High-energy collision-induced dissociation tandem mass spectrometry of regioisomeric lactose palmitic acid monoesters using matrix-assisted laser desorption/ionization

Research Article

Received: 26 August 2013 Revised: 6 October 2013 Accepted: 18 October 2013 Published online in Wiley Online Library

Rapid Commun. Mass Spectrom. 2014, 28, 169–177

High-energy collision-induced dissociation tandem massspectrometry of regioisomeric lactose palmitic acid monoestersusing matrix-assisted laser desorption/ionization

Mina R. Narouz1,2, Sameh E. Soliman3,4, Travis D. Fridgen1, Mina A. Nashed3

and Joseph H. Banoub1,5*1Department of Chemistry, Memorial University of Newfoundland, St. John’s, Newfoundland, A1B 3V6, Canada2Department of Chemistry, Faculty of Science, Damanhour University, Damanhour, Egypt3Department of Chemistry, Faculty of Science, Alexandria University, Ibrahimia, PO Box 426, Alexandria 21321, Egypt4NIDDK, LBC, National Institutes of Health, Bethesda, MD, 20892-0815, USA5Fisheries and Oceans Canada, Science Branch, Environmental Sciences Division, Special Projects, P.O. Box 5667, St John’s,Newfoundland, A1C 5X1, Canada

RATIONALE: Structural characterization and differentiation of three newly synthesized lactose monopalmitateregioisomers at positions O-3, O-3’ and O-6’ were realized by single-stage matrix-assisted laser desorption/ionizationtime-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) in the positive ion mode and by high-energycollision-induced dissociation tandem mass spectrometry (CID-MS/MS).METHODS: A MALDI-TOF/TOF analyzer was utilized for the analysis of these isobaric lactose monopalmitateregioisomers. The CID-MS/MS spectra were acquired using high-energy cid with a 2 kV potential difference betweenthe source acceleration voltage and the collision cell.RESULTS: High-energy (CID) tandem mass spectrometry (MS/MS) analyses of the sodiated molecules, [M+Na]+,showed distinguishing cross-ring product ions and characteristic fingerprint product ions, which allowed the straight-forward mass spectrometric characterization of these different regiosiomers.CONCLUSIONS: This investigation allowed us to unravel the novel fragmentation behavior of the sodiated regioisoimermolecules obtained from the mono-substituted D-lactose fatty acid esters using high-energy CID-TOF/TOF-MS/MSanalyses. The high-energy CID of the [M+Na]+ ions from the isobaric lactose monopalmitate regioiosmers promotedthe formation of characteristic 0,2A2 cross-ring cleavage product ions. Copyright © 2013 John Wiley & Sons, Ltd.

(wileyonlinelibrary.com) DOI: 10.1002/rcm.6770

Amphiphilic carbohydrate fatty acid ester molecules belongto the non-ionic surfactants class of compounds, and arecomposed of a carbohydrate moiety, which is considered asthe hydrophilic head, and fatty chainswhich form the lipophilictails. These compounds are completely biodegradable, non-toxic, non-irritating to the skin, odorless and tasteless, whichfacilitates their use in the food, cosmetic, and pharmaceuticalindustries.[1–3] Furthermore, these non-ionic surfactants possessa wide range of hydrophilic–hydrophobic balance (HLB),depending obviously on the nature of the sugar and the degreeof esterification with the fatty acids.[4] Carbohydrate fatty acidmonoesters possess better water solubility than the di-, tri-and higher ester derivatives.[5] On the other hand, it was foundthat the surfactant and antimicrobial properties of theregioisomeric sugar monoesters depend on the position of thefatty acyl group.[6–8]

* Correspondence to: J. H. Banoub, Fisheries and OceansCanada, Science Branch, Special Projects, P.O. Box 5667,St John’s, Newfoundland, A1C 5X1, Canada.E-mail: [email protected]

Rapid Commun. Mass Spectrom. 2014, 28, 169–177

16

Narouz et al. have recently reported the chemicalregioselective synthesis of lactose monopalmitate regioisomersat different O-3’, O-6’ and O-3 positions (Fig. 1) employingsmart protecting group strategy and the chemoselectivereactivity of cyclic tin alkoxides.[9] The structural characterizationof these well-defined regioisomers was accomplished using1H NMR spectra, 1H–1H COSY experiments, 13C-NMRspectra, 13C–1H correlation, and attached proton test (APT)experiments.[9]

It is well known that collision-induced dissociation tandemmass spectrometry (CID-MS/MS) of protonated disaccharidesand/or oligosaccharides is initiated by glycosidic bondcleavage between the sugar ring units. These cleavagesprovide outstanding information on the sequence of theglycosyl units.

Recently, it has been established that matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS) analyses of carbohydrates usually affordthe sodiated molecules, [M+Na]+. This process differs fromthe MALDI analysis of peptides in which the protonatedmolecules usually prevail.[10] Moreover, it was recognizedthat high-energy CID-MS/MS analysis of alkali metal ionadducts, such as the [M+Na]+ ions, enhances sugar

Copyright © 2013 John Wiley & Sons, Ltd.

9

Figure 1. Structures of lactose monopalmitate regioisomersat positions O-3, O-3’ and O-6’.

M. R. Narouz et al.

170

cross-ring fragmentations, which are very informative for thedetermination of linkage positions of the composingglycosyl units.[10–16]

Many investigators have explored the high-energyCID-MS/MS analyses of sodiated oligosaccharides. Amongthese, Tryfona et al. analyzed oligosaccharides released byenzymatic hydrolysis of the arabinogalactan carbohydrate,[11]

Maslen et al. analyzed arabinoxylan isomers,[12] Mechref et al.analyzed neutral and structural isomers of permethylatedsialylated glycans,[13,14] Spina et al. analyzed complexcarbohydrates from human milk[15] and, finally, Stephenset al. analyzed high-mannose and hybrid-type N-glycanspurified from avidin.[16] In all these examples, theauthors observed, in their respective analysis, the presenceof the structurally informative carbohydrate cross-ringcleavages.[10–16]

Nevertheless, it is interesting to mention that while manystudies using soft ionization mass spectrometric methods tocharacterize carbohydrate esters have been reported,[17–21]

very few have appeared on the mass spectrometriccharacterization of mono-substituted carbohydrate fatty acidesters. For that reason, Perez-Victoria and coworkers havecharacterized and differentiated the structures of severalregioisomeric monolaurate esters of the non-reducingtrisaccharides raffinose and melezitose, and the non-reducingtetrasaccharide stachyose, using electrospray ionization(ESI)-MS with ’in-source CID’ fragmentation of thecorresponding [M+Na]+ ions.[22] De Koster and coworkershave studied the fragmentation of [M+Na]+ ions formed byfast-atom bombardment (FAB) of sucrose monocaprate andsucrose monolaurate by low-energy collision-induceddissociation tandem mass spectrometry (CID-MS/MS).[23] Onthe other hand, Banoub and coworkers have also studiedregioisomeric fatty acid monoesters of sucrose by electrosprayionization tandem mass spectrometry, establishing thatlow-energy CID-MS/MS analyses of the [M+H]+ ions providecharacteristic fingerprint patterns, and permit differentiation ofthe various regioisomers.[24]

To date, no MALDI-TOF-MS and high-energy CID-MS/MSanalyses have been reported for the series of novelsynthesized (isobaric) non-ionic surfactants compounds.Herein, we describe the structural characterization anddifferentiation of a novel group of isobaric regioisomericmonopalmitate esters of the reducing D-lactose disaccharidesugar using MALDI-TOF-MS (+ve ion mode) and high-energy CID-MS/MS analyses. High-energy CID-MS/MSwas shown to be a valuable method for generating structuralinformation on the gas-phase fragmentation of the precursorions formed from these molecules, especially as the primaryMALDI-ionization did not impart enough internal energyfor spontaneous fragmentation to occur during conventionalsingle-stage mass spectrometry.

wileyonlinelibrary.com/journal/rcm Copyright © 2013 John Wi

EXPERIMENTAL

Materials and matrix

The lactose monopalimtates studied, 3-O-palmitoyl-D-lactose(1), 3’-O-palmitoyl-D-lactose (2) and 6’-O-palmitoyl-D-lactose(3), illustrated in Fig. 1, were prepared by the chemicalregioselective synthesis of the corresponding lactosederivatives as recently reported.[9] All chemicals were usedwithout further purification. Acetonitrile and trifluoroaceticacid (TFA) were from ACP (Montreal, Canada).2,5-Dihydroxybenzoic acid (DHB) matrix was from Sigma-Aldrich (St. Louis, MO, USA). Milli-Q water (Milli-Q plussystem, Millipore, Bedford, MA, USA), with a specificresistance of 18.2 M Ω cm–1, was used to prepare all solutions.

Sample preparation

The DHB matrix was used at a concentration of 10 mg/mLin a 1:1 mixture (v/v) of acetonitrile and 0.1% TFA/pureMilli-Q water. Sugars were used at a concentration of1 mg/mL in a 1:1 mixture (v/v) of acetonitrile and water.The samples were prepared by mixing equal volumes ofDHB solution and sugar solutions. 1 μL of this mixedsolution was spotted onto a MALDI plate. Samples were leftto crystallize and dry in desiccators before being loaded intothe MALDI-MS instrument.

Mass spectrometry

Amodel 4800 MALDI TOF/TOFmass spectrometer (AppliedBiosystems/MDS SCIEX, Foster City, CA, USA) was utilizedin this study. This instrument has a mass accuracy of 5 ppmand a mass resolution of 15000–25000 FWHM and isequipped with an Nd:YAG laser with 355 nm wavelengthof <500 ps pulse, 3 to 7 ns pulse width, and 200 Hz firing ratein both MS and MS/MS modes. All measurements weremade in automatic mode. In MS mode, 400 shots wereaccumulated and in MS/MS mode 1000 were accumulated.All spectra shown in this manuscript were obtained usingDHB as the matrix and air as the collision gas. The potentialdifference between the source acceleration voltage and thecollision cell was set as 2 kV. All experiments were performedin positive (+ve) ion mode. The internal calibration wasperformed in the range m/z 100–1000 using known pureleucine enkephalin with a monoisotopic protonated molecule[M+H]+ at m/z 556.2771.

RESULTS AND DISCUSSION

The conventional full scan MALDI mass spectra of theisobaric 3-O-palmitoyl-D-lactose (1), 3’-O-palmitoyl-D-lactose(2) and 6’-O-palmitoyl-D-lactose (3) were characterized by thepresence of abundant [M+Na]+ ions at m/z 603.3374,603.3301 and 603.3387, respectively (Table 1). The differencesbetween the calculated (C28H52O12Na+, mass 603.3351) andmeasured values of the [M+Na]+ ions for the regioisomers1, 2 and 3 were 3.81, –8.29 and 5.97 ppm, respectively(Table 1). In addition, the [M+K]+ ions were also observedwith much lower relative abundance at m/z 619.3090,619.3018 and 619.3104 for 1, 2 and 3, respectively. The[M+K]+ ions have a theoretical exact mass of 619.3090 and

ley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2014, 28, 169–177

Table 1. Different [M+K]+ and [M+Na]+ ions in MALDI-TOF/TOF-MS (+ve ion mode) of the different regioisomers

RegioisomerTheoretical massof [M+Na]+

Experimental massof [M+Na]+ (%)*

Error(ppm)

Theoretical massof [M+K]+

Experimental massof [M+K]+ (%)

Error(ppm)

3-O-palmitoyl-D-lactose (1) 603.3351 603.3374 (100) 3.81 619.3090 619.3090 (6) 0.003’-O-palmitoyl-D-lactose (2) 603.3351 603.3301 (100) �8.29 619.3090 619.3018 (40) �11.626’-O-palmitoyl-D-lactose (3) 603.3351 603.3387 (100) 5.97 619.3090 619.3104 (8) 2.26

MS/MS of regioisomeric lactose palmitic acid monoesters

the errors between theoretical and experimental masses for theregioisomers 1, 2 and 3 were 0.00, –11.62 and 2.26 ppm,respectively (Table 1). The mass differences between the[M+K]+ and [M+Na]+ ions could help to confirm the tentativeassignment of the molecular weight of the compounds. It isimportant to note that no protonated molecules [M+H]+ wereobserved in the MALDI-MS spectra of this series of syntheticregioisomers.High-energy CID-MS/MS experiments on the precursor

[M+Na]+ ions were undertaken and it was noted that theCID gas-phase fragmentations involved both glycosidic andcross-ring cleavages of the sodiated disaccharide molecules.The resulting product ions were very informative about theinitial molecular structures of the studied disaccharides. Inthis work we have also identified the formation of a uniquediagnostic product ion E, formed by a concerted six-atomcyclic rearrangement connecting the 1→ 4 linked disaccharideprecursor ions. This mechanism was originally described bySpina et al. as a new fragmentation in the MALDI-TOF/TOF-MS of carbohydrates (Scheme 1).[15]

It is important to note that the CID fragmentations of thisseries of non-ionic surfactant compounds were analogous tothat of the sodium-cationized lactose sugar in a quadrupoleion trap, as reported by Asam and Glish.[25] Furthermore, itis noteworthy that the secondary CID fragmentations of thisseries of product ions usually occurred by neutral losses ofmolecules in either a concerted or a consecutive fashion. Thefragmentation routes of these sugar derivatives have beenrationalized using the scheme for systematic nomenclaturefor carbohydrate fragmentation proposed by Domon andCostello.[26]

Furthermore, it is essential to report that the distinguishingpresence of the 0,2A2 cross-ring product ions, created from thedifferent isobaric regioisomer precursor ions, allowed us toconfirm the positions of the acyl groups which acylate thespecific different hydroxyl groups of the disaccharide unit.It is also interesting to mention that, in the case of theregioisomer disaccharide 1, the presence of the 0,2A2 productions was rationalized as follows: the precursor [M+Na]+

ion initially loses the C14H29CHCO ketene molecule to formthe [M+Na–C14H29CHCO]+ product ion. This latter production loses a molecule of glycolaldehyde C2H4O2 (60 Da) to

Scheme 1. E fragmentation mechanism observed inet al.[15]

Copyright © 2013 JoRapid Commun. Mass Spectrom. 2014, 28, 169–177

afford the secondary 0,2A2 product ion. This loss has beenrationalized by migration of the hydrogen atom of theanomeric hydroxyl group of the reducing disaccharide tothe oxygen atom of the C-5-O-C-1 sugar ring. Consequently,this results in the opening of the pyranose ring followed byneutral loss of the C2H4O2 molecule from C-1-C-2, to providethe 0,2A2 product ions (Scheme 2).

The high-energy CID-MS/MS of the [M+Na]+ ions from3-O-palmitoyl-D-lactose (1) (Fig. 2), 3’-O palmitoyl-D-lactose(2) (Fig. 3) and 6’-O-palmitoyl-D-lactose (3) (Fig. 4) suggestedthe formation of a series of product ions whose formation hasbeen tentatively presented in Schemes 3, 4 and 5, and inTable 2.

The CID-fragmentation pathways observed in theseMS/MS spectra were produced mainly from the glycosidiccleavages (B- and Y-types) associated with the neutral lossof molecules such as water (18 Da) or the neutral loss of thepalmitoyl group (C16:0) as a ketene (C14H29CHCO,C16H30O, 238 Da), cross-ring cleavages, E fragmentation andγ-cleavage at the fatty acid chain.

In the proposed CID-fragmentation routes of sodiated3-O-palmitoyl-D-lactose (1), the precursor ion at m/z 603.3936loseswater (18Da) to form the base peak [B2 +Na]+ product ionat m/z 585.4435 (Fig. 2 and Scheme 3). The [B2 +Na]+ ion thenloses the palmitoyl ketene molecule (C16H30O) to form thesecondary product ion [B2 +Na–C16H30O]+ at m/z 347.1619(30%). The formation of the sodiated anhydrogalactose ion,[B1+Na]+ at m/z 185.0884 (33%), occurred via the loss of the3-O-palmitoyl-D-glucose moiety. This ion subsequently lost ofa molecule of water (18 Da) to afford the product ion[B1+Na–H2O]+ at m/z 167.0846.

The [Y1+Na]+ product ion atm/z 441.3679 (20%)was formedvia the synchronized losses of an anhydrogalactose residue anda palmitoyl ketene molecule (C16H30O) to create the secondaryproduct ion [Y1 +Na–C16H30O]+ at m/z 203.1010 (30%).

The product ion at m/z 305.1439 (11%) was assignedas [M+Na–C16H30O/0,2A2]

+ which was produced by theconsecutive loss of the palmitoyl ketene molecule (C16H30O),followed by the 0,2A2 cross-ring cleavage which is unique anddiagnostic for 1→4 linked disaccharides.[15] For that reason, thisindicates that the palmitoyl chain was attached to the O-3 ofthe glucose reducing end moiety and it also suggests, as

1→ 4 linked carbohydrates as proposed by Spina

wileyonlinelibrary.com/journal/rcmhn Wiley & Sons, Ltd.

171

Scheme 2. Loss of the ketene molecule at O-3 prior to the occurrence of the 0,2A2 fragmentationconfirming that the presence of the proton at O-3 is necessary for the 0,2A2 mechanism.

Figure 2. MALDI-TOF/TOF-MS/MS of the [M+Na]+ ion atm/z 603.3936 of compound 1.

M. R. Narouz et al.

172

six-atom rearrangement mechanism within the precursor ioninvolving the atoms at positions C-4 and C-1’-C-2’.The [M+Na–C13H28]

+ product ion at m/z 419.2355 (9%)was created via the formation of a relatively stableα,β-unsaturated ester due to the γ-cleavage with respect tothe carbonyl group of the long-chain fatty acyl group, whichis unique for long-chain esters.[23]

In the proposed CID-MS/MS fragmentation routes ofsodiated 3’-O-palmitoyl-D-lactose (2) (Fig. 3 and Scheme 4),the precursor ion at m/z 603.4136 afforded the [B2 +Na]+

product ion at m/z 585.4619 (base peak) by loss of a

wileyonlinelibrary.com/journal/rcm Copyright © 2013 John Wi

water molecule (18 Da). Subsequently, the [B2 +Na]+ ioneliminated the palmitoyl ketene molecule (C16H30O) toform the secondary product ion [B2 +Na–C16H30O]+ atm/z 347.1904 (17%).

The sodiated 3-O-palmitoyl-anhydrogalactose product ion[B1+Na]+ atm/z 423.3725 (5%)was produced by loss of glucosemoiety from the [M+Na]+ precursor ion. This [B1+Na]+ ionthen loses a palmitoyl ketene molecule (C16H30O) to form thesecondary product ion [B1 +Na–C16H30O]+ at m/z 185.1049(8%). This latter ion then loses a water molecule toafford the secondary product ion [B1 +Na–C16H30O–H2O]+

at m/z 167.0751.The [Y1 +Na]+ product ion at m/z 203.1094 (47%) was

formed via the loss of 3-O-palmitoyl-anhydrogalactose fromthe [M+Na]+ precursor ion. The 0,2A2 cross-ring cleavageoccurred directly from the [M+Na]+ precursor ion to afford

ley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2014, 28, 169–177

Figure 3. MALDI-TOF/TOF-MS/MS of the [M+Na]+ ion atm/z 603.4136 of compound 2.

Figure 4. MALDI-TOF/TOF-MS/MSof the [M+Na]+ ion atm/z 603.3848 fromcompound 3.

MS/MS of regioisomeric lactose palmitic acid monoesters

17

the [0,2A2 +Na]+ ion at m/z 543.4318 (70%), indicating that thepalmitoyl group is attached to the galactose moiety. We havealso noticed the formation of the cross-ring product ion[1,5X1 +Na]+ at m/z 231.1234 (9%).Interestingly, the formation of the diagnostic E product

ion created by CID fragmentation was initiated by the lossof a palmitoyl ketene molecule (C16H30O) to afford the[E1 +Na-C16H30O]+ product ion at m/z 169.1008.

Copyright © 2013 JoRapid Commun. Mass Spectrom. 2014, 28, 169–177

Lastly, the formation of the [M+Na–C13H28]+ product

ion at m/z 419.2482 (21%) can be attributed to theelimination of a neutral (C13H28) molecule from theprecursor ion to form the sodiated α,β-unsaturated esterat m/z 419.2482.

In the CID-fragmentation routes of the precursor ion atm/z 603.3848 of sodiated 6’-O-palmitoyl-D-lactose (3), weobserved the formation of the product ion [B2 +Na]+ at

wileyonlinelibrary.com/journal/rcmhn Wiley & Sons, Ltd.

3

Scheme 3. Proposed fragmentation routes obtained during MALDI-TOF/TOF-MS/MS of the[M+Na]+ ion at m/z 603.3936 of compound 1.

Scheme 4. Proposed fragmentation routes obtained during MALDI-TOF/TOF-MS/MS of the[M+Na]+ ion at m/z 603.4136 of compound 2.

M. R. Narouz et al.

174

m/z 585.4570 (base peak). This latter ion lost a palmitoylketene (C16H30O) molecule to afford the product ion[B2+Na–C16H30O]+ atm/z 347.2003 (7%) (Fig. 4 and Scheme 5).The sodiated 6-O-palmitoyl-anhydrogalactose product ion

[B1+Na]+ at m/z 423.3652 (10%), which originated from thenon-reducing end of the disaccharide precursor ion, wasproduced by loss of the glucose moiety. This latter [B1+Na]+ ionsubsequently lost a palmitoyl ketenemolecule (C16H30O) to createthe secondary product ion [B1+Na–C16H30O]+ at m/z 185.1108.The product ion [Y1 +Na]+ ion at m/z 203.1052 (35%) was

formed by loss of the 6-O-palmitoyl-anhydrogalactose moietyfrom the [M+Na]+ precursor ion.

wileyonlinelibrary.com/journal/rcm Copyright © 2013 John Wi

As indicated previously for disaccharide 2, the 0,2A2 cross-ring cleavage was produced directly from the [M+Na]+

precursor ion, to afford the product ion [0,2A2 +Na]+ atm/z 543.4327 (56%). This indicates that in this instancethe specific attachment of the palmitoyl group was to thegalactose moiety.

Moreover, the cross-ring product ion [1,5X1 +Na]+ of the[M+Na]+ precursor ion was found at m/z 231.1277 (9%).Once more, we noted the presence of the E fragmentationfrom the precursor ion, following elimination of a palmitoylketene molecule (C16H30O) to afford the [E1 +Na–C16H30O]+

product ion at m/z 169.1150.

ley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2014, 28, 169–177

Scheme 5. Proposed fragmentation routes obtained during MALDI-TOF/TOF-MS/MS of the[M+Na]+ ion at m/z 603.3848 from compound 3.

Table 2. Characteristic product ions obtained during MALDI-TOF/TOF-MS/MS of the [M+Na]+ ions from compounds 1, 2and 3

Characteristic ions

3-O-palmitoyl- 3’-O-palmitoyl- 6’-O-palmitoyl-

D-lactose (1) D-lactose (2) D-lactose (3)

m/z % m/z % m/z %

[M+Na]+ 603.3936 21% 603.4136 10% 603.3848 19%[B2 +Na]+ 585.4435 100% 585.4619 100% 585.4570 100%[B2 +Na–C16H30O ]+ 347.1619 30% 347.1904 17% 347.2003 7%[B1 +Na]+ 185.0884 33% 423.3725 5% 423.36652 10%[B1 +Na–C16H30O ]+ * 185.1049 8% 185.1108 3%[B1 +Na–C16H30O–H2O]+ – 167.0751 6% –[B1 +Na–H2O]+ 167.0846 4% * *[Y1 +Na]+ 441.3679 20% 203.1094 47% 203.1052 35%[Y1 +Na–C16H30O ]+ 203.1010 30% * *[1,5X1 +Na]+ – 231.1234 9% 231.1277 9%[0,2A2 +Na]+ – 543.4318 70% 543.4327 56%[M+Na–C16H30O/0,2A2]

+ 305.1439 11% – –[E1 +Na]+ 169.1008 8% – –[E1 +Na-C16H30O]+ * 169.1008 6% 169.1150 5%[M+Na-C13H28]

+ γ-Cleavage 419.2355 9% 419.2482 21% 419.2478 21%

*The formation of this product ion is not possible from the structure of the precursor compound.– The product ion was not observed.

MS/MS of regioisomeric lactose palmitic acid monoesters

17

The [M+Na–C13H28]+ product ion at m/z 419.2478 (21%)

was formed from the precursor ion by elimination of a neutral(C13H28) molecule to form the sodiated α,β-unsaturated ester.In the following part, we will attempt to make

comparisons between the CID-MS/MS spectra of theprecursor sodiated molecules [M+Na]+ obtained from 1, 2and 3. The product ion scans of all the precursor ionsobtained from the three regioisomeric esters showed thatthe [B2 +Na]+ product ions were the base peaks. Obviously,

Copyright © 2013 JoRapid Commun. Mass Spectrom. 2014, 28, 169–177

these product ions were produced by loss of watermolecules from the C-1 anomeric carbon of the reducingend glucose units.

Likewise, we have noted that the most common glycosidiccleavages afforded the [B1 +Na]+ and [Y1 +Na]+ product ions,along with other characteristic cross-ring product ions, whichwere very useful to confirm the attachment of the acyl groupsto the glucose moiety or the galactose moiety. Consequently,in the case of compound 1, the presence of the product ion

wileyonlinelibrary.com/journal/rcmhn Wiley & Sons, Ltd.

5

M. R. Narouz et al.

176

[B1 +Na]+ at m/z 185.0884 indicated that the galactose moietywas free, having no acyl group, whereas the product ion at[Y1 +Na]+ at m/z 441.3679 indicated that the acyl group wasattached to the glucose moiety. In addition, in the case ofcompounds 2 and 3, the product ions [B1 +Na]+ were isobaricat m/z (423.3725, 423.36652) indicating that only the galactosemoiety carried the palmitoyl group, whereas the [Y1 +Na]+

product ions formed were also isobaric at m/z 203.1094,203.1052, representing the sodiated glucose moiety.The benefit of the highly energetic collision inside the CID

collision cell of theMALDI-TOF/TOF instrument is that it providesdistinguishing cross-ringproduct ions,which areprimordial for theconfirmation of the position of the acyl at the hexose unit.Therefore, we have discerned that the 0,2A2 and

1,5X1 productions are created from cross-ring fragmentations. The 0,2A2-typegas-phase fragmentationwas found to be highly discriminatingfor these three regioisomers as it confirmed the presence or theabsence of the acyl group at O-3 of the glucose moiety. Wepropose that, in compound 1, the 0,2A2 fragmentation occurredafter the [M+Na]+ ion lost the palmitoyl ketene molecule(C16H30O) at the O-3 position from the glucose moiety,affording the product ion [M+Na–C16H30O]+ with a freeOH-3, followed by 0,2A2 cleavage to afford the product ion[M+Na–C16H30O/0,2A2]

+ at m/z 305.1439. On the otherhand, this diagnostic [M+Na–C16H30O/0,2A2]

+ product ion atm/z 305.1439 was not observed in the CID spectra ofcompounds 2 and 3. Nevertheless, as mentioned previously,the product ion [0,2A2 +Na]+ was not formed from theCID-MS/MS of the sodiated molecule of the 3-O-palmitoyl-D-lactose 1 but was present in both sodiated 3’-O-palmitoyl-D-lactose (2) and sodiated 6’-O-palmitoyl-D-lactose (3).Definitely, the formation of the isobaric distinguishing[0,2A2 +Na]+ product ions obtained for compounds 2 and 3 atm/z (543.4318, 543.4327) with different relative intensities, 70%and 56%, respectively, may be useful to differentiate betweencompounds 2 and 3. It is important tomention that these resultswere shown to be reproducible by repeating the experimentsseveral times under the same conditions. This indicated thatthe OH-3 in the reducing end of the disaccharide precursorion was free and that the attachment of the palmitoyl groupwas on the galactose moiety. The relative intensity differencesbetween these isobaric products ions resulting from the 0,2A2

cleavage were attributed to the relatively high concentrationsof the precursor intermediates containing the free C-3-OHhydroxyl group.The cross-ring product ions 1,5X1 were not observed in the

MS/MS spectra of the sodiated palmitic acid ester 1, whereasfor both 2, 3 they were observed in relatively low intensity(9%). Moreover, the presence of the isobaric product ionsobtained from 1,5X1 fragmentation at m/z (231.1234, 231.1277)established that the acyl groups were attached either to theglucose moiety or to the galactose moiety. Nonetheless, theisobaric product ions at m/z (419.2355, 419.2482 and 419.2478)were highly differentiating for all the palmitoyl lactose esterspectra. These isobaric ions appeared in 1 with a 9% relativeintensity, in 2 with a 21% relative intensity, and in 3 with a21% relative intensity.Another interesting feature was the appearance of the

product ions formed by the γ-cleavage of the long-chain fattyacyl group of the precursor ions. This resulted in theformation of the distinguishing relatively stable sodiatedα,β-unsaturated ester ions.[23]

wileyonlinelibrary.com/journal/rcm Copyright © 2013 John Wi

Finally, the presence of the diagnostic CID fragmentation,typical for the E six-atom rearrangement, was specific forthe 1→ 4 linkage and, as expected, produced isobaric productions at m/z (169.1008, 169.1008 and 169.1150), whichoriginated from the galactose moiety. Moreover, we postulatethat the CID-fragmentation route of 1 involved the [E1 +Na]+

product ion, whereas in compounds 2 and 3 this ion wasformed from the [E1 +Na-C16H30O]+ product ion afterelimination of the palmitoyl ketene molecule (C16H30O).

CONCLUSIONS

In this MALDI-TOF/TOF-MS study, we have takenadvantage of the presence of the palmitoyl substituent inpositions O-3, O-3’ and O-6’ of the D-lactose disaccharide,as a reporter group to unequivocally differentiate betweenthese isobaric structural isomers.

The gas-phase fragmentation pathways of the precursorsodiated molecules obtained from the lactose monoesterdisaccharides were investigated by high-energy collision-induced dissociation tandem mass spectrometry. We haveobserved that CID-MS/MS analysis was highly advantageoussince it allowed for the determination of the 0,2A2 cross-ringfragmentations, whichwere characteristic for the three differentisobaric regioisomers.

Furthermore, the occurrence of the isobaric product ions, dueto the γ-cleavage with respect to the carbonyl group in the long-chain fatty acyl group, was also distinct for all the regioisomers.Another important observation was that the gas-phaseglycosidic cleavages allowed us to establish the differentpositions of the acyl groups on the disaccharide precursor ions.In addition, it also allowed us to observe the formation of theE product ions, which highlighted the presence of the 1→ 4linkage of the disaccharide sugar.

Finally, this investigation established a better understandingof the CID-fragmentation behavior of sodiated mono-substituted D-lactose fatty acid ester regioisomers.

REFERENCES

[1] I. Perez-Victoria, F. J. Perez-Victoria, S. Roldan-Vargas,R. Garcia-Hernandez, L. Carvalho, S. Castanys, F. Gamarro,J.C. Morales, J.M. Perez-Victoria. Non-reducing trisaccharidefatty acid monoesters: novel detergents in membranebiochemistry. Biochim. Biophys. Acta 2011, 1808, 717.

[2] M. Habulin, S. Sabeder, Z. Knez. Enzymatic synthesis ofsugar fatty acid esters in organic solvent and in supercriticalcarbon dioxide and their antimicrobial activity. J. Supercrit.Fluid. 2008, 45, 338.

[3] M. Ferrer, J. Soliveri, F. J. Plou, N. Lopez-Cortes,D. Reyes-Duarte, M. Christensen, J.L. Copa-Patino,A. Ballesteros. Synthesis of sugar esters in solvent mixturesby lipases from Thermomyces lanuginosus and Candidaantarctica B, and their antimicrobial properties. EnzymeMicrob. Technol. 2005, 36, 391.

[4] M. Ferrer, F. Comelles, F. J. Plou, M. A. Cruces, G. Fuentes,J. L. Parra, A. Ballesteros. Comparative surface activities ofdi- and trisaccharide fatty acid esters. Langmuir 2002, 18, 667.

[5] I. Perez-Victoria, A. Zafraa, J. C. Moralesa. Determinationof regioisomeric distribution in carbohydrate fatty acidmonoesters by LC–ESI-MS. Carbohydr. Res. 2007, 342, 236.

ley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2014, 28, 169–177

MS/MS of regioisomeric lactose palmitic acid monoesters

[6] P. Nobmann, A. Smith, J. Dunne, G. Henehan, P. Bourke.The antimicrobial efficacy. and structure activityrelationship of novel carbohydrate fatty acid derivativesagainst Listeria spp. and food spoilage microorganisms.Int. J. Food Microbiol. 2009, 128, 440.

[7] T. Polat, R. J. Lindhardt. Syntheses and applications ofsucrose-based esters. J. Surfactants Deterg. 2001, 4, 415.

[8] G. Garofalakis, B. S. Murria, D. B. Sarney. Surface activityand critical aggregation concentration of pure sugar esterswith different sugar headgroups. J. Colloid Interface Sci.2000, 229, 391.

[9] M. R. Narouz, S. E. Soliman, R. W. Bassily, R. I. El-Sokkary,A. Z. Nasr, M. A. Nashed. Regioselective synthesis of novelmono-substituted D-lactose fatty acid ester derivatives. Lett.Org. Chem. 2013, 10, 502. DOI: 10.2174/15701786113109990018.

[10] M. T. Cancilla, S. G. Penn, J. A. Carroll, C. B. Lebrilla.Coordination of alkali metals to oligosaccharides dictatesfragmentation behavior in matrix assisted laser desorptionionization/Fourier transform mass spectrometry. J. Am.Chem. Soc. 1996, 118, 6736.

[11] T. Tryfona, H.-C. Liang, T. Kotake, S. Kaneko, J. Marsh,H. Ichinose, A. Lovegrove, Y. Tsumuraya, P. R. Shewry,E. Stephens, P. Dupree. Carbohydrate structural analysisof wheat flour arabinogalactan protein. Carbohydr. Res.2010, 345, 2648.

[12] S. L. Maslen, F. Goubet, A. Adam, P. Dupreeb, E. Stephensa.Structure elucidation of arabinoxylan isomers by normalphase HPLC–MALDI-TOF/TOF-MS/MS. Carbohydr. Res.2007, 342, 724.

[13] Y. Mechref, P. Kang, M. V. Novotny. Differentiatingstructural isomers of sialylated glycans by matrix-assistedlaser desorption/ionization time-of-flight/time-of-flighttandem mass spectrometry. Rapid. Commun. Mass Spectrom.2006, 20, 1389.

[14] E. Stephens, S. L. Maslen, L. G. Green, D. H. Williams.Fragmentation characteristics of neutral N-linked glycansusing a MALDI-TOF/TOF tandemmass spectrometer. Anal.Chem. 2004, 76, 2343.

[15] E. Spina, L. Sturiale, D. Romeo, G. Impallomeni, D.Garozzo,D. Waidelich, M. Glueckmann. New fragmentationmechanisms in matrix-assisted laser desorption/ionizationtime-of-flight/time-of-flight tandem mass spectrometry ofcarbohydrates. Rapid Commun. Mass Spectrom. 2004, 18, 392.DOI: 10.1002/rcm.1350.

Copyright © 2013 JoRapid Commun. Mass Spectrom. 2014, 28, 169–177

[16] Y. Mechref, M. V. Novotny, C. Krishnan. Structuralcharacterization of oligosaccharides using MALDI-TOF/TOF tandem mass spectrometry. Anal. Chem. 2003, 75, 4895.

[17] E. Gaquerel, S. Heiling,M. Schoettner, G. Zurek, I. T. Baldwin.Development and validation of a liquid chromatography-electrospray ionization-time-of-flight mass spectrometrymethod for induced changes in Nicotiana attenuataleaves during simulated herbivory. J. Agric. Food Chem.2010, 58, 9418.

[18] M. R. M. Domingues, P. Domingues, A. Reis, A. J. F. Correia,J. P. CTome,A.C. Tome,M.G. P.M. S.Neves, J. A. S. Cavaleiro.Structural characterization of glycoporphyrins by electrospraytandem mass spectrometry. J. Mass Spectrom. 2004, 39, 158.

[19] A. Zamfir, D. G. Seidler, H. Kresse, J. Peter-Katalinic.Structural investigation of chondroitin/dermatan sulfateoligosaccharides from human skin fibroblast decorin.Glycobiology 2003, 13, 733.

[20] A. K. Ganguly, G. Chenb, B. N. Pramanik, I. Daarob, E. Lukb,P. L. Bartnerb, A. K. Saksenab, V. M. Girijavallabhanb.Negative ion multiple-stage mass spectrometric analysis ofcomplex oligosaccharides (everninomicins) in a quadrupoleion trap: implications for charge-remote fragmentation.ARKIVOC 2003, 3, 31.

[21] I. Jeric, C. Versluis, S. Horvat, A. J. R. Heck. Tracingglycoprotein structures: electron ionization tandem massspectrometric analysis of sugar–peptide adducts. J. MassSpectrom. 2002, 37, 803.

[22] I. Perez-Victoria, A. Zafra, J. C.Morales. Positive-ion ESI massspectrometry of regioisomeric nonreducing oligosaccharidefatty acid monoesters: In-source fragmentation of sodiumadducts. J. Mass Spectrom. 2008, 43, 633. DOI: 10.1002/jms.1363.

[23] C. G deKoster, A. M. Pajarron, W. Heerma, J. Haverkamp.Fast atom bombardment mass spectrometry of sucrosemonocaprate and sucrose monolaurate. Biol. Mass Spectrom.1993, 22, 277. DOI: 10.1002/bms.1200220503.

[24] C. Chauvin, P. Thibault, D. Plusquellec, J. Banoub.Differentiation of regioisomeric esters of sucrose by ionspraytandem mass spectrometry. J. Carbohydr. Chem. 1993, 12, 459.

[25] M. R. Asam, G. L. Glish. Tandem mass spectrometry of alkalicationized polysaccharides in a quadrupole ion trap. J. Am.Soc. Mass Spectrom. 1997, 8, 987. DOI: 10.1016/S1044-0305(97)00124-4.

[26] B. Domon, C. E. Costello. A systematic nomenclature forcarbohydrate fragmentations in FAB-MS/MS spectra ofglycoconjugates. Glycoconjugate J. 1988, 5, 397.

wileyonlinelibrary.com/journal/rcmhn Wiley & Sons, Ltd.

177