Introduction

Carbohydrates play an important part in numerous biological processes, such as molecular recognition and protein-protein interactions. Some carbohydrates are found free in solution (for example, human milk oligosaccharides) and others are attached to a lipid or peptide to form glycoconjugates. Changes in carbohydrate or glycosylation patterns can affect biological processes and thereby have a significant influence in different diseases and disease states.

The huge number of possible structures that can be built from different monosaccharides makes analysis of carbohydrates challenging. In addition to monosaccharide composition and sequence, variations in branching, linkages, and anomeric configurations must be elucidated to fully characterize a carbohydrate1.

In MS analysis of carbohydrates, low-energy CID fragmentation of protonated oligosaccharides leads to glycosidic bond cleavages (to produce B- and Y-ions), providing information about composition and sequence. To obtain further details of branching and linkages, cross-ring cleavages are mandatory. Different kinds of cross-ring fragments are generated by CID fragmentation of metal-adducted (positive ionization mode) or deprotonated

Authors

Kristina Marx, Andrea Kiehne und Markus MeyerBruker Daltonics, Bremen, Germany

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Application Note # LCMS-90

amaZon ion trap: An all-rounder for in-depth structure elucidation of carbohydrates

Keywords Instrumentation and Software

Glycans amazon ion trap series

oligosaccharides ProteinScape

MSn GlycoQuest

positive/ negative ion mode

Data processing and structural elucidation

Data were processed using DataAnalysis 4.2. After peak picking and charge deconvolution, MS/MS spectra were exported to ProteinScape 3.1. For carbohydrate identification, a glycan database search was performed against CarbBank using the integrated search engine GlycoQuest. Additional manual validation of MS and MSn spectra was carried out in DataAnalysis 4.2 using the Annotation Tool. Results

1. Comparison of signal intensities of protonated and metal-adducted precursor ions

It is well known that glycans and oligosaccharides can be stabilized by adduct formation with various ions. Harvey demonstrated this effect for released N -linked glycans in negative ionization mode (Harvey, 2005). In this study, the ionization efficiency of difucosyllacto-N -hexaose was investigated in positive and negative ionization mode using different additives, such as formic acid, ammonium bicarbonate (NH4HCO3), sodium acetate (NaOAc), and calcium chloride (CaCl2).

Figure 2 shows an overview of the MS spectra obtained in positive mode. Using pure water/methanol as solvent, (Figure 2A) mainly sodium and potassium adducts are detected in the form of singly or doubly charged ions.

precursor ions (negative ionization mode). Subsequent MSn experiments can support the verification of the proposed structure.

This study describes the analysis of difucosyllacto-N-hexaose — a typical branched milk sugar (see Figure 1) — using the amazon ion trap MS system, which enables fast polarity switching and MSn experiments for structural elucidation.

Experimental

Sample preparation

A 1 nmol/µL dilution of difucosyllacto-N -hexaose (Carbosynth, UK) was prepared in water. This stock solution was further diluted to a final concentration of 100 pmol/µl using different additives (see Table 1).

Mass spectrometric analysis

The diluted samples were introduced using a syringe pump (2.5 µL/min) into an amaZon ion trap mass spectrometer (Bruker Daltonics) using electrospray ionization (ESI).The default application method “tuning mix.m” was selected as starting point for mass spectrometric analysis. MS settings were as follows: capillary voltage 4500 V, end plate off-set 500 V, nebulizer 0.5 bar, dry gas temperature 200 °C at a flow rate of 3 L/min. Additional acquisition parameters are shown in Table 2 and Table 3.

Difucosyllacto-N-hexaose

α-antenna

β-antennaChemical Formula: C52H88N2O39Exact Mass: 1364.496

Fuc

Fuc

Gal

Gal

GlcNAc

GlcNAc

Gal Glc

OOH

OH

OH

CH2OH

O

O

NHAc

CH2OH

O

OH

OH

OH

H3C

O

O

OOH

OH

CH2

O

O

NHAc

CH2OH

O

O

OH

OH

OH

H3C

OOOH

OH

OH

CH2OH

O

O

OH

OH

CH2OH

OH

Figure 1: Difucosyllacto-N-hexaose illustrated in Haworth projection (left, Gal=Galactose, Fuc= Fucose, GlcNAc=N-Acetylglucosamine, Glc=Glucose) and with glycan symbols (right); nomenclature from Consortium for Functional Glycomics (CFG).

The addition of formic acid induces the generation of protonated ions, but also leads to the formation of additional adducts (Figure 2B). The same is true for the addition of NH4HCO3, with ammonium adducts being the most abundant ions (Figure 2C). A reduction in the complexity of the spectrum can be achieved by adding alkaline or alkaline earth metal salts. Figure 2D and 2E show the formation of sodium and calcium adducts: other species are virtually absent. This leads to improved sensitivity, because the signal intensity is not distributed over multiple adducts, such as protonated ions.

To enhance signal intensities in negative ionization mode, formic acid or NH4HCO3 can be added. Figure 3 shows the respective MS spectra. The extent of adduct formation with formic acid or neutral exchange with sodium can be altered by adjustment of acid and/or buffer concentrations.

2. CID fragmentation in positive ion mode

A. Protonated precursor ions

Low-energy activation applied to protonated species results predominantly in glycosidic bond cleavages (abundant B- and Y-ion series). These patterns can be used for the elucidation of oligosaccharide composition and sequence (Figure 4A). For automated identification of the structure, the spectrum was transferred to ProteinScape and searched against the CarbBank database using the integrated GlycoQuest serach engine. Figure 5 gives an overview over the result. The identified oligosaccharide composition and structure as well as a list of the detected B- and Y-fragments are shown. The corresponding annotated MS/MS spectrum is also shown. GlycoQuest supports the identification of protonated as well as metal-adducted oligosaccharides and calculation of cross-ring fragments.

B. Metal-adducted precursor ions

For further structural characterization, such as detailed branching analysis, cross-ring cleavages are mandatory. This can be achieved by introducing alkaline or alkaline earth metal ions. Alkaline metal adducts can undergo three different CID reactions: First, loss of a cation from

No additive 0.05% Formic acid 10 mM NH4HCO3 300 µM Na Acetate 300 µM CaCl2

Difucosyllacto-N-hexaose(Stock solution: 1 nmol/µL)

5 µL 5 µL 5 µL 5 µL 5 µL

H2O 25 µL 23 µL 15 µL 10 µL 10 µL

Methanol 20 µL 20 µL 20 µL 20 µL 20 µL

Additive -2 µL HCOOH (1%) 10 µL NH4HCO3

(50 mM)15 µL CH3COONa (1 mM)

15 µL CaCl2 (1 mM)

Table 1: Dilutions of Difucosyllacto-N-hexaose using different additives.

Acquisition parameters

Ionization ESI positive and negative

Scan mode Enhanced Resolution mode (8,100 m/z s-1) or UltraScan (32,500 m/z s-1)

Scan range 100-1,600 m/z

ICC target Positive mode: 200,000-300,000Negative mode: 70,000

Spectra averages 10

Rolling Averaging 2

Tuning Target Mass adjusted according to MSn precusor

Isolation width 2.5-4.0 m/z

CID Fragmentation Cut-off: Default (27%)SmartFrag Enhanced Amplitude Ramping 80-120%

Table 2: General MS and MS/MS parameters.

Precursor [m/z]

Precursor Ion

MSn stage Fragmentation Amplitude

683.31 [M+2H]2+ MS2 45%

1387.59 [M+Na]+ MS2 95%

702.22 [M+Ca]2+ MS2 50%

1363.63 [M-H]– MS2 80%

528.05 [M-H]– MS3 40%

600.19 [M-H]– MS3 60%

672.27 [M-H]– MS3 70%

Table 3: Detailed MS/MS settings.

Ionization of Difucosyllacto-N-hexaose using different additives in positive ionization mode

705.282+

876.30 1241.471+

1387.541+

702.302+

786.252+

1365.461+

691.762+

927.96

1382.661+

705.322+

1241.511+

1387.601+

702.232+

2

4

6x10Intens.

2

4

66x10

2

4

6x10

0.5

1.0

1.5

7x10

0

2

4

6

7x10

600 700 800 900 1000 1100 1200 1300 m/z

+ Formic acid

+ NaOAc

+ CaCl2

A

B

C

D

[M+Na] +

[M+NH3] +

[M+2Na]2 +

[M+Ca]2 +E

[M+H] +

[M+Na] +

+ NH4HCO3

No additive

694.272+

705.282+

713.232+

674.28

683.312+

694.30

702.302+

713.772+

683.282+

691.762+

700.292+

713.252+

1

2

3

4

5

6x10Intens.

1

2

3

6x10

0

1

2

3

4

5

66x10

680 690 700 710 720 m/z

[M+Na+H]2+

[M+2Na]2+

[M+K+H]2+

[M+Na+K]2+

[M+2H]2+

[M+K+H]2+ [M+Na+K]2+

[M+2H]2+

[M+NH4+H]2+

[M+2NH4]2+

Zoom:No additive

Zoom:Formic Acid

Zoom:NH4HCO3

Figure 2: Left: Comparison of MS full scan spectra in positive ionization mode using different solvent additives (spectra averaged over 1 min). Right: Zoom into spectrum A, B, and C. Target mass for tuning m/z 700.

Ionization of Difucosyllacto-N-hexaose using different additives in negative ionization mode

1217.551–

1363.641–

681.282–

1217.581–

1363.681–

0.25

0.50

0.75

1.00

1.256x10

Intens.

0

1

2

3

4

5x10

700 800 900 1000 1100 1200 1300 1400 m/z

+ Formic acid A

B

[M-H]–

[M-2H]2– + NH4HCO3[M-H] –

[M+Na-3H]2–

[M+HCOOH-H] –

Figure 3: Comparison of full scan MS spectra in negative ionization mode using different solvent additives (spectra averaged over 1 min) . Target mass for tuning m/z 700.

Effect of the nanoBooster

multiply charged precursors; second, glycosidic bond cleavage and third, cross-ring cleavage. Figure 4B shows the fragmentation of the singly sodiated precursor ion [M+Na]+=1387.59 m/z. The prominent fragments arise from glycosidic bond cleavages whereas only minor cross-ring fragments are observed (for example, –60 Da / –120 Da). Typical cleavage sites are illustrated in Figure 6 using the nomenclature according to Domon and Costello (Domon & Costello, 1988).

Table 4 summarizes abundant and characteristic fragments observed after CID fragmentation of the sodiated precursor. The major fragments m/z 1241.48 and 1095.43 originate from the cleavage of the two very labile fucose units. The fragment Y3X’’ (–146 Da) indicates the loss of one fucose, either from the α- or β-antenna whereas Y3α’’/Y3β’’ represents the loss of both fucose units (–292 Da). Other Y fragments can be explained by losses of hexose (–162 Da) and N -acetylhexosamine (203 Da). The loss of a complete antenna is also observed in the Y2 fragment detected at m/z 876.34. Cross-ring fragments are formed by cleavage of the

sugar at the reducing end, for example m/z 1327.48 (0,2A4, –60 Da) and m/z 1181.45 (0,2A4/Y3X‘‘). Further interpretations can be found in the study by Penn et al. (Penn, Cancilla, & Lebrilla, 1996).

Alkaline earth metal ions can also be used to induce cross-ring cleavages. However, interpretation of these fragment spectra is more difficult due to the presence of bivalent cations (see Figure 4C).

A comprehensive overview of the differences in CID fragmentation when using protonated or metal-adducted precursor ions can be found in Joseph Zaia’s review (Zaia, 2003).

3. CID fragmentation in negative ion mode

CID fragmentation in negative ion mode represents a complementary technique for structure elucidation of oligosaccharides. It yields sequence and branching sites as well as linkage information. Unlike sodiated ions in positive mode, the fucose residues of [M-H]– ions show a relatively high level of stability and therefore, a determination of their

CID fragmentation of Difucosyllacto-N-hexaose in positive ionization mode

Figure 4: CID spectra in positive ion mode. A) Fragmentation of protonated precursor (+ formic acid).B) Fragmentation of sodium adduct (+NaOAc). For reasons of clarity all fragments are calculated as singly charged ions. C) Fragmentation of calcium adduct (+CaCl2).

GlycoQuest search result

Figure 5: GlycoQuest search result for the doubly charged protonated precursor at m/z 683.31.

Fragmentation scheme of sodiated Difucosyllacto-N-hexaose

Figure 6: Positive ion mode CID fragmentation scheme of the sodiated precursor of difucosyllacto-N-hexaose (m/z 1387.59). Gal=Galactose, Fuc= Fucose, GlcNAc=N-Acetylglucosamine, Glc=Glucose.

Project Navigator

Identifi ed Oligosaccharide

Identifi ed Fragments

Annotated MS/MS SpectrumIdentifi ed Structure

Fragment Structure View

m/z Fragment

1327.48 0,2A4

1241.48 Y3x‘‘

1225.49 Y3X‘ (1-3 or 1-4)

1181.45 0,2A4/Y3X‘‘

1121.44 Y3X‘‘/2,4A4

1095.43 Y3α‘‘/Y3β‘‘

1079.43 Y3X‘/Y3X‘‘

1061.42 Y3X‘/Y3X‘‘/-H2O

1035.42 Y3α‘‘/Y3β‘‘/0,2A4

975.38 Y3α‘‘/Y3β‘‘/2,4A4

933.38 Y3α‘‘/Y3β‘‘/Y3X’

915.36 Y3α‘‘/Y3β‘‘/Z3X’

876.34 Y2

730.28 Y2/Y3X‘‘

Table 4: Assignment of difucosyllacto-N-hexaose fragments observed after CID fragmentation of the sodiated precursor at m/z 1387.59 (see also Figure 4B).

CID fragmentation of Difucosyllacto-N-hexaose in negative ionization mode

600.191-

672.271-

1201.511-

1243.561-

1285.581-

6078

120HexHex

72

144

364.08

529

547

0.0

0.5

1.0

1.5

5x10Intens.

400 500 600 700 800 900 1000 1100 1200 1300 m/z

363.961-

600.08

0

1000

2000

3000

Intens.

200 300 400 500 600 m/z

345.971-

654.141-

1-178.79

363.961-

528.01

0

500

1000

1500

2000

2500

Intens.

200 300 400 500 m/z

1-

1-345.97

1-

363.961-

0

200

400

600

800

Intens.

200 300 400 m/z

178.791-

347.921-

528.051-

1-

A

B C D

Figure 7: CID MSn spectra in negative ionization mode: A) MS/MS of the deprotonated precursor.B), C) and D) MS3 fragmentation of m/z 528.05, 600.19 and 672.27. Sample preparation with formic acid.

MS/MS: [M-H]- = 1363.63 m/z

MS3: [M-H]- = 528.05 m/z

MS3: [M-H]- = 600.19 m/z

MS3: [M-H]- = 672.27 m/z

position is possible. Figure 7 shows the CID MSn spectra of the deprotonated precursor with m/z 1363.63. Main fragments are C- and A-type ions as well as internal D-ions (Figure 8). Table 5 summarizes the observed fragments and their assignment according to the proposition made by Pfenninger et al. (Pfenninger, Karas, Finke, & Stahl, 2002) and Chai et al (Chai, Lawson, & Piskarev, 2002).The presence of a 1,4-linked glucose at the reducing end is confirmed by the unique cross-ring 0,2A4 doublet (m/z 1303.50 and 1285.58), corresponding to a mass difference of –60 and –78 Da respectively, and the additional cross-ring fragment 2,4A4 at m/z 1243.56 (Δm = –120 Da).The branching point can be identified by the loss of 529 Da, indicating the loss of one N -acetyllactosamine and one fucose. Such a large mass difference is only observed in branched structures and represents the loss of a complete antenna. The successive diagnostic fragmentation pattern Δm = –144 Da/–72 Da indicates the presence of an anhydro hexose. The fragment m/z 528.13 (C2α or C2β) derives from the cleavage of anhydro hexose, which is typically found at branching sites, and confirms the bi-antennary structure. The second signal at m/z 600.19 corresponds to the cross-ring fragment 0,3A3 that is characteristic for a 1,6-linked anhydro galactose. The linkage of the fucose at position 3 of N -acetylglucosamine (α-antenna) can be deduced from the fragment D1α-2α at m/z 364.13.

4. CID MSn fragmentation in negative ion mode

MSn analyses were used for the final verification of selected linkages. An example is shown in Figure 7B – 7D. For the unambiguous confirmation of the fucose linkages, MS3 experiments were performed on D2β-3 (m/z 672.27), 0,3A3 (m/z 600.19), and C2α or C2β (m/z 528.12). The first two MS3 precursors have already lost the β-antenna and therefore reflect the fucose linkage at the α-antenna. The corresponding MS3 spectra show the diagnostic fragment D1α-2α at m/z 363.96 (Figure 7C and 7D) which contains a 1,3-linked fucose (compare with Table 5). The precursor at m/z 528.05 is presumed to be a mixture of C2α and C2β fragments. In this case both, the diagnostic D1α-2α fragment representing the 1,3-linked fucose at the α-antenna as well as the diagnostic D1β-2β fragment at m/z 347.92 indicating the 1,4-linked fucose at the β-antenna are observed (Figure 7B).Further examples of structural elucidation of O -glycans by MS3 can be found in the application note LCMS-84.

Fragmentation scheme of deprotonated Difucosyllacto-N-hexaose

Figure 8: Negative ion mode CID fragmentation scheme of the [M-H]- of difucosyllacto-N-hexaose (m/z 1363.63). Gal=Galactose, Fuc= Fucose, GlcNAc=N-Acetylglucosamine, Glc=Glucose.

Conclusion

Detailed characterization of oligosaccharides and glycans requires different MS techniques for the unambiguous identification of composition, sequence, branching, and linkages. Ion traps are extremely versatile MS instruments that offer several advantages for structure elucidation, including fast polarity switching and MSn capabilities. The latter is of particular interest for linkage analysis, where selected fragments can undergo subsequent MS3 analysis for confirmation of the linkage positions.Data processing is supported by the GlycoQuest search engine, which enables structure identification using a variety of databases and is especially useful when working with complex oligosaccharide mixtures. All data can be submitted at the same time to an automated search, leading to an enormous time saving for data processing. An alternative fragmentation technique for the structural elucidation of oligosaccharides and glycans is Electron Transfer Dissociation (ETD) in combination with multivalent cations, for example Mg2+ (Han & Costello, 2011). This approach is currently under investigation using the amaZon speed ETD.

Table 5: Structural assignment of difucosyllacto-N-hexaose fragments observed after CID fragmentation of the deprotonated precursor at m/z 1363.63 (see also Figure 7A).

m/z Fragment

1303.50 0,2A4

1285.58 0,2A4-H2O

1243.56 2,4A4

1201.51 C3

672.27 D2β-3

654.25 D2β-3-H2O

600.19 0,3A3

528.12 C2α or C2β

364.13 D1α-2α

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