skip to main content

Explore Research Products in the NSF-PAR

Title: Differentiating aspartic acid isomers and epimers with charge transfer dissociation mass spectrometry (CTD-MS)
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.  more » « less
Award ID(s):
1710376
NSF-PAR ID:
10345477
Author(s) / Creator(s):
; ; ;
Date Published:
Journal Name:
The Analyst
Volume:
147
Issue:
6
ISSN:
0003-2654
Page Range / eLocation ID:
1159 to 1168
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; ( , Rapid Communications in Mass Spectrometry)
    Rationale

    The function of a protein or the binding affinity of an antibody can be substantially altered by the replacement of leucine (Leu) with isoleucine (Ile), and vice versa, so the ability to identify the correct isomer using mass spectrometry can help resolve important biological questions. Tandem mass spectrometry approaches for Leu/Ile (Xle) discrimination have been developed, but they all have certain limitations.

     Methods

    Four model peptides and two wild‐type peptide sequences containing either Leu or Ile residues were subjected to charge transfer dissociation (CTD) mass spectrometry on a modified three‐dimensional ion trap. The peptides were analyzed in both the 1+ and 2+ charge states, and the results were compared to conventional collision‐induced dissociation spectra of the same peptides obtained using the same instrument.

     Results

    CTD resulted in 100% sequence coverage for each of the studied peptides and provided a variety of side‐chain cleavages, includingd,wandvions. Using CTD, reliabledandwions of Xle residues were observed more than 80% of the time. When present,dions are typically greater than 10% of the abundance of the correspondingaions from which they derive, andwions are typically more abundant than thezions from which they derive.

     Conclusions

    CTD has the benefit of being applicable to both 1+ and 2+ precursor ions, and the overall performance is comparable to that of other high‐energy activation techniques like hot electron capture dissociation and UV photodissociation. CTD does not require chemical modifications of the precursor peptides, nor does it require additional levels of isolation and fragmentation.

     
    more » « less
  2. ; ( , The Analyst)
    Recent studies have illuminated connections between spontaneous chemical reactions that cause isomerization at specific protein residues and various age-related diseases including cataracts and Alzheimer's. These discoveries provide impetus for better analytical methods to detect and characterize isomerization in proteins, which will enable a more complete understanding of the underlying relationship between these modifications and biology. Herein we employ a two-dimensional approach for identification of peptides isomers that also includes pinpointing of the modified residue. Collision-induced dissociation is used to fragment ions in the first dimension, followed by separation of the fragments with travelling-wave ion mobility. By comparing data obtained from both isomers, differences in either fragment-ion intensities or arrival-time distributions can be used to identify isomeric forms and the specific site of modification within the peptides. Synthetic peptide standards with sequences derived from long-lived proteins in the eye lens and isomerization at serine, aspartic acid, and glutamic acid were examined. Although both dimensions are capable of isomer identification, ion mobility is much better at determining the site of modification. In general, separation of isomeric forms by ion mobility is possible but does not follow predictable trends dictated by sequence or fragment-ion length. In most cases, however, the site of isomerization can be precisely determined. 
    more » « less
  3. ; ( , Glycobiology)
    Abstract The combination of helium charge transfer dissociation mass spectrometry (He–CTD–MS) with ultrahigh performance liquid chromatography (UHPLC) is presented for the analysis of a complex mixture of acidic and neutral human milk oligosaccharides (HMOs). The research focuses on the identification of the monosaccharide sequence, the branching patterns, the sialylation/fucosylation arrangements, and the differentiation of isomeric oligosaccharides in the mixture. Initial studies first optimized the conditions for the UHPLC separation and the He–CTD–MS conditions. Results demonstrate that He–CTD is compatible with UHPLC timescales and provides unambiguous glycosidic and cross-ring cleavages from both the reducing and the nonreducing ends, which is not typically possible using collision-induced dissociation. He–CTD produces informative fragments, including 0,3An and 0,4An ions, which have been observed with electron transfer dissociation, electron detachment dissociation, and ultraviolet photodissociation (UVPD) and are crucial for differentiating the α-2,3- versus α-2,6-linked sialic acid (Neu5Ac) residues present among sialyllacto-N-tetraose HMOs. In addition to the linkage positions, He–CTD is able to differentiate structural isomers for both sialyllacto-N-tetraoses and lacto-N-fucopentaoses structures by providing unique, unambiguous cross-ring cleavages of types 0,2An, 0,2Xn, and 1,5An while preserving most of the labile Neu5Ac and fucose groups. 
    more » « less
  4. ; ; ; ( , Chemical Science)
    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
  5. ; ; ; ; ; ; ; ; ; ; et al ( , Chemical Science)
    Growing evidence supports the confident association between distinct amyloid beta (Aβ) isoforms and Alzheimer's Disease (AD) pathogenesis. As such, critical investigations seeking to uncover the translational factors contributing to Aβ toxicity represent a venture of significant value. Herein, we comprehensively assess full-length Aβ42 stereochemistry, with a specific focus on models that consider naturally-occurring isomerization of Asp and Ser residues. We customize various forms of d -isomerized Aβ as natural mimics, ranging from fragments containing a single d residue to full length Aβ42 that includes multiple isomerized residues, systematically evaluating their cytotoxicity against a neuronal cell line. Combining multidimensional ion mobility-mass spectrometry experimental data with replica exchange molecular dynamics simulations, we confirm that co- d -epimerization at Asp and Ser residues within Aβ42 in both N-terminal and core regions effectively reduces its cytotoxicity. We provide evidence that this rescuing effect is associated with the differential and domain-specific compaction and remodeling of Aβ42 secondary structure. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email