ESI,APCI, and MALDI tandem mass spectrometry of poly(methyl acrylate)s: A comparison study for the structural characterization of polymers synthesized via CRP techniques and the software application to analyze MS/MS data

Research in polymer science and engineering is moving from classical methodologies to advanced analytical strategies in which mass spectrometry (MS)‐based techniques play a crucial role. The molecular complexity of polymers requires new characterization tools and approaches to elucidate the detailed structural information. In this contribution, a comparison study of poly(methyl acrylate)s (PMA) using different tandem mass spectrometry techniques (ESI, APCI, and MALDI MS/MS) is reported to provide insights into the macromolecular structure with the aid of a special MS/MS data interpretation software. Collision‐induced dissociation (CID) was utilized to examine the fragmentation pathways of PMAs synthesized via various controlled radical polymerization techniques. All three mass spectrometry techniques are used to analyze structural details of PMAs and the labile end‐groups are determined based on the fragmentation behavior in CID. Fragmentation products were identified which are characteristics for the cleavage between the polymer chain and the end‐group. The application of a tailor‐made software is shown to analyze complex MS/MS data, and it is proven that this kind of software will be helpful for polymer scientists to identify fragmentation products obtained by tandem mass spectrometry similar to the fields of proteomics, metabolomics, genomics, and glycomics. © 2013 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2013     

Characterization of different poly(2‐ethyl‐2‐oxazoline)s via matrix‐assisted laser desorption/ionization time‐of‐flight tandem mass spectrometry

Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) coupled with CID (collision‐induced dissociation) has been used for the detailed characterization of two poly(2‐ethyl‐2‐oxazoline)s as part of a continuing study of synthetic polymers by MALDI‐TOF MS/MS. These experiments provided information about the variety of fragmentation pathways for poly(oxazoline)s. It was possible to show that, in addition to the eliminations of small molecules, like ethene and hydrogen, the McLafferty rearrangement is also a possible fragmentation route. A library of fragmentation pathways for synthetic polymers was also constructed and such a library should enable the fast and automated data analysis of polymers in the future. Copyright © 2009 John Wiley & Sons, Ltd.     

Automated interpretation of low-energy collision-induced dissociation spectra by SeqMS, a software aid for de novo sequencing by tandem mass spectrometry

SeqMS, a software aid for de novo sequencing by tandem mass spectrometry (MS/MS), which was initially developed for the automated interpretation of high-energy collision-induced dissociation (CID) MS/MS spectra of peptides, has been applied to the interpretation of low-energy CID and post-source decay (PSD) spectra of peptides. Based on peptide backbone fragmented ions and their related ions, which are the dominant ions observed in the latter two techniques, the types of ions and their propensities to be observed have been optimized for efficient interpretation of the spectra. In a typical example, the modified SeqMS allowed the complete sequencing of a 31-amino acid synthetic peptide, except for the isobaric amino acids (Leu or Ile, and Lys or Gln), based on only the low-energy CID-MS/MS spectrum.     

Electron Transfer Dissociation of Milk Oligosaccharides

For structural identification of glycans, the classic collision-induced dissociation (CID) spectra are dominated by product ions that derived from glycosidic cleavages, which provide only sequence information. The peaks from cross-ring fragmentation are often absent or have very low abundances in such spectra. Electron transfer dissociation (ETD) is being applied to structural identification of carbohydrates for the first time, and results in some new and detailed information for glycan structural studies. A series of linear milk sugars was analyzed by a variety of fragmentation techniques such as MS/MS by CID and ETD, and MS(3) by sequential CID/CID, CID/ETD, and ETD/CID. In CID spectra, the detected peaks were mainly generated via glycosidic cleavages. By comparison, ETD generated various types of abundant cross-ring cleavage ions. These complementary cross-ring cleavages clarified the different linkage types and branching patterns of the representative milk sugar samples. The utilization of different MS(3) techniques made it possible to verify initial assignments and to detect the presence of multiple components in isobaric peaks. Fragment ion structures and pathways could be proposed to facilitate the interpretation of carbohydrate ETD spectra, and the main mechanisms were investigated. ETD should contribute substantially to confident structural analysis of a wide variety of oligosaccharides.     

The rapid characterisation of poly(ethylene glycol) oligomers using desorption electrospray ionisation tandem mass spectrometry combined with novel product ion peak assignment software

A rapid method for the characterisation of polyglycol esters and ethers is described which uses accurate mass desorption electrospray ionisation (DESI) quadrupole time-of-flight mass spectrometry (Q-ToFMS). The results are combined with newly developed software which aids the interpretation of product ions produced using collision-induced dissociation (CID) of selected precursor ions. The poly(ethylene glycol) (PEG) samples analysed were PEG dibenzoate, PEG monooleate, PEG butyl ether, PEG bis(2-ethyl hexanoate) and PEG diacrylate. Lithium metal was used for cationisation of the PEG oligomers since it yielded the most useful structural information compared with other group I metals. The full scan mass spectra and product ion mass spectra were all obtained in <5 s. Interpretation of the MS/MS product ion spectra, using the product ion interpretation software which incorporates previously developed fragmentation rules, was carried out in <1 s.     

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.     

A case study of de novo sequence analysis of N-sulfonated peptides by MALDI TOF/TOF mass spectrometry

The simplicity and sensitivity of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry have increased its application in recent years. The most common method of "peptide mass fingerprint" analysis often does not provide robust identification. Additional sequence information, obtained by post-source decay or collision induced dissociation, provides additional constraints for database searches. However, de novo sequencing by mass spectrometry is not yet common practice, most likely because of the difficulties associated with the interpretation of high and low energy CID spectra. Success with this type of sequencing requires full sequence coverage and demands better quality spectra than those typically used for data base searching. In this report we show that full-length de novo sequencing is possible using MALDI TOF/TOF analysis. The interpretation of MS/MS data is facilitated by N-terminal sulfonation after protection of lysine side chains (Keough et al., Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 7131-7136). Reliable de novo sequence analysis has been obtained using sub-picomol quantities of peptides and peptide sequences of up to 16 amino acid residues in length have been determined. The simple, predictable fragmentation pattern allows routine de novo interpretation, either manually or using software. Characterization of the complete primary structure of a peptide is often hindered because of differences in fragmentation efficiencies and in specific fragmentation patterns for different peptides. These differences are controlled by various structural parameters including the nature of the residues present. The influence of the presence of internal Pro, acidic and basic residues on the TOF/TOF fragmentation pattern will be discussed, both for underivatized and guanidinated/sulfonated peptides.     

Analysis of different synthetic homopolymers by the use of a new calculation software for tandem mass spectra

The manual interpretation of tandem mass spectra of synthetic polymers is very time-consuming. Therefore, a new software tool was developed to accelerate the interpretation of spectra obtained without requiring any further knowledge about the polymer class or the fragmentation behavior under high-energy collision-induced dissociation (CID) conditions. The software only requires an alphabetical list of elements and a peak list of the measured substance as an xml file for the evaluation of the chosen mass spectrum. Tandem mass spectra of different homopolymers, like poly(2-oxazoline)s, poly(ethylene glycol) and poly(styrene), were interpreted by the new software tool. This contribution describes a fast and automated software tool for the rapid analysis of homopolymers.     

Energy-dependent collision-induced dissociation study of buprenorphine and its synthetic precursors

The collision-induced dissociation (CID) of protonated buprenorphine ([M+H](+) ) and four related compounds was studied by electrospray quadrupole/time-of-flight mass spectrometry (ESI-QTOF MS). The fragmentation pathways were investigated by using energy-dependent CID and pseudo-MS(3) (in-source CID combined with tandem mass spectrometry (MS/MS)) methods. The first steps of the fragmentation are the parallel losses of the substituents from the non-aromatic ring moieties. Depending on the applied collision energies, a large number of further fragment ions arising from the cross-ring cleavages of the core-ring structure were observed. Based on the experimental results, a generalized fragmentation scheme was developed for the five buprenorphine derivatives highlighting the differences for the alternatively substituted compounds. The collision-energy-dependent fragmentation profile of buprenorphine is visualized in a two-dimensional plot to aid its fingerprint identification.     

High mass accuracy in-source collision-induced dissociation tandem mass spectrometry and multi-step mass spectrometry as complementary tools for fragmentation studies of quaternary ammonium herbicides

Fragmentation studies using both an ion-trap mass analyzer and a hybrid quadrupole time-of-flight (Q-TOF) mass spectrometer were performed in order to establish the fragmentation pathways of organic molecules. A general strategy combining MSn data (n = 1-4) in an ion-trap analyzer with tandem mass spectrometry and in-source collision-induced dissociation tandem mass spectrometry (CID MS/MS) in a Q-TOF instrument was applied. The MSn data were used to propose a tentative fragmentation pathway following genealogical relationships. When several assignments were possible, MS/MS and in-source CID MS/MS (Q-TOF) allowed the elemental compositions of the fragments to be confirmed. Quaternary ammonium herbicides (quats) were used as test compounds and their fragmentation pathways were established. The elemental composition of the fragments was confirmed using the TOF analyzer with relative errors <0.0023 Da. Some fragments previously reported in the literature were reassigned taking advantage of the high mass resolution and accuracy of the Q-TOF instrument, which made it possible to solve losses where nitrogen was involved.     

Improving confidence in detection and characterization of protein N-glycosylation sites and microheterogeneity

Protein glycosylation is one of the most common post-translational modifications, estimated to occur in over 50% of human proteins. Mass spectrometry (MS)-based approaches involving different fragmentation mechanisms have been frequently used to detect and characterize protein N-linked glycosylations. In addition to the popular Collision-Induced Dissociation (CID), high-energy C-trap dissociation (HCD) fragmentation, which is a feature of a linear ion trap orbitrap hybrid mass spectrometer (LTQ Orbitrap), has been recently used for the fragmentation of tryptic N-linked glycopeptides in glycoprotein analysis. The oxonium ions observed with high mass accuracy in the HCD spectrum of glycopeptides can be combined with characteristic fragmentation patterns in the CID spectrum resulting from consecutive glycosidic bond cleavages, to improve the detection and characterization of N-linked glycopeptides. As a means of automating this process, we describe here GlypID 2.0, a software tool that implements several algorithmic approaches to utilize MS information including accurate precursor mass and spectral patterns from both HCD and CID spectra, thus allowing for an unequivocal and accurate characterization of N-linked glycosylation sites of proteins.     

An experimental approach to enhance precursor ion fragmentation for metabolite identification studies: application of dual collision cells in an orbital trap

Recent advancements in mass spectrometry including data-dependent scanning and high-resolution mass spectrometry have aided metabolite profiling for non-radiolabeled xenobiotics. However, narrowing down a site of metabolism is often limited by the quality of the collision-induced dissociation (CID)-based precursor ion fragmentation. An alternative dissociation technique, higher energy collisional dissociation (HCD), enriches compound fragmentation and yields 'triple-quadrupole-like fragmentation'. Applying HCD along with CID and data-dependent scanning could enhance structural elucidation for small molecules. Liquid chromatography/multi-stage mass spectrometry (LC/MS(n) ) experiments with CID and HCD fragmentation were carried out for commercially available compounds on a hybrid linear ion trap orbital trap mass spectrometer equipped with accurate mass measurement capability. The developed method included stepped normalized collision energy (SNCE) parameters to enhance MS fragmentation without tuning for individual compounds. All the evaluated compounds demonstrated improved fragmentation under HCD as compared with CID. The results suggest that an LC/MS(n) method that incorporated both SNCE HCD- and CID-enabled precursor ion fragmentation afforded comprehensive structural information for the compounds under investigation. A dual collision cell approach was remarkably better than one with only CID MS(n) in an orbital trap. It is evident that such an acquisition method can augment the identification of unknown metabolites in drug discovery by improving fragmentation efficiency of both the parent compound and its putative metabolite(s).     

Tandem mass spectrometry of poly(ethylene imine)s by electrospray ionization (ESI) and matrix‐assisted laser desorption/ionization (MALDI)

In this contribution, linear poly(ethylene imine) (PEI) polymers, which are of importance in gene delivery, are investigated in detail by using electrospray ionization‐quadrupole‐time of flight (ESI‐Q‐TOF) and matrix‐assisted laser desorption/ionization‐time of flight (MALDI‐TOF) mass spectrometry (MS). The analyzed PEIs with different end groups were synthesized using the polymerization of substituted 2‐oxazoline via a living cationic ring‐opening polymerization (CROP) and a subsequent hydrolysis under acidic conditions. The main goal of this study was to identify linear PEI polymers in a detailed way to gain information about their fragmentation pathways. For this purpose, a detailed characterization of three different linear PEIs was performed by using ESI‐Q‐TOF and MALDI‐TOF MS in combination with collision‐induced dissociation (CID) experiments. In ESI‐MS as well as MALDI‐MS analysis, the obtained spectra of PEIs resulted in fitting mass distributions for the investigated PEIs. In the tandem MS analysis, a 1,2‐hydride shift with a charge‐remote rearrangement via a four‐membered cyclic transition state, as well as charge‐induced fragmentation reactions, was proposed as the main fragmentation mechanisms according to the obtained fragmentation products from the protonated parent peaks. In addition, heterolytic and homolytic cleavages were proposed as alternative fragmentation pathways. Moreover, a 1,4‐hydrogen elimination was proposed to explain different fragmentation products obtained from the sodiated parent peaks. Copyright © 2012 John Wiley & Sons, Ltd.     

Characterization of glycopeptides by combining collision‐induced dissociation and electron‐transfer dissociation mass spectrometry data

Structural characterization of a glycopeptide is not easily attained through collision‐induced dissociation (CID), due to the extensive fragmentation of glycan moieties and minimal fragmentation of peptide backbones. In this study, we have exploited the potential of electron‐transfer dissociation (ETD) as a complementary approach for peptide fragmentation. Model glycoproteins, including ribonuclease B, fetuin, horseradish peroxidase, and haptoglobin, were used here. In ETD, radical anions transfer an electron to the peptide backbone and induce cleavage of the N–Cα bond. The glycan moiety is retained on the peptide backbone, being largely unaffected by the ETD process. Accordingly, ETD allows not only the identification of the amino acid sequence of a glycopeptide, but also the unambiguous assignment of its glycosylation site. When data acquired from both fragmentation techniques are combined, it is possible to characterize comprehensively the entire glycopeptide. This is being achieved with a mass spectrometer capable of alternating between CID and ETD on‐the‐fly during an LC/MS/MS analysis. This is demonstrated here with several tryptic glycopeptides. Copyright © 2008 John Wiley & Sons, Ltd.     

Tandem mass spectrometric sequence characterization of synthetic thymidine-rich oligonucleotides

Tandem mass spectrometry (MS/MS) can provide direct and accurate sequence characterization of synthetic oligonucleotide drugs, including modified oligonucleotides. Multiple factors can affect oligonucleotide MS/MS sequencing, including the intrinsic properties of oligonucleotides (i.e., nucleotide composition and structural modifications) and instrument parameters associated with the ion activation for fragmentation. In this study, MS/MS sequencing of a thymidine (T)-rich and phosphorothioate (PS)-modified DNA oligonucleotide was investigated using two fragmentation techniques: trap-type collision-induced dissociation (“CID”) and beam-type CID also termed as higher-energy collisional dissociation (“HCD”), preceded by a hydrophilic interaction liquid chromatography (HILIC) separation. A low to moderate charge state (−4), which predominated under the optimized HILIC-MS conditions, was selected as the precursor ion for MS/MS analysis. Comparison of the two distinctive ion activation mechanisms on the same precursor demonstrated that HCD was superior to CID in promoting higher sequence coverage and analytical sensitivity in sequence elucidation of T-rich DNA oligonucleotides. Specifically, HCD provided more sequence-defining fragments with higher fragment intensities than CID. Furthermore, the direct comparison between unmodified and PS-modified DNA oligonucleotides demonstrated a loss of MS/MS fragmentation efficiency by PS modification in both CID and HCD approaches, and a resultant reduction in sequence coverage. The deficiency in PS DNA sequence coverage observed with single collision energy HCD, however, was partially recovered by applying HCD with multiple collision energies. Collectively, this work demonstrated that HCD is advantageous to MS/MS sequencing of T-rich PS-modified DNA oligonucleotides.     

Effects of Electron-Transfer Coupled with Collision-Induced Dissociation (ET/CID) on Doubly Charged Peptides and Phosphopeptides

Electron-transfer dissociation (ETD) is a useful peptide fragmentation technique that can be applied to investigate post-translational modifications (PTMs), the sequencing of highly hydrophilic peptides, and the identification of large peptides and even intact proteins. In contrast to traditional fragmentation methods, such as collision-induced dissociation (CID), ETD produces c- and z^·-type product ions by randomly cleaving the N–Cα bonds. The disappointing fragmentation efficiency of ETD for doubly charged peptides and phosphopeptide ions has been improved by ETcaD (supplemental activation). However, the ETD data derived from most database search algorithms yield low confidence scores due to the presence of unreacted precursors and charge-reduced ions within MS/MS spectra. In this work, we demonstrate that eight out of ten standard doubly charged peptides and phosphopeptides can be effortlessly identified by electron-transfer coupled with collision-induced dissociation (ET/CID) using the SEQUEST algorithm without further spectral processing. ET/CID was performed with the further dissociation of the charge-reduced ions isolated from ETD ion/ion reactions. ET/CID had high fragmentation efficiency, which elevated the confidence scores of doubly charged peptide and phosphospeptide sequencing. ET/CID was found to be an effective fragmentation strategy in “bottom-up” proteomic analysis.     

Electrospray ionization collision‐induced dissociation mass spectrometry: a tool to characterize synthetic polyaminocarboxylate ferric chelates used as fertilizers

Fertilizers based on synthetic polyaminocarboxylate ferric chelates have been known since the 1950s to be successful in supplying Fe to plants. In commercial Fe(III)‐chelate fertilizers, a significant part of the water‐soluble Fe‐fraction consists of still uncharacterized Fe byproducts, whose agronomical value is unknown. Although collision‐induced dissociation (CID) tandem mass spectrometry (MS/MS) is a valuable tool for the identification of such compounds, no fragmentation data have been reported for most Fe(III)‐chelate fertilizers. The aim of this study was to characterize the CID‐MS² fragmentation patterns of the major synthetic Fe(III)‐chelates used as Fe‐fertilizers, and subsequently use this technique for the characterization of commercial fertilizers. Quadrupole‐time‐of‐flight (QTOF) and spherical ion trap mass analyzers equipped with an electrospray ionization (ESI) source were used. ESI‐CID‐MS² spectra obtained were richer when using the QTOF device. Specific differences were found among Fe(III)‐chelate fragmentation patterns, even in the case of positional isomers. The analysis of a commercial Fe(III)‐chelate fertilizer by high‐performance liquid chromatography (HPLC) coupled to ESI‐MS(QTOF) revealed two previously unknown, Fe‐containing compounds, that were successfully identified by a comprehensive comparison of the ESI‐CID‐MS²(QTOF) spectra with those of pure chelates. This shows that HPLC/ESI‐CID‐MS²(QTOF), along with the Fe(III)‐chelate fragmentation patterns, could be a highly valuable tool to directly characterize the water‐soluble Fe fraction in Fe(III)‐chelate fertilizers. This could be of great importance in issues related to crop Fe‐fertilization, both from an agricultural and an environmental point of view. Copyright © 2009 John Wiley & Sons, Ltd.     

Collision-induced fragmentation pathways including odd-electron ion formation from desorption electrospray ionisation generated protonated and deprotonated drugs derived from tandem accurate mass spectrometry

The rapid desorption electrospray ionisation (DESI) of some small molecules and their fragmentation using a triple-quadrupole and a hybrid quadrupole time-of-flight mass spectrometer (Q-ToF) have been investigated. Various scanning modes have been employed using the triple-quadrupole instrument to elucidate fragmentation pathways for the product ions observed in the collision-induced dissociation (CID) spectra. Together with accurate mass tandem mass spectrometry (MS/MS) measurements performed on the hybrid Q-ToF mass spectrometer, unequivocal product ion identification and fragmentation pathways were determined for deprotonated metoclopramide and protonated aspirin, caffeine and nicotine. Ion structures and fragmentation pathway mechanisms have been proposed and compared with previously published data. The necessity for elevated resolution for the differentiation of isobaric ions are discussed.     

Nano-electrospray ionization time-of-flight mass spectrometry of gangliosides from human brain tissue

A general approach for the detection and structural elucidation of brain ganglioside species GM1, GD1 and GT1 by nano-electrospray ionization quadrupole time-of-flight (nanoESI-QTOF) mass spectrometry (MS), using combined data from MS and MS/MS analysis of isolated native ganglioside fractions in negative ion mode and their permethylated counterparts in the positive ion mode is presented. This approach was designed to detect and sequence gangliosides present in preparatively isolated ganglioside fractions from pathological brain samples available in only very limited amounts. In these fractions mixtures of homologue and isobaric structures are present, depending on the ceramide composition and the position of the sialic acid attachment site. The interpretation of data for the entire sequence, derived from A, B, C and Y ions by nanoESI-QTOFMS/MS in the negative ion mode of native fractions, can be compromised by ions arising from double and triple internal cleavages. To distinguish between isobaric carbohydrate structures in gangliosides, such as monosialogangliosides GM1a and GM1b, disialogangliosides GD1a, GD1b and GD1c or trisialogangliosides GT1b, GT1c and GT1d, the samples were analysed after permethylation in the positive ion nanoESI-QTOFMS/MS mode, providing set of data, which allows a clear distinction for assignment of outer and inner fragment ions according to their m/z values. The fragmentation patterns from native gangliosides obtained by low-energy collision induced dissociation (CID) by nanoESI-QTOF show common behaviour and follow inherent rules. The combined set of data from the negative and positive ion mode low-energy CID can serve for the detection of structural isomers in mixtures, and to trace new, not previously detected, components.