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Home arrow Health arrow Analysis of Protein Post-Translational Modifications by Mass Spectrometry
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[M-H]- and [M+Anion]- Ions

CID spectra of negative ions from neutral N-glycans tend to be simpler than the corresponding positive ion spectra, but they carry considerably more specific structural information [254, 304-306] (Figure 3.7c). Negative ion spectra are associated with [M-H]- ions, and indeed these are formed from the glycans but tend to be rather prone to fragmentation in ESI ion sources. In order to produce stable ions, the glycans can be adducted with anions such as chloride, bromide, iodide, phosphate, sulfate, or nitrate by addition of the appropriate ammonium salt to the electrospray solvent. Normally, samples derived from biological sources naturally contain phosphate and some chloride anions, but in order to avoid splitting the signal, it is advantageous to convert all gly- cans to their phosphate adducts. These adducts, together with the chloride and

(a) CID spectrum of the [M+H]+ ion from the high-mannose N-linked glycan, Man5GlcNAc2

Figure 3.7 (a) CID spectrum of the [M+H]+ ion from the high-mannose N-linked glycan, Man5GlcNAc2. (b) CID spectrum of the [M+Na]+ ion from Ma^GlcNAc2. Ions marked with a star are cross-ring products. (c) CID spectrum of the [M-H]- ion from MansGlcNAc2.

nitrate adducts, all fragment similarly [238, 250, 254, 307-309] by first eliminating the anion with a proton to give what is essentially the [M-H]- ion. Bromide adducts fragment poorly, but sulfate and iodide adducts do not fragment at all. Doubly charged negative ions of the type [M+(H2PO4)2]2- from neutral carbohydrates behave similarly [310].

It is the abstraction of a proton from different hydroxyl groups that is responsible for the diagnostic fragments, most of which are A-type cross-ring fragmentation products. Glycosidic fragments are also formed but often tend to be less abundant. In Q-TOF-type instruments, these tend to be mainly of the C rather than the B or Y type [311-314], whereas, more B- and Y-type cleavages appear to be produced in TOF/TOF instruments [297]. These fragment ions are very diagnostic of specific structural features such as the branching pattern of the glycan, the location of fucose residues, and the presence of a bisecting GlcNAc residue (Figure 3.8), which are difficult to determine by techniques such as exoglycosidase digestion.

One of the most prominent fragments in these spectra is a 2,4AR cleavage of the reducing-terminal GlcNAc residue (m/z 1154 in Figure 3.8 as an example), which can be used to indicate the presence or absence of 6-linked fucose on the core GlcNAc because fucose is eliminated in the neutral fragment (Figure 3.8c). The proposed mechanism is initiated by abstraction of the proton from the OH group in the 3-position. If a fucose residue occupies this position, the 2,4AR

ion is missing. This ion is also absent from the spectra of glycans derivatized at the reducing terminus by reductive amination, because of the open nature of the GlcNAc ring. Abstraction of the corresponding proton from the 3-position of the penultimate GlcNAc residue leads to the production of the 2,4AR-1 ion, and cleavage between the two GlcNAc residues produces a prominent BR-1 ion.

These three ions are typical of all N-linked glycans with a trimannosyl-chitobi- ose core and confirm the pi ^ 4 linkage between them.

Two of the most useful ions are named D and D-18 and are formed by loss of the chitobiose core and 3-antenna, followed, in the case of the D-18 ion, by loss of water. These ions specify the composition of the 6-antenna and are usually accompanied by 0,3A and 0,4A cross-ring fragments of the core mannose residue (see Table 3.4 for masses).

When a bisecting GlcNAc residue is present, the D ion, which contains the bisecting GlcNAc residue, eliminates this GlcNAc as a neutral molecule (221 u) to give what is usually a very abundant ion (Figure 3.8c). Antenna composition of complex glycans is revealed by a cross-ring cleavage of the outer mannose residues to give an ion consisting of the antenna plus -O-CH=CH-O- (59 u), for example, Gal-GlcNAc-O-CH=CH-O- (m/z 424), and, consequently, the presence of substituents such as fucose or a-galactose can easily be spotted. Common fragments of this type are listed in Table 3.4. The monosaccharide residue terminating the chain is revealed by the mass of the C1 fragment as listed in Table 3.4. Another useful feature of these negative ion spectra is their ability to distinguish the branching pattern of triantennary glycans [315]. In glycans containing branching of the 3-antenna (with Gal-GlcNAc groups), a 0,4A cleavage of the mannose residue gives a prominent fragment containing both the 2- and 4-linked chains (m/z 831). The triantennary glycan branched on the 6-antenna contains its branches at the 2- and 6-positions; consequently, this ion is missing. The structures of these glycans can be confirmed by prominent D, D-18, and D-36 ions. An additional advantage of negative ion fragmentation is that isomeric compounds yield ions with differing m/z values that are much more useful than the differences in abundance of the same mass ion that frequently characterize isomers in positive ion spectra [256].

Negative ion MS/MS spectra of selected N-glycans

Figure 3.8 Negative ion MS/MS spectra of selected N-glycans. (a) The position of the core fucose is defined by the masses of the 2,4A4 and 2,4A5 ions. The C ion at m/z 220 indicates GlcNAc at the ends of the antennae, and the D and [D-18]- ions at m/z 526 and 508, respectively, show that the 6-antenna contains only mannose and GlcNAc. (b) The masses of the 2,4A6 and 2,4A5 ions show the absence of fucose at the core. The C, ion at m/z 179 shows hexose (mannose) at the nonreducing termini, and the D, D-18, 0,3A3 ions show the presence of the four mannose residues on the 6-antenna. Furthermore, the D' ion at m/z 585 shows two of these mannose residues on the 6-branch. (c) The C, ion at m/z 179 in this and spectra d-f indicates hexose (galactose) at the ends of the antennae, and the D and [D-18]- ions at m/z 688 and 670, respectively, show that the 6-antenna contains Gal-GlcNAc. The 0,3A3 ion at m/z 424 is a cross-ring ion (Gal-GlcNAc-O-CH=CH-O-). (d) The presence of the bisecting GlcNAc residue produces the abundant [M-221]- ion at m/z 670. (e) The branched 3-antenna gives rise to the abundant E ion at m/z 831. The D and [D-18]- ions remain at m/z 688 and 670, respectively. (f) The branching pattern is revealed by the absence of the E ion at m/z 831 and the presence of D and [D-18]- ions at m/z 1053 and 1035. A third diagnostic ion is present at m/z 1017 ([D-36]-) [315].

Table 3.4 Ions defining structural features in the negative ion spectra of N-linked glycans.

Structural feature

Ion

Ionic composition

m/z

Antenna sequence

C

Gal, Man, Glc

179

GlcNAc, GalNAc

220

[Fuc]Gal

325

Gal-[Fuc]GlcNAc

528

aGal-Gal,

Man-Man

341

Man-[Man]Man

503

GalNAc-GlcNAc

423

Antenna composition

F

Man

262

GlcNAc

303

Gal-GlcNAc

424

Gal-[Fuc]GlcNAc

570

aGal-Gal

586

GalNAc-GlcNAc

627

(Gal-GlcNAc)2

789

(Gal-GlcNAc)2Fuc

935

(Gal-GlcNAc)3

1154

Fucose at 6-position

2,4 A

ar

[M-Cl-307]-

[M-342]-

of reducing terminus

[M-NO3-307]-

[M-369]-

[M-H2PO4-307]-

[M-405]-

Absence of fucose at 6-position of reducing terminus

2,4 * AR

[M-Cl-161]-

[M-NO3-161]-

[M-196]-

[M-223]-

[M-H2PO4-161]-

[M-259]-

Composition of

D and [D-18]- ([D-36]-)

GlcNAc

526, 508

6-antenna

Gal-GlcNAc

688, 670

Gal-[Fuc]GlcNAc

834, 816

(Gal-GlcNAc)2

  • 1053,
  • 1035 (1017)

(GalGlcNAc)2Fuc

  • 1199,
  • 1181 (1163)

Man3

647, 629

Man4

808, 791

Man5

971, 953

Table 3.4 (Continued)

Structural feature

Ion

Ionic composition

m/z

0,3Ar-2 and 0,4Ar-2

GlcNAc

292, 262

GalGlcNAc

454, 424

Gal-[Fuc]GlcNAc

600, 570

(Gal-GlcNAc)2

819, 789

(GalGlcNAc)2Fuc

965, 935

Man3

251, 221

Man4

413, 383

Man5

575, 545

Composition of

0,4Ar-3 (E) ion

Gal-GlcNAc

466

3-antenna

GlcNAc2

507

Gal-GlcNAc2

669

(Gal-GlcNAc)2

831

Gal-[Fuc]GlcNAc

977

Presence of bisect

Abundant [D-221]- ion

GlcNAc

508

Gal-GlcNAc

670

Gal-[Fuc]GlcNAc

816

(Gal-GlcNAc)2

1035

(Gal-GlcNAc)2Fuc

1181

Man3

629

Presence of sialic acid

B1

Neu5Ac

290

Neu5Gc

306

Presence of a2 6-

O,4A2-CO2

Neu5Ac

306

linked sialic acid

Neu5Gc

322

Unfortunately, acidic carbohydrates, such as those containing sialic acid or carboxyl-containing derivatives such as 2-AA [316], ionize by loss of one or more protons from acidic groups to give [M-иИ]”- ions with localized charges and, consequently, restricted fragmentation. 2-AA derivatives should, therefore, be avoided in this context. The spectra of sialylated glycans, however, do yield useful, although restricted, fragmentation [314, 317]. The type of sialic acid attached to the glycan is revealed by the mass of the prominent B1 fragment (m/z 290 for Neu5Ac and 306 for Neu5Gc), and the presence of a2 ^ 6-linked sialic acids can be determined by the presence of fragments at m/z 306 and 322 for these two sialic acids, respectively. Glycans containing sulfated GalNAcGlcNAc moieties fragment to give two very prominent ions at m/z 282 and 485.

Negative ion fragmentation spectra of O-linked glycans appear to offer similar specificity to that seen in the spectra of the N-linked glycans. Karlsson et al. [318] have investigated negative ion spectra of O-linked alditols from salivary mucin MUC5B and found major Z- and Y-type fragments. C- and A-type cleavages provided information on the structure of the reducing termini. However, cross-ring fragments were not as abundant as in the spectra of the N-linked glycans.

 
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