De novo sequencing of highly modified therapeutic oligonucleotides by hydrophobic tag sequencing coupled with LC–MS

Correct sequences are prerequisite for quality control of therapeutic oligonucleotides. However, there is no definitive method available for determining sequences of highly modified therapeutic RNAs, and thereby, most of the oligonucleotides have been used clinically without direct sequence determination. In this study, we developed a novel sequencing method called ‘hydrophobic tag sequencing’. Highly modified oligonucleotides are sequenced by partially digesting oligonucleotides conjugated with a 5′‐hydrophobic tag, followed by liquid chromatography–mass spectrometry analysis. 5′‐Hydrophobic tag‐printed fragments (5′‐tag degradates) can be separated in order of their molecular masses from tag‐free oligonucleotides by reversed‐phase liquid chromatography. As models for the sequencing, the anti‐VEGF aptamer (Macugen) and the highly modified 38‐mer RNA sequences were analyzed under blind conditions. Most nucleotides were identified from the molecular weight of hydrophobic 5′‐tag degradates calculated from monoisotopic mass in simple full mass data. When monoisotopic mass could not be assigned, the nucleotide was estimated using the molecular weight of the most abundant mass. The sequences of Macugen and 38‐mer RNA perfectly matched the theoretical sequences. The hydrophobic tag sequencing worked well to obtain simple full mass data, resulting in accurate and clear sequencing. The present study provides for the first time a de novo sequencing technology for highly modified RNAs and contributes to quality control of therapeutic oligonucleotides. Copyright © 2016 John Wiley & Sons, Ltd.     

Electron transfer dissociation of dipositive uranyl and plutonyl coordination complexes

Reported here is a comparison of electron transfer dissociation (ETD) and collision‐induced dissociation (CID) of solvent‐coordinated dipositive uranyl and plutonyl ions generated by electrospray ionization. Fundamental differences between the ETD and CID processes are apparent, as are differences between the intrinsic chemistries of uranyl and plutonyl. Reduction of both charge and oxidation state, which is inherent in ETD activation of [An^VIO₂(CH₃COCH₃)₄]²⁺, [An^VIO₂(CH₃CN)₄]², [U^VIO₂(CH₃COCH₃)₅]²⁺ and [U^VIO₂(CH₃CN)₅]²⁺ (An = U or Pu), is accompanied by ligand loss. Resulting low‐coordinate uranyl(V) complexes add O₂, whereas plutonyl(V) complexes do not. In contrast, CID of the same complexes generates predominantly doubly‐charged products through loss of coordinating ligands. Singly‐charged CID products of [U^VIO₂(CH₃COCH₃)_4,5]²⁺, [U^VIO₂(CH₃CN)_4,5]²⁺ and [Pu^VIO₂(CH₃CN)₄]²⁺ retain the hexavalent metal oxidation state with the addition of hydroxide or acetone enolate anion ligands. However, CID of [Pu^VIO₂(CH₃COCH₃)₄]²⁺ generates monopositive plutonyl(V) complexes, reflecting relatively more facile reduction of Pu^VI to Pu^V. Copyright © 2011 John Wiley & Sons, Ltd.     

Toward a suitable structural analysis of gene delivery carrier based on polycationic carbohydrates by electron transfer dissociation tandem mass spectrometry

Polycationic carbohydrates represent an attractive class of biomolecules for several applications and particularly as non viral gene delivery vectors. In this case, the establishment of structure-biological activity relationship requires sensitive and accurate characterization tools to both control and achieve fine structural deciphering. Electrospray-tandem mass spectrometry (ESI-MS/MS) appears as a suitable approach to address these questions. In the study herein, we have investigated the usefulness of electron transfer dissociation (ETD) to get structural data about five polycationic carbohydrates demonstrated as promising gene delivery agents. A particular attention was paid to determine the influence of charge states as well as both fluoranthene reaction time and supplementary activation (SA) on production of charge reduced species, fragmentation yield, varying from 2 to 62%, as well as to obtain the most higher both diversity and intensity of fragments, according to charge states and targeted compounds. ETD fragmentation appeared to be mainly directed toward pending group rather than carbohydrate cyclic scaffold leading to a partial sequencing for building blocks when amino groups are close to carbohydrate core, but allowing to complete structural deciphering of some of them, such as those including dithioureidocysteaminyl group which was not possible with CID only. Such findings clearly highlight the potential to help the rational choice of the suitable analytical conditions, according to the nature of the gene delivery molecules exhibiting polycationic features. Moreover, our ETD-MS/MS approach open the way to a fine sequencing/identification of grafted groups carried on various sets of oligo-/polysaccharides in various fields such as glycobiology or nanomaterials, even with unknown or questionable extraction, synthesis or modification steps.     

Comparison of CID,ETD and metastable atom‐activated dissociation (MAD) of doubly and triply charged phosphorylated tau peptides

The fragmentation behavior of the 2+ and 3+ charge states of eleven different phosphorylated tau peptides was studied using collision‐induced dissociation (CID), electron transfer dissociation (ETD) and metastable atom‐activated dissociation (MAD). The synthetic peptides studied contain up to two known phosphorylation sites on serine or threonine residues, at least two basic residues, and between four and eight potential sites of phosphorylation. CID produced mainly b‐/y‐type ions with abundant neutral losses of the phosphorylation modification. ETD produced c‐/z‐type ions in highest abundance but also showed numerous y‐type ions at a frequency about 50% that of the z‐type ions. The major peaks observed in the ETD spectra correspond to the charge‐reduced product ions and small neutral losses from the charge‐reduced peaks. ETD of the 2+ charge state of each peptide generally produced fewer backbone cleavages than the 3+ charge state, consistent with previous reports. Regardless of charge state, MAD achieved more extensive backbone cleavage than CID or ETD, while retaining the modification(s) in most cases. In all but one case, unambiguous modification site determination was achieved with MAD. MAD produced 15–20% better sequence coverage than CID and ETD for both the 2+ and 3+ charge states and very different fragmentation products indicating that the mechanism of fragmentation in MAD is unique and complementary to CID and ETD. Copyright © 2012 John Wiley & Sons, Ltd.     

De novo prediction of ground state multiplicity and structural isomerism for transition metal complexes

Prediction of the ground state geometries and multiplicities for 33 transition metal tetrachlorides has been carried out using two different levels of quantum mechanics: semiempirical and density functional theory. All data regarding geometry and spin state provided by both computational methods were compared with experimental data when available. The calculations were performed for all possible spin multiplicities. The most important geometries for coordination number four (tetrahedral, square-planar, dodecahedral, and disphenoidal), as well as less symmetric structural isomers, were evaluated. A match between both computational methods in terms of predicted ground state multiplicity and geometry was found for 26 species, which translated into almost 80% agreement. Even though the PM3(tm) geometry prediction protocol involved more steps for isolating a feasible global minimum, the aggregate of these calculations was still orders of magnitude faster than DFT calculations using extended basis sets. The calculations indicate that caution is needed in the application of the PM3(tm) method to very high-spin transition metal complexes, but point to the suitability of very rapid semiempirical methods for reliable prediction of structural and ‘spin’ isomers, and hence their use in an efficient de novo design protocol for transition metal complexes.     

Sequence analysis of peptide:oligonucleotide heteroconjugates by electron capture dissociation and electron transfer dissociation

Mass spectrometry analysis of protein-nucleic acid cross-links is challenging due to the dramatically different chemical properties of the two components. Identifying specific sites of attachment between proteins and nucleic acids requires methods that enable sequencing of both the peptide and oligonucleotide component of the heteroconjugate cross-link. While collision-induced dissociation (CID) has previously been used for sequencing such heteroconjugates, CID generates fragmentation along the phosphodiester backbone of the oligonucleotide preferentially. The result is a reduction in peptide fragmentation within the heteroconjugate. In this work, we have examined the effectiveness of electron capture dissociation (ECD) and electron-transfer dissociation (ETD) for sequencing heteroconjugates. Both methods were found to yield preferential fragmentation of the peptide component of a peptide:oligonucleotide heteroconjugate, with minimal differences in sequence coverage between these two electron-induced dissociation methods. Sequence coverage was found to increase with increasing charge state of the heteroconjugate, but decreases with increasing size of the oligonucleotide component. To overcome potential intermolecular interactions between the two components of the heteroconjugate, supplemental activation with ETD was explored. The addition of a supplemental activation step was found to increase peptide sequence coverage over ETD alone, suggesting that electrostatic interactions between the peptide and oligonucleotide components are one limiting factor in sequence coverage by these two approaches. These results show that ECD/ETD methods can be used for the tandem mass spectrometry sequencing of peptide:oligonucleotide heteroconjugates, and these methods are complementary to existing CID methods already used for sequencing of protein-nucleic acid cross-links.     

Improved sequencing of oxidized cysteine and methionine containing peptides using electron transfer dissociation

Oxidative modifications to the side chains of sulfur-containing amino acids often limit the number of product ions formed during collision-induced dissociation (CID) and thus make it difficult to obtain sequence information for oxidized peptides. In this work, we demonstrate that electron-transfer dissociation (ETD) can be used to improve the sequence information obtained from peptides with oxidized cysteine and methionine residues. In contrast to CID, ETD is found to be much less sensitive to the side-chain chemistry, enabling extensive sequence information to be obtained in cases where CID fails to provide this information. These results indicate that ETD is a valuable technique for studying oxidatively modified peptides and proteins. In addition, we report a unique and very abundant product ion that is formed in the CID spectra of peptides having N-terminal cysteine sulfinic acid residues. The mechanism for this unique dissociation pathway involves a six-membered cyclic intermediate and leads to the facile loss of NH(3) and SO(2), which corresponds to a mass loss of 81 Da. While the facile nature of this dissociation pathway limits the sequence information present in CID spectra of peptides with N-terminal cysteine sulfinic acid residues, extensive sequence information for these peptides can be obtained with ETD.     

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

Electron detachment dissociation (EDD) of peptide poly-anions is gentle towards post-translational modifications (PTMs) and produces predictable and interpretable fragment ion types (a., x ions). However, EDD is considered an inefficient fragmentation technique and has not yet been implemented in large-scale peptide characterization strategies. We successfully increased the EDD fragmentation efficiency (up to 9%), and demonstrate for the first time the utility of EDD-MS/MS in liquid chromatography time-scale experiments. Peptides and phosphopeptides were analyzed in both positive- and negative-ion mode using electron capture/transfer dissociation (ECD/ETD) and EDD in comparison. Using approximately 1 pmol of a BSA tryptic digest, LC-EDD-MS/MS sequenced 14 peptides (27% aa sequence coverage) and LC-ECD-MS/MS sequenced 19 peptides (39% aa sequence coverage). Seven peptides (18% aa sequence coverage) were sequenced by both EDD and ECD. The relative small overlap of identified BSA peptides demonstrates the complementarity of the two dissociation modes. Phosphopeptide mixtures from three trypsin-digested phosphoproteins were subjected to LC-EDD-MS/MS resulting in the identification of five phospho-peptides. Of those, one was not found in a previous study using a similar sample and LC-ETD-MS/MS in the positive-ion mode. In this study, the ECD fragmentation efficiency (15.7% av.) was superior to the EDD fragmentation efficiency (3.6% av.). However, given the increase in amino acid sequence coverage and extended PTM characterization the new regime of EDD in combination with other ion-electron fragmentation techniques in the positive-ion mode is a step towards a more comprehensive strategy of analysis in proteome research.     

A review of electron-capture and electron-transfer dissociation tandem mass spectrometry in polymer chemistry

Mass spectrometry (MS)-based studies of synthetic polymers often characterise detected polymer components using mass data alone. However when mass-based characterisations are ambiguous, tandem MS (MS/MS) offers a means by which additional analytical information may be collected. This review provides a synopsis of two particularly promising methods of dissociating polymer ions during MS/MS: electron-capture and electron-transfer dissociation (ECD and ETD, respectively). The article opens with a summary of the basic characteristics and operating principles of ECD and ETD, and relates these techniques to other methods of dissociating gas-phase ions, such as collision-induced dissociation (CID). Insights into ECD- and ETD-based MS/MS, gained from studies into proteins and peptides, are then discussed in relation to polymer chemistry. Finally, ECD- and ETD-based studies into various classes of polymer are summarised; for each polymer class, ECD- and ETD-derived data are compared to CID-derived data. These discussions identify ECD and ETD as powerful means by which unique and diagnostically useful polymer ion fragmentation data may be generated, and techniques worthy of increased utilisation by the polymer chemistry community.     

De novo sequencing of proteolytic peptides by a combination of C-terminal derivatization and nano-electrospray/ collision-induced dissociation mass spectrometry

A series of synthetic peptides (3-15 residues), C-terminally derivatized with 4-aminonaphthalenesulfonic acid (ansa), have been analyzed on a hybrid magnetic sector-orthogonal acceleration time-of-flight tandem mass spectrometer, fitted with a nano-electrospray (nano-ES) interface. Deprotonated molecules generated by negative-ion ES were subjected to collision-induced dissociation (CID) using either methane or xenon as the collision gas, at a collision energy of 400 eV (laboratory frame of reference). As a consequence of charge localization on the sulfonate group, only C-terminal fragment ions were formed, presumably by charge-remote fragmentation mechanisms. Interpretable CID spectra were obtained from fmol amounts of the small peptides (up to 6 residues), whereas low pmol amounts were required for the larger peptides. CID spectra were also recorded of derivatized, previously noncharacterised peptides obtained by proteolysis of cytosolic hamster liver aldehyde dehydrogenase. Interpretation of these CID spectra was based on rules established for the fragmentation of the synthetic peptides. This study shows that derivatization with ansa may be useful in the de novo sequencing of peptides.     

Characterization and de novo sequencing of snow crab tropomyosin enzymatic peptides by both electrospary ionization and matrix‐assisted laser desorption ionization QqToF tandem mass spectrometry

The protein tropomyosin (TM) is a known major allergen present in shellfish causing frequent food allergies. TM is also an occupational allergen generated in the working environment of snow crab (Chionoecetes opilio) processing plants. The TM protein was purified from both claw and leg meats of snow crab and analyzed by electrospray ionization and matrix‐assisted laser desorption/ionization (MALDI) using hybrid quadruple time‐of‐flight tandem mass spectrometry (QqToF‐MS). The native polypeptide molecular weight of TM was determined to be 32 733 Da. The protein was further characterized using the ‘bottom‐up’ MS approach. A peptide mass fingerprinting was obtained by two different enzymatic digestions and de novo sequencing of the most abundant peptides performed. Any post‐translational modifications were identified by searching their calculated and predicted molecular weights in precursor ion spectra. The immunological reactivity of snow crab extract was evaluated using specific antibodies and allergenic reactivity assessed with serum of allergic patients. Subsequently, a signature peptide for TM was identified and evaluated in terms of identity and homology using the basic local alignment search tool (BLAST). The identification of a signature peptide for the allergen TM using MALDI‐QqToF‐MS will be critical for the sensitive and specific quantification of this highly allergenic protein in the work place. Copyright © 2010 John Wiley & Sons, Ltd.     

A perspective on MALDI alternatives—total solvent‐free analysis and electron transfer dissociation of highly charged ions by laserspray ionization

Progress in research is hindered by analytical limitations, especially in biological areas in which sensitivity and dynamic range are critical to success. Inherent difficulties of characterization associated with complexity arising from heterogeneity of various materials including topologies (isomeric composition) and insolubility also limit progress. For this reason, we are developing methods for total solvent‐free analysis by mass spectrometry consisting of solvent‐free ionization followed by solvent‐free gas‐phase separation. We also recently constructed a novel matrix‐assisted laser desorption ionization (MALDI) source that provides a simple, practical and sensitive way of producing highly charged ions by laserspray ionization (LSI) or singly charged ions commonly observed with MALDI by choice of matrix or matrix preparation. This is the first ionization source with such freedom—an extremely powerful analytical ‘switch’. Multiply charged LSI ions allow molecules exceeding the mass‐to‐charge range of the instrument to be observed and permit for the first time electron transfer dissociation fragment ion analysis. Copyright © 2010 John Wiley & Sons, Ltd.     

De novo sequencing,peptide composition analysis,and composition-based sequencing: A new strategy employing accurate mass determination by fourier transform ion cyclotron resonance mass spectrometry

A new strategy is described for the determination of amino acid sequences of unknown peptides. Different from the well-known but often inefficient de novo sequencing approach, the new method is based on a two-step process. In the first step the amino acid composition of an unknown peptide is determined on the basis of accurate mass values of the peptide precursor ion and a small number of accurate fragment ion mass values, and, as in de novo sequencing, without employing protein database information or other pre-information. In the second step the sequence of the found amino acids of the peptide is determined by scoring the agreement between expected and observed fragment ion signals of the permuted sequences. It was found that the new approach is highly efficient if accurate mass values are available and that it easily outstrips common approaches of de novo sequencing being based on lower accuracies and detailed knowledge of fragmentation behavior. Simple permutation and calculation of all possible amino acid sequences, however, is only efficient if the composition is known or if possible compositions are at least reduced to a small list. The latter requires the highest possible instrumental mass accuracy, which is currently provided only by fourier transform ion cyclotron resonance mass spectrometry. The connection between mass accuracy and peptide composition variability is described and an example of peptide compositioning and composition-based sequencing is presented.     

De novo sequencing of a 21-kDa cytochrome c4 from Thiocapsa roseopersicina by nanoelectrospray ionization ion-trap and Fourier-transform ion-cyclotron resonance mass spectrometry

We have determined the primary structure of cytochrome c(4) from Thiocapsa roseopersicina by de novo protein sequencing using the 'bottom up' approach. Three different enzymes (trypsin, endoproteinase Lys-C, and endoproteinase Glu-C) were employed to prepare four different sets of proteolytic digests. The digestion strategy was designed to permit a gradual buildup of smaller peptides into larger ones that were overlapped to yield the complete protein sequence. In this way we countered the main problem: peptides larger than about 1500 Da were difficult to sequence fully by tandem mass spectrometry. Direct infusion and online liquid chromatography were used on a linear ion trap Fourier-transform ion-cyclotron resonance hybrid instrument. The high resolving power, high mass accuracy and the availability of electron capture dissociation and collision-induced dissociation were essential to achieve full sequence coverage. The software DeNovoX complemented by manual interpretation was used to generate sequence information from tandem mass spectra. The predominantly automated nature of data acquisition and handling allowed for a relatively straightforward and fast procedure, which could compete with the mainstream alternative of nucleotide sequence determination.     

Effects of acidic peptide size and sequence on trivalent praseodymium adduction and electron transfer dissociation mass spectrometry

Using the lanthanide ion praseodymium, Pr(III), metallated ion formation and electron transfer dissociation (ETD) were studied for 25 biological and model acidic peptides. For chain lengths of seven or more residues, even highly acidic peptides that can be difficult to protonate by electrospray ionization will metallate and undergo abundant ETD fragmentation. Peptides composed of predominantly acidic residues form only the deprotonated ion, [M + Pr ‐ H]²⁺; this ion yields near complete ETD sequence coverage for larger peptides. Peptides with a mixture of acidic and neutral residues generate [M + Pr]³⁺, which cleaves between every residue for many peptides. Acidic peptides that contain at least one residue with a basic side chain also produce the protonated ion, [M + Pr + H]⁴⁺; this ion undergoes the most extensive sequence coverage by ETD. Primarily metallated and non‐metallated c‐ and z‐ions form for all peptides investigated. Metal adducted product ions are only present when at least half of the peptide sequence can be incorporated into the ion; this suggests that the metal ion simultaneously attaches to more than one acidic site. The only site consistently lacking dissociation is at the N‐terminal side of a proline residue. Increasing peptide chain length generates more backbone cleavage for metal‐peptide complexes with the same charge state. For acidic peptides with the same length, increasing the precursor ion charge state from 2+ to 3+ also leads to more cleavage. The results of this study indicate that highly acidic peptides can be sequenced by ETD of complexes formed with Pr(III). Copyright © 2017 John Wiley & Sons, Ltd.     

Activation of freons by cascade electron transfer from a nucleophile using two mutually complementary mediators—Methylviologen and sulfur dioxide

It was shown that a system consisting of two mutually complementary electron transfer mediators (methylviologen and sulfur dioxide) is capable of bringing about cascade electron transfer from thiophenols to Freons (CF₃Br, CF₂ClCCl₂F), activating the latter under mild conditions, and bringing them into fluoroalkylation. L. V. Pisarzhevskii Institute of Physical Chemistry, National Academy of Sciences of Ukraine, 31 Prospekt Nauki, Kiev 252039, Ukraine. Translated from Teoreticheskaya i éksperimental'naya Khimiya, Vol. 34, No. 6, pp. 347–350, November–December, 1998.     

A simultaneous determination method for 5‐fluorouracil and its metabolites in human plasma with linear range adjusted by in‐source collision‐induced dissociation using hydrophilic interaction liquid chromatography–electrospray ionization–tandem mass spectrometry

We applied a new technique for quantitative linear range shift using in‐source collision‐induced dissociation (CID) to complex biological fluids to demonstrate its utility. The technique was used in a simultaneous quantitative determination method of 5‐fluorouracil (5‐FU), an anticancer drug for various solid tumors, and its metabolites in human plasma by liquid chromatography–electrospray ionization–tandem mass spectrometry (LC/ESI‐MS/MS). To control adverse effects after administration of 5‐FU, it is important to monitor the plasma concentration of 5‐FU and its metabolites; however, no simultaneous determination method has yet been reported because of vastly different physical and chemical properties of compounds. We developed a new analytical method for simultaneously determining 5‐FU and its metabolites in human plasma by LC/ESI‐MS/MS coupled with the technique for quantitative linear range shift using in‐source CID. Hydrophilic interaction liquid chromatography using a stationary phase with zwitterionic functional groups, phosphorylcholine, was suitable for separation of 5‐FU from its nucleoside and interfering endogenous materials. The addition of glycerin into acetonitrile‐rich eluent after LC separation improved the ESI‐MS response of high polar analytes. Based on the validation results, linear range shifts by in‐source CID is the reliable technique even with complex biological samples such as plasma. Copyright © 2016 John Wiley & Sons Ltd.     

Trace determination of peptides in water samples using packed capillary liquid chromatography with UV and MS detection and characterization of peptide oxidation products by MS

A capillary liquid chromatographic column switching method has been developed for fast and sensitive determination of peptides in water samples. Sample volumes of 1 mL were loaded onto a (320 m I.D. ×30 mm) 10 m Kromasil C₁₈ pre-column, providing on-line analyte enrichment, prior to back-flushed elution onto a (320 m I.D. ×150 mm) 3.5 m Kromasil C₁₈ analytical column. Loading flow rates of 250 L/min and a mobile phase composition of acetonitrile/water/trifluoroacetic acid (22/77.9/0.1, v/v) provided a total analysis time of less than 25 minutes for the test peptides angiotensin II, bombesin, bradykinin, corazonin, neurotensin and substance P, using temperature programmed elution. In addition, solvent gradient elution and combined solvent gradient elution and temperature programming were explored. Using on-capillary UV detection at 210 nm resulted in a concentration limit of detection (cLOD) of about 1 ng/mL. The method was validated over the concentration range 1–100 ng/mL, yielding a coefficient of correlation of 0.997 or better. The within-assay (n=6) and between-assay (n=6) precisions of peak areas were on average 6% RSD and 5% RSD, respectively.When the method was applied to spiked chlorinated tap water samples, it was found that peptides containing methionine, tryptophan and cystine were oxidized. Identification of the oxidation products of the peptides in hypochlorite-treated water was done with positive electrospray ionization time-of-flight mass spectrometric detection.