Protein Modifications and Their MSMS Reactions

So far, >100 covalent modifications of proteins have been described [e.g., 20-22]. The annotated protein database SwissProt [23] is a rich source of well established covalent protein modifications. Many covalent modifications are accompanied by an increase in molecular weight and an increase in acidity (lower pi value), including deamidation, phosphorylation, sulfation, N-acylation, and cysteic acid formation. A selection of covalent modifications is given in Table 6.1, together with information about signature fragmentation reactions after CID.

The stability of the covalent modifications under the CID conditions can vary considerably. As a demonstration, Figure 6.7 shows survey MS spectra of two peptides of identical sequence, which carry a phosphotyrosine or a sulfotyrosine residue at the third position from the C terminus. Although the phosphotyrosine peptide shows a stable molecular ion signal, the sulfotyrosine peptide exhibits strong loss of SO3 from the sulphate ester group even under standard (usually nonfragmenting) conditions.

Following CID, the sulfotyrosine peptide exhibits a spectrum identical to that of the unmodified peptide, since loss of SO3 results in the formation of a tyrosine residue. In contrast, the phosphotyrosine residue is so stable that a phosphotyrosine im-monium ion is formed, which can be used as a marker ion - Figure 6.8 shows the low-mass region of the CID spectra of these two tyrosine-modified peptides. These MS/MS spectra, recorded under identical conditions, show the Y immonium ion at m/z 163 for the sulfated peptide and the pY immonium ion at m/z 216 for the phos-phorylated peptide. Those fragment ions that do not contain the modified tyrosine residue are observed at identical m/z values for both peptides.

Fig. 6.7 Positive ion survey spectra of two modified peptides, carrying (a) a sulfotyrosine residue or (b) a phosphotyrosine residue. The phosphotyrosine peptide shows a stable molecular ion signal, whereas the sulfotyrosine peptide exhibits a strong loss of SO3 even without the CID step.

Tab. 6.1 Selection of abundant covalent protein modifications and their signature reaction (if one occurs) in positive ion electrospray MS after collision-induced dissociation.

Protein modification

Mass shift

CID signature ions or reactions (m/z values)

N-terminal demethionylation

-131 Da


Thiazole formation between Cys and Ser

-20 Da

Succinimide formation at Asn

-18 Da


pyro-Glu formation from Glu

-18 Da

pyro-Glu formation from Gln

-17 Da


Disulfide formation Cys-Cys

-2 Da

doubled 18O content after digestion in H218O

Deamidation at Asn, Gln

+ 1 Da


Methylation at His

+ 14 Da

meHis immonium ion at 124

Methyl ester formation at the C terminus

+ 14 Da


Hydroxylation at proline

+ 16 Da

hydroxy-P immonium ion at 86

Oxidation at Met

+ 16 Da

loss of64= CH3-SOH

Dimethylation at Arg

+ 28 Da

neutral loss of45, 87

dimethylammonium ion at 46,

dimethylcarbodiimidium ion at m/z 71

Formylation at Lys

+ 28 Da

formyl-b1 ion for N-terminal formylation


+ 42 Da

acetyl-b1 ion for N-terminal acetylation

acetyl-Lys-(-NH3) immonium ion at 126

Trimethylation at Lys

+ 42 Da

neutral loss of59

me-Lys and dime-Lys immonium ions at 98,

1 1 T


+ 43 Da

carbamoyl-b1 ions for N-terminal carba-


Nitration at Tyr

+ 45 Da

nitroTyr immonium ion at 181

Oxidation of Cys to Cysteic Acid

+ 48 Da


Phosphorylation at Ser,Thr

+ 80 Da

loss of H3PO4 = 98 Da

69 Da or 83 Da distance in fragment ion


Phosphorylation at Tyr

+ 80 Da

loss of HPO3 = 80 Da, pY immonium ion

at 216

Sulfation at Tyr

+ 80 Da

loss of SO3 = 80 Da (strong)

Phosphorylation at hydroxyproline

+ 96 Da

dehydro-P immonium ion at 68

Iodination at Tyr

+ 126 Da

I-Tyr immonium ion at 262

N-terminal gluconoylation

+ 178 Da

N-terminal myristoylation to myrG

+ 210 Da

b0 at 211, a1 at 240, b1 at 268

O-GlcNAc addition at Ser,Thr

+ 203 Da

GlcNac-H2O ion at 204

Palmitoylation at Cys

+ 238 Da

acyl ion at 239

Dioidination at Tyr

+ 252 Da

I2-Tyr immonium ion at 388

N-terminal 6-phosphogluconoylation

+ 258 Da


+ 305 Da


+ 541 Da


+ >1 kDa

oxonium ions at 163, 204, 366


+ ca. 8 kDa

internal K-GG unit after tryptic digestion

Fig. 6.8 Low-mass CID spectra of the two peptides shown in Figure 6.6. (a) The MS/MS spectrum of the sulfopeptide shows abundant Yimmonium ion at m/z 136; (b) the corresponding spectrum of the phosphotyrosine peptide instead shows an abundant pYimmonium ion at m/z 216.

Modifications lost as neutral entities can be specifically detected by neutral loss scanning. Those giving rise to specific marker ions can be detected by precursor ion scanning or by extraction of mass-specific ion traces from a pool of MS/MS spectra.

0 0

Post a comment