplished through a chiral discrimination factor, R , which compares an ion ratio in a spectrum of one epimer or isomer to the same ion ratio in the spectrum of a different epimer or isomer. The R values obtained using CTD are as robust and statistically significant as other fragmentation techniques, like radical directed dissociation (RDD). In summary, the extent of backbone and side-chain fragments produced by CTD enabled the differentiation of isomers and epimers of Asp in a variety of peptides.
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Capillary Zone Electrophoresis-Electron-Capture Collision-Induced Dissociation on a Quadrupole Time-of-Flight Mass Spectrometer for Top-Down Characterization of Intact Proteins
Mass spectrometry (MS)-based denaturing top-down proteomics (dTDP) requires high-capacity separation and extensive gas-phase fragmentation of proteoforms. Herein, we coupled capillary zone electrophoresis (CZE) to electron-capture collision-induced dissociation (ECciD) on an Agilent 6545 XT quadrupole time-of-flight (Q-TOF) mass spectrometer for dTDP for the first time. During ECciD, the protein ions were first fragmented using ECD, followed by further activation and fragmentation by applying a CID potential. In this pilot study, we optimized the CZE-ECciD method for small proteins (lower than 20 kDa) regarding the charge state of protein parent ions for fragmentation and the CID potential applied to maximize the protein backbone cleavage coverage and the number of sequence-informative fragment ions. The CZE-ECciD Q-TOF platform provided extensive backbone cleavage coverage for three standard proteins lower than 20 kDa from only single charge states in a single CZE-MS/MS run in the targeted MS/MS mode, including ubiquitin (97%, +7, 8.6 kDa), superoxide dismutase (SOD, 87%, +17, 16 kDa), and myoglobin (90%, +16, 17 kDa). The CZE-ECciD method produced comparable cleavage coverage of small proteins (i.e., myoglobin) with direct-infusion MS studies using electron transfer dissociation (ETD), activated ion-ETD, and combinations of ETD and collision-based fragmentation on high-end orbitrap mass spectrometers. The results render CZE-ECciD a new tool for dTDP to enhance both separation and gas-phase fragmentation of proteoforms.
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- Award ID(s):
- 1846913
- NSF-PAR ID:
- 10225398
- Date Published:
- Journal Name:
- Journal of the American Society for Mass Spectrometry
- ISSN:
- 1044-0305
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Rationale Free fatty acids and lipid classes containing fatty acid esters are major components of lipidome. In the absence of a chemical derivatization step, FA anions do not yield all of the structural information that may be of interest under commonly used collision‐induced dissociation (CID) conditions. A line of work that avoids condensed‐phase derivatization takes advantage of gas‐phase ion/ion chemistry to charge invert FA anions to an ion type that provides the structural information of interest using conventional CID. This work was motivated by the potential for significant improvement in overall efficiency for obtaining FA chain structural information.
Methods A hybrid triple quadrupole/linear ion‐trap tandem mass spectrometer that has been modified to enable the execution of ion/ion reaction experiments was used to evaluate the use of 4,4′,4″‐tri‐tert‐butyl‐2,2′:6′,2″‐terpyridine (ttb‐Terpy) as the ligand in divalent magnesium complexes for charge inversion of FA anions.
Results Mg(ttb‐Terpy)22+complexes provide significantly improved efficiency in producing structurally informative products from FA ions relative to Mg(Terpy)22+complexes, as demonstrated for straight‐chain FAs, branched‐chain FAs, unsaturated FAs, and cyclopropane‐containing FAs. It was discovered that most of the structurally informative fragmentation from [FA‐H + Mg(ttb‐Terpy)]+results from the loss of a methyl radical from the ligand followed by radical‐directed dissociation (RDD), which stands in contrast to the charge‐remote fragmentation (CRF) believed to be operative with the [FA‐H + Mg(Terpy)]+ions.
Conclusions This work demonstrates that a large fraction of product ions from the CID of ions of the form [FA‐H + Mg(ttb‐Terpy)]+are derived from RDD of the FA backbone, with a very minor fraction arising from structurally uninformative dissociation channels. This ligand provides an alternative to previously used ligands for the structural characterization of FAs via CRF.
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Ion dissociation is the usual basis for tandem MS analysis but a significant limitation is that only charged fragments from ion dissociation events are detected while neutral fragments are simply lost. This study reports our continued effort to solve this problem by developing atmospheric pressure neutral reionization mass spectrometry (APNR). In APNR, analyte ions are thermally dissociated (atmospheric pressure thermal dissociation, APTD) followed by soft reionization using electrosonic spray ionization (ESSI). Our results show that APNR is a powerful method for structural analysis of various biomolecules such as peptides, saccharides and nucleotides, as well as for elucidating unimolecular ion dissociation mechanisms. It was found that APNR provides extensive fragment ions including a series of y ions in peptides, which benefit sequencing and provide complementary information to collision induced dissociation (CID). In particular, direct cleavage of disulfide bonds of peptides occurs during APTD, facilitating peptide sequencing and disulfide bond mapping. In addition, many cross-ring cleavage fragments are detected during APNR analysis of oligosaccharides, indicating that the APTD dissociation process is energetic and potentially useful for identifying glycan linkage sites. Fragmentation patterns of oligosaccharide isomers can be used for their differentiation. Furthermore, in the cases of dissociation of nucleotides and synthetic naphthoylindole drugs, the putative neutral, phosphorylated riboses and indoles, were successfully detected using APNR, providing strong evidence to confirm previously proposed unimolecular ion dissociation mechanisms. We believe this APNR technique along with APTD should be of high value in structure determination of biomolecules.more » « less
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Abstract We report a collision‐induced dissociation (CID) based gas phase rearrangement study using quadrupole time‐of‐flight mass spectrometry coupled with liquid chromatography on a novel endothelin and angiotensin II receptor antagonist, sparsentan. We performed tandem mass spectrometry to identify precursor and fragment ion relationships and assigned structures for major fragment ions. We propose a benzyl migration mechanism based on bond length measurements in density functional theory (B3LYP/6‐31+G*) optimized geometries of protonated sparsentan and its
m/z 547 fragment. Protonated sparsentan undergoes loss of ethanol, which yields a resonance‐stabilized benzylic cation withm/z 547, which further fragments intom/z 353 via benzyl migration, where the benzylic cation migrates to one of the nucleophilic nitrogen atoms followed by proton transfer from the sulfonamide nitrogen to a carbonyl oxygen, resulting in a neutral loss of mass 194. Further fragmentation ofm/z 353 results inm/z 258, which undergoes radical and neutral loss to yieldm/z 193 and 194, respectively. The proposed mechanism of generation ofm/z 353 was confirmed by CID of deuterated sparsentan. Considering the importance of gas phase rearrangements of organic molecules in structural identifications as well as the novelty of the molecule, these findings will be helpful for future studies to predict gas phase benzyl migration in sparsentan analogs and for degradation product and metabolite identification of sparsentan and its analogs using LC–MS. -
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Rationale Tandem‐ion mobility spectrometry/mass spectrometry methods have recently gained traction for the structural characterization of proteins and protein complexes. However, ion activation techniques currently coupled with tandem‐ion mobility spectrometry/mass spectrometry methods are limited in their ability to characterize structures of proteins and protein complexes.
Methods Here, we describe the coupling of the separation capabilities of tandem‐trapped ion mobility spectrometry/mass spectrometry (tTIMS/MS) with the dissociation capabilities of ultraviolet photodissociation (UVPD) for protein structure analysis.
Results We establish the feasibility of dissociating intact proteins by UV irradiation at 213 nm between the two TIMS devices in tTIMS/MS and at pressure conditions compatible with ion mobility spectrometry (2–3 mbar). We validate that the fragments produced by UVPD under these conditions result from a radical‐based mechanism in accordance with prior literature on UVPD. The data suggest stabilization of fragment ions produced from UVPD by collisional cooling due to the elevated pressures used here (“UVnoD2”), which otherwise do not survive to detection. The data account for a sequence coverage for the protein ubiquitin comparable to recent reports, demonstrating the analytical utility of our instrument in mobility‐separating fragment ions produced from UVPD.
Conclusions The data demonstrate that UVPD carried out at elevated pressures of 2–3 mbar yields extensive fragment ions rich in information about the protein and that their exhaustive analysis requires IMS separation post‐UVPD. Therefore, because UVPD and tTIMS/MS each have been shown to be valuable techniques on their own merit in proteomics, our contribution here underscores the potential of combining tTIMS/MS with UVPD for structural proteomics.
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1021/jasms.0c00484
