Structural determination of cerebrosides isolated from Asterias amurensis starfish eggs using high-energy collision-induced dissociation of sodium-adducted molecules

Six cerebrosides were isolated from the eggs of the starfish Asterias amurensis using solvent extraction, silica gel column chromatography, and reversed‐phase high‐performance liquid chromatography. This study demonstrated that the structures of cerebrosides could be completely characterized, based on their sodium‐adducted molecules, using fast atom bombardment (FAB) tandem mass spectrometry. The high‐energy collision‐induced dissociation of the sodium‐adducted molecule, [M + Na]⁺, of each cerebroside molecular species generated abundant ions, providing information on the compositions of the 2‐hydroxy fatty acids and long‐chain sphingoid bases, as well as the sugar moiety polar head group. Each homologous ion series along the fatty acid and aliphatic chain of the sphingoid base was useful for locating the double‐bond positions of both chains and the methyl branching position of the long‐chain base. The N‐fatty acyl portions were primarily long‐chain saturated or monoenoic acids (C16 to C24) with an α‐hydroxy group. The sphingoid long‐chain base portions were aliphatic chains (C18 or C22) with two or three degrees of unsaturation and with or without methyl branching. Copyright © 2011 John Wiley & Sons, Ltd.     

Structural determination of glucosylceramides isolated from marine sponge by fast atom bombardment collision‐induced dissociation linked scan at constant B/E

Five glucosylceramides (GlcCers) were isolated by reversed phase high‐performance liquid chromatography from the MeOH extracts of a marine sponge, Haliclona (Reniera) sp., collected from the coast of Ulleung Island, Korea, and analyzed by fast atom bombardment mass spectrometry (FAB–MS) in positive‐ion mode. FAB‐mass spectra of these compounds included protonated molecules [M + H]⁺ and abundant sodiated molecules [M + Na]⁺ from a mixture of m‐NBA and NaI. The structures of these GlcCers, which were similar, were elucidated by FAB‐linked scan at constant B/E. To find diagnostic ions for their characterization, the GlcCers were analyzed by collision‐induced dissociation (CID) linked scan at constant B/E. The CID‐linked scan at constant B/E of [M + H]⁺ and [M + Na]⁺ precursor ions resulted in the formation of numerous characteristic product ions via a series of dissociative processes. The product ions formed by charge‐remote fragmentation provided important information for the characterization of the fatty N‐acyl chain moiety and the sphingoid base, commonly referred to as the long‐chain base. The product ions at m/z 203 and 502 were diagnostic for the presence of a sodiated sugar ring and β‐D ‐glucosylsphinganine, respectively. For further confirmation of the structure of the fatty N‐acyl chain moiety in each GlcCer, fatty acid methyl esters were obtained from the five GlcCers by methanolysis and analyzed by FAB–MS in positive‐ion mode. On the basis of these dissociation patterns, the structures of the five GlcCers from marine sponge were elucidated. In addition, the accurate mass measurement was performed to obtain the elemental composition of the GlcCers isolated from marine sponge. Copyright © 2009 John Wiley & Sons, Ltd.     

A lipidomic platform establishment for structural identification of skin ceramides with non-hydroxyacyl chains

The stratum corneum (SC) is the outermost layer of skin that functions as a barrier and protects against environmental influences and transepidermal water loss. Its unique morphology consists of keratin-enriched corneocytes embedded in a distinctive mixture of lipids containing mainly ceramides, free fatty acids, and cholesterol. Ceramides are sphingolipids consisting of sphingoid bases, which are linked to fatty acids by an amide bond. Typical sphingoid bases in the skin are composed of dihydrosphingosine (dS), sphingosine (S), phytosphingosine (P), and 6-hydroxysphingosine (H), and the fatty acid acyl chains are composed of non-hydroxy fatty acid (N), α-hydroxy fatty acid (A), ω-hydroxy fatty acid (O), and esterified ω-hydroxy fatty acid (E). The 16 ceramide classes include several combinations of sphingoid bases and fatty acid acyl chains. Among them, N-type ceramides are the most abundant in the SC. Mass spectrometry (MS)/MS analysis of N-type ceramides using chip-based direct infusion nanoelectrospray-ion trap mass spectrometry generated the characteristic fragmentation pattern of both acyl and sphingoid units, which could be applied to structural identification of ceramides. Based on the MS/MS fragmentation patterns of N-type ceramides, comprehensive fragmentation schemes were proposed. In addition, mass fragmentation patterns, which are specific to the sphingoid backbone of N-type ceramides, were found in higher m/z regions of tandem mass spectra. These characteristic and general fragmentation patterns were used to identify N-type ceramides in human SC. Based on established MS/MS fragmentation patterns of N-type ceramides, 52 ceramides (including different classes of NS, NdS, NP, and NH) were identified in human SC. The MS/MS fragmentation patterns of N-type ceramides were characterized by interpreting their product ion scan mass spectra. This information may be used to identify N-type ceramides in the SC of human, rat, and mouse skin.     

Identification of hydroxyl radical oxidation products of N‐hexanoyl‐homoserine lactone by reversed‐phase high‐performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A reversed‐phase high‐performance liquid chromatography/electrospray tandem mass spectrometry method was developed for the characterization of hydroxyl radical oxidation products of N‐hexanoyl‐homoserine lactone (C6‐HSL), a member of the N‐acylhomoserine lactone (AHL) class of microbial quorum‐sensing signaling molecules identified in many Gram‐negative strains of bacteria. Six products were identified: four with molecular weight (MW) of 213 and two with MW of 260. The characteristic product ions formed through collision‐induced dissociation (CID) provided diagnostic structural information. One of the photolysis products was determined to be N‐(3‐oxohexanoyl)homoserine lactone (3OC6‐HSL), a highly active quorum‐sensing signal, by comparison with a reference standard. Three structural isomers with the same mass as 3OC6‐HSL were identified as acyl side chain oxidized C6‐HSL (keto/enol functionalized) by accurate mass measurement and the structures of these products were proposed from CID spectral interpretation. Two structural isomers formed from concurrent oxidation and nitration of C6‐HSL were also observed and their structures were postulated based on CID spectra. In addition to the six hydroxyl radical oxidation products formed from the C6‐HSL precursor, five additional compounds generated from combined oxidation and lactonolysis of C6‐HSL were identified and structures were postulated. Copyright © 2009 John Wiley & Sons, Ltd.     

Electron‐induced dissociation (EID) for structure characterization of glycerophosphatidylcholine: determination of double‐bond positions and localization of acyl chains

Glycerophospholipids are a highly abundant and diverse collection of biologically relevant lipids, and distinction between isomeric and isobaric species is a fundamental aspect for confident identification. The ability to confidently assign a unique structure to a glycerophospholipid of interest is dependent on determining the number and location of the points of unsaturation and assignment of acyl chain position. The use of high‐energy electrons (>20 eV) to induce gas‐phase dissociation of intact precursor ions results in diagnostic product ions for localizing double‐bond positions and determining acyl chain assignment. We describe a high‐resolution, tandem mass spectrometry method for structure characterization of glycerophospholipids using electron‐induced dissociation (EID). Furthermore, the inclusion of nomenclature to systematically assign bond cleavage sites with acyl chain position and double‐bond location enables a uniform platform for lipid identification. The EID methodology detailed here combines novel application of an electron‐based dissociation technique with high‐resolution mass spectrometry that facilitates a new experimental approach for lipid biomarker discovery and validation. Copyright © 2015 John Wiley & Sons, Ltd.     

Identification of 1‐palmitoyl‐2‐linoleoyl‐phosphatidylethanolamine modifications under oxidative stress conditions by LC‐MS/MS

Phosphatidylethanolamines are a major class of phospholipids found in cellular membranes. Identification of the alterations in these phospholipids, induced by free radicals, could provide new tools for in vivo diagnosis of oxidative stress. In this study, 1‐palmitoyl‐2‐linoleoyl‐phosphatidylethanolamine oxidation products, induced by the hydroxyl radical, were studied using LC‐MS and LC‐MS/MS. Data obtained allowed the identification and separation of isomeric oxidative products with modifications in the sn‐2 acyl chain, attributed to long‐ and short‐chain products. Among long‐chain products keto, keto‐hydroxy, hydroxy, poly‐hydroxy, peroxy and hydroxy–peroxy derivatives were identified. Product ions formed by loss of two H₂O molecules vs loss of HOOH, allowed the identification of, respectively, di‐ (or poli‐) hydroxy vs peroxy derivatives. Location of functional groups was determined by the product ions formed by cleavage of C–C bonds, in the vicinity of the oxidation positions, allowing the identification of C9, C12 and C13 as the predominant substituted positions. Short‐chain products identified comprised aldehydes, hydroxy‐aldehydes and carboxylic derivatives, with modified sn‐2 acyl lengths of C7–C9 and C11, C12. Among the short‐chain products identified, C9 products showed higher relative abundance. Copyright © 2009 John Wiley & Sons, Ltd.     

Structural elucidation of diglycosyl diacylglycerol and monoglycosyl diacylglycerol from Streptococcus pneumoniae by multiple‐stage linear ion‐trap mass spectrometry with electrospray ionization

The cell wall of the pathogenic bacterium Streptococcus pneumoniae contains glucopyranosyl diacylglycerol (GlcDAG) and galactoglucopyranosyldiacylglycerol (GalGlcDAG). The specific GlcDAG consisting of vaccenic acid substituent at sn‐2 was recently identified as another glycolipid antigen family recognized by invariant natural killer T‐cells. Here, we describe a linear ion‐trap multiple‐stage (MSⁿ) mass spectrometric approach towards structural analysis of GalGlcDAG and GlcDAG. Structural information derived from MSⁿ (n = 2, 3) on the [M + Li]⁺ adduct ions desorbed by electrospray ionization affords identification of the fatty acid substituents, assignment of the fatty acyl groups on the glycerol backbone, as well as the location of double bond along the fatty acyl chain. The identification of the fatty acyl groups and determination of their regio‐specificity were confirmed by MSⁿ (n = 2, 3) on the [M + NH₄]⁺ ions. We establish the structures of GalGlcDAG and GlcDAG isolated from S. pneumoniae, in which the major species consists of a 16:1‐ or 18:1‐fatty acid substituent mainly at sn‐2, and the double bond of the fatty acid is located at ω‐7 (n‐7). More than one isomers were found for each mass in the family. This mass spectrometric approach provides a simple method to achieve structure identification of this important lipid family that would be very difficult to define using the traditional method. Copyright © 2012 John Wiley & Sons, Ltd.     

Effects of end groups on phase transition and segmental mobility of poly(N‐isopropylacrylamide) chains in D2O

Phase transition and mobility of poly(N‐isopropylacrylamide) (PNIPA) chains with three different types of end groups (hydroxyl, carbon–carbon double bond, and camphoric sulfonic groups) have been studied by measurements of the normal ¹H NMR spectrum, spin–spin relaxation time, and 2D NOESY spectrum. It is found that at room temperature not only the end group parts but also the part of the PNIPA chain with hydroxyl end group have higher mobility than corresponding parts of PNIPA with double bond and camphoric sulfonic end groups. The lower critical solution temperatures (LCST) of PNIPAs modified with hydrophilic hydroxyl and hydrophobic double bond end groups are inversely dependent and directly dependent on the molecular weight of polymer respectively, whereas the LCST of PNIPA with the camphoric sulfonic end group bearing both hydrophobic and hydrophilic structures is independent of the molecular weight. The double bond end groups collapse simultaneously with inner segments of the PNIPA chain, whereas the hydroxyl and camphoric sulfonic end groups still exhibit higher mobility and do not shrink tightly after heating‐induced collapsing of inner segments. It is suggested that the hydroxyl and camphoric sulfonic end groups locate on the surface of globules, but the double bond end groups are probably buried inside the globules. © 2011 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys, 2011     

Structural determination of glycopeptidolipids of Mycobacterium smegmatis by high‐resolution multiple‐stage linear ion‐trap mass spectrometry with electrospray ionization

Glycopeptidolipids (GPLs) are abundant in the cell walls of different species of mycobacteria and consist of tripeptide‐amino‐alcohol core of D‐Phe‐D‐allo‐Thr‐D‐Ala‐L‐alaninol linked to 3‐hydroxy or 3‐methoxy C_26–34 fatty acyl chain at the N‐terminal of D‐Phe via amide linkage, and a 6‐deoxytalose (6‐dTal) and an O‐methyl rhamnose residues, respectively, attach to D‐allo‐Thr and the terminal L‐alaninol. They are important cell‐surface antigens that are implicated in the pathogenesis of opportunistic mycobacteria belonging to the Mycobacterium avium complex. In this contribution, we described multiple‐stage linear ion trap in conjunction with high‐resolution mass spectrometry towards structural characterization of complex GPLs as [M + Na]⁺ ions isolated from Mycobacterium smegmatis, a fast‐growing and non‐pathogenic mycobacterial species. Following resonance excitation in an ion trap, MSⁿ spectra of the [M + Na]⁺ ions of GPLs contained mainly b and y series ions that readily determine the peptide sequence. Fragment ions from MSⁿ also afford locating the 6‐dTal and O‐methyl rhamnose residues linked to the D‐allo‐Thr and terminal L‐alaninol of the peptide core, respectively, as well as recognizing the modifications of the glycosides, including their acetylation and methylation states and the presence of succinyl group. The GPL families consisting of 3‐hydroxy fatty acyl and of 3‐methoxy fatty acyl substituents are readily distinguishable. The MS profiles of the GPLs from cells are dependant on the conditions they were grown, and several isobaric isomers were identified for many of the molecular species. These multiple‐stage mass spectrometric approaches give detailed structures of GPL in complex mixtures of which the isomeric structures are difficult to define using other analytical methods. Copyright © 2012 John Wiley & Sons, Ltd.     

Development of a tandem time‐of‐flight mass spectrometer with an electrospray ionization ion source

A new tandem time‐of‐flight mass spectrometer with an electrospray ionization ion source ‘ESI‐TOF/quadTOF’ was designed and constructed to achieve the desired aim of structural elucidation via high‐energy collision‐induced dissociation (CID), and the simultaneous detection of all fragment ions. The instrument consists of an orthogonal acceleration‐type ESI ion source, a linear TOF mass spectrometer, a collision cell, a quadratic‐field ion mirror and a microchannel plate detector. High‐energy CID spectra of doubly protonated angiotensin II and bradykinin were obtained. Several fragment ions such as a‐, d‐, v‐ and w‐type ions, characteristic of high‐energy CID, were clearly observed in these spectra. These high‐energy CID fragment ions enabled confirmation of the complete sequence, including leucine–isoleucine determinations. It was demonstrated that high‐energy CID of multiply protonated peptides could be achieved in the ESI‐TOF/quadTOF. Copyright © 2010 John Wiley & Sons, Ltd.     

Gas‐phase fragmentation reactions of protonated benzofuran‐ and dihydrobenzofuran‐type neolignans investigated by accurate‐mass electrospray ionization tandem mass spectrometry

We have investigated gas‐phase fragmentation reactions of protonated benzofuran neolignans (BNs) and dihydrobenzofuran neolignans (DBNs) by accurate‐mass electrospray ionization tandem and multiple‐stage (MSⁿ) mass spectrometry combined with thermochemical data estimated by Computational Chemistry. Most of the protonated compounds fragment into product ions B ([M + H–MeOH]⁺), C ([ B –MeOH]⁺), D ([ C –CO]⁺), and E ([ D –CO]⁺) upon collision‐induced dissociation (CID). However, we identified a series of diagnostic ions and associated them with specific structural features. In the case of compounds displaying an acetoxy group at C‐4, product ion C produces diagnostic ions K ([ C –C₂H₂O]⁺), L ([ K –CO]⁺), and P ([ L –CO]⁺). Formation of product ions H ([ D –H₂O]⁺) and M ([ H –CO]⁺) is associated with the hydroxyl group at C‐3 and C‐3′, whereas product ions N ([ D –MeOH]⁺) and O ([ N –MeOH]⁺) indicate a methoxyl group at the same positions. Finally, product ions F ([ A –C₂H₂O]⁺), Q ([ A –C₃H₆O₂]⁺), I ([ A –C₆H₆O]⁺), and J ([ I –MeOH]⁺) for DBNs and product ion G ([ B –C₂H₂O]⁺) for BNs diagnose a saturated bond between C‐7′ and C‐8′. We used these structure‐fragmentation relationships in combination with deuterium exchange experiments, MSⁿ data, and Computational Chemistry to elucidate the gas‐phase fragmentation pathways of these compounds. These results could help to elucidate DBN and BN metabolites in in vivo and in vitro studies on the basis of electrospray ionization ESI‐CID‐MS/MS data only.     

Localization of Fatty Acyl and Double Bond Positions in Phosphatidylcholines Using a Dual Stage CID Fragmentation Coupled with Ion Mobility Mass Spectrometry

A high content molecular fragmentation for the analysis of phosphatidylcholines (PC) was achieved utilizing a two-stage [trap (first generation fragmentation) and transfer (second generation fragmentation)] collision-induced dissociation (CID) in combination with travelling-wave ion mobility spectrometry (TWIMS). The novel aspects of this work reside in the fact that a TWIMS arrangement was used to obtain a high level structural information including location of fatty acyl substituents and double bonds for PCs in plasma, and the presence of alkali metal adduct ions such as [M?+?Li]⁺ was not required to obtain double bond positions. Elemental compositions for fragment ions were confirmed by accurate mass measurements. A very specific first generation fragment ion m/z 577 (M-phosphoryl choline) from the PC [16:0/18:1 (9Z)] was produced, which by further CID generated acylium ions containing either the fatty acyl 16:0 (C₁₅H₃₁CO⁺, m/z 239) or 18:1 (9Z) (C₁₇H₃₃CO⁺, m/z 265) substituent. Subsequent water loss from these acylium ions was key in producing hydrocarbon fragment ions mainly from the α-proximal position of the carbonyl group such as the hydrocarbon ion m/z 67 (+H₂C-HC?=?CH-CH?=?CH₂). Formation of these ions was of important significance for determining double bonds in the fatty acyl chains. In addition to this, and with the aid of ¹³C labeled lyso-phosphatidylcholine (LPC) 18:1 (9Z) in the ω-position (methyl) TAP fragmentation produced the ion at m/z 57. And was proven to be derived from the α-proximal (carboxylate) or distant ω-position (methyl) in the LPC.     

Multiple‐stage linear ion‐trap with high resolution mass spectrometry towards complete structural characterization of phosphatidylethanolamines containing cyclopropane fatty acyl chain in Leishmania infantum

The structures of phosphatidylethanolamine (PE) in Leishmania infantum are unique in that they consist of a rare cyclopropane fatty acid (CFA) containing PE subfamily, including CFA‐containing plasmalogen PE species. In this contribution, we applied multiple‐stage linear ion‐trap combined with high‐resolution mass spectrometry to define the structures of PEs that were desorbed as [M – H]^? and [M – H + 2Li]⁺ ions by ESI, respectively. The structural information arising from MSⁿ on both the molecular species are complimentary, permitting complete determination of PE structures, including the identities of the fatty acid substituents and their location on the glycerol backbone, more importantly, the positions of the double bond(s) and of the cyclopropane chain of the fatty acid chain, directing to the realization of the CFA biosynthesis pathways that were reported previously. We also uncovered the presence of a minor dimethyl‐PE subclass that has not been previously reported in L. infantum. This LIT MSⁿ mass spectrometric approach led to unambiguous identification of PE molecules including many isomers in complex mixture that would otherwise be very difficult to define using other analytical approaches. Copyright © 2014 John Wiley & Sons, Ltd.     

Ortho effects in organic molecules on electron impact part 22. Competing oxygen transfers from the nitro group to sulphur and the olefinic double bond in 2-nitrophenyl styryl sulphides

Double oxygen migration to sulphur from the ortho-nitro group leading to eliminations of SO₂ and ^·SO₂H from the molecular ions and single oxygen transfer to the olefinic double bond in the side-chain giving rise to the most abundant ion at m/z 138 have been observed in 2-nitrophenyl styryl sulphides on electron impact. The proposed fragmentation mechanisms and the product ion structures were confirmed with the aid of high-resolution data, B/E linked scan and CID spectra.     

Collision‐induced dissociation of 3‐keto anabolic steroids and related compounds after electrospray ionization. Considerations for structural elucidation

The collision‐induced dissociation of forty‐one 3‐keto anabolic steroids and related compounds has been studied using both triple quadrupole (QqQ) and hybrid quadrupole‐time of flight (QTOF) instruments. Due to the complexity of the product ion spectra of these analytes, which generate a large number of ions, only two specific regions were studied in depth: the product ions near the precursor ion (m/z ≥M–100) and the most abundant product ions at a collision energy of 30 eV. Accurate mass measurements were used in order to obtain an unequivocal assignment of the empirical formula and the origin of each selected product ion. Analytes have been divided into eight groups according to the number and position of double bonds and the presence of functional groups such as hydroxyl‐ or nitrogen‐containing rings. A correlation between the steroid structure and the product ions obtained has been postulated. The application of these correlations can be useful in the elucidation of feasible structures for unknown steroids and/or their metabolites. Copyright © 2008 John Wiley & Sons, Ltd.     

Investigation of collision‐induced dissociations involving odd‐electron ion formation under positive electrospray ionization conditions using accurate mass

Collision induced dissociation (CID) has been extensively used for structure elucidation. CID in the electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) modes has been found to generate mostly even‐electron fragment ions while it has been occasionally reported to form odd‐electron free radical ions. However, the structural requirements and the fragmentation mechanisms for free‐radical CIDs have not been well characterized in the literature. For this purpose, we studied a series of aromatic and non‐aromatic compounds such as sulfonamides, N‐aryl amides, tert‐butyl‐substituted aromatic compounds, aryl alkyl ethers, and O‐alkyl aryl oximes using the LTQ? and LTQ Orbitrap? linear ion trap mass spectrometers. The accurate measurement of the fragment ion masses established the unambiguous assignment of the fragment structures resulting from the test compounds. Our results showed that free radical fragmentation is structure dependent and is to a large extent correlated with the neighboring groups in the structures that stabilize the newly formed free radical ions. Copyright © 2010 John Wiley & Sons, Ltd.     

Sulfonamide bond cleavage in benzenesulfonamides and rearrangement of the resulting p‐aminophenylsulfonyl cations: application to a 2‐pyrimidinyloxybenzylaminobenzenesulfonamide herbicide

The gas‐phase fragmentation/rearrangement reactions of compound 1, [2‐(4,6‐dimethoxypyrimidin‐2‐yloxy)‐benzyl]‐[4‐(piperidine‐1‐sulfonyl)phenyl]amine, have been examined by Fourier transform ion cyclotron resonance mass spectrometry (FTICR‐MS). The analyses reveal that under sustained off‐resonance irradiation collision‐induced dissociation (SORI‐CID) conditions in the FTICR cell, protonated 1 undergoes two competitive pathways initiated by different protonation positions. The first pathway is initiated by protonation on the amino group and yields only one fragment ion due to loss of the entire benzenesulfonamide moiety. In the second pathway, protonation of the sulfonamide group leads to cleavage of a sulfonamide bond with loss of the neutral piperidine, followed by loss of SO via a sulfonyl cation rearrangement. An intramolecular S_NAr mechanism is proposed to rationalize the rearrangement of the p‐aminophenylsulfonyl cation and the resulting SO loss. To test the generality of this process, SORI‐CID spectra of protonated sulfamethoxazole and of the p‐aminophenylsulfonyl cation (SBN) were obtained. For the SBN ion, SORI‐CID experiments as well as density functional theory (B3LYP) calculations show that rearrangement, assigned as a S_NAr reaction of the sulfonyl cation group, can account for the observed SO loss process. Candidate transition state structures were optimized at the B3LYP/6‐31+G (d, p) level of theory using the Gaussian98 molecular modeling package. The computational results show that the barrier for SO loss from SBN is much lower than that for SO₂ loss, which satisfactorily rationalizes the SORI‐CID experimental results for SBN. Moreover, it is proposed that a fragment ion at m/z 196 in the MS/MS spectrum of protonated 1 is formed via the ion resulting from SO loss via a second intramolecular S_NAr mechanism. Copyright © 2005 John Wiley & Sons, Ltd.     

Sulfate migration in oligosaccharides induced by negative ion mode ion trap collision‐induced dissociation

Migration of sulfate groups between hydroxyl groups was identified after collision‐induced dissociation (CID) of sulfated oligosaccharides in an ion trap mass spectrometer in negative ion mode. Analysis of various sulfated oligosaccharides showed that this was a common phenomenon and was particularly prominent in sulfated oligosaccharides also containing sialic acid. It was also shown that the level of migration was increased when the sulfate was positioned on the flexible areas of the oligosaccharides not involved in the pyranose ring, such as the extra‐cyclic C‐6 carbon of hexoses or N‐acetylhexosamines, or on reduced oligosaccharide. This suggested that migration is dependent on the spatial availability of the sulfate in the ion trap during collision. It is proposed that the migration is initiated when the negatively charged ‐SO₃^– residue attached to the oligosaccharide precursor becomes protonated by a CID‐induced proton transfer. This is supported by the CID fragmentation of precursor ions depleted of acidic protons such as doubly charged [M – 2H]^2– ions or the sodiated [M + Na – 2H]^– ions of oligosaccharides containing one sulfate and one sialic acid in the same molecule. Compared to the CID fragmentation of their monocharged [M – H]^– ions, no migration was observed in CID of proton depleted precursors. Alternative fragmentation parameters to suppress migration of sulfated oligosaccharides also showed that it was not present when sulfated oligosaccharides were fragmented by HCD (High‐Energy C‐trap Dissociation) in an Orbitrap mass spectrometer. Copyright © 2011 John Wiley & Sons, Ltd.