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Home arrow Health arrow Analysis of Protein Post-Translational Modifications by Mass Spectrometry
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Acetylation- and Methylation-Specific Diagnostic Ions in MS Analysis

The generation of PTM-specific and thus diagnostic ions upon peptide fragmentation is a key feature used for the assignment of acetylated and methylated peptides. PTM diagnostic ions include immonium ions, side-chain fragment ions, and those resulting from neutral loss (NL). Details of acetyl and methyl PTM diagnostic ions, their mass, and fragment type are listed in Table 4.1, together with specific references in which their application was described. These ions can be used to discriminate between acetylation and trimethylation [21, 30] and also between methyl-arginine and methyl-lysine forms [11]. IM ions indicate the presence of acetylation or methylation in a peptide but do not provide information on site localization. Many of the diagnostic masses were first determined from CID spectra in lower resolution instruments in the early 2000s, typically cited to first or no decimal place of mass value. These low-mass diagnostic ions can also be observed in HCD spectra that are measured using high-resolution MS instrumentation [31]. HCD offers an advantage over CID since the beam-type energy deposited during fragmentation improves the generation of both IM and other sequence-related ions [31]. Including information

Table 4.1 PTM diagnostic ions for the assignment of acetylation and methylation status.

PTM

Mass of ion or neutral loss

Type

Mode of fragmentation

References

Acetyl-E- lysine side chain, N-Acetyl

126.091

IM-NH3

CID

[34, 48, 51]

Monomethyl-

lysine

98.0964

IM-NH3

CID

[30, 37]

Dimethyl-

lysine

112.1

IM

CID

[30, 37, 44]

45.0578

NL

Trimethyl-

lysine

59.0735

NL

CID

[30, 37, 44]

Monomethyl-

arginine

73.064

NL monomethyl- guanidine

ETD

[28, 37, 39, 40, 42, 44]

31.0422

NL monomethylamine

ETD

Dimethyl-

arginine

46.0651

Side-chain

dimethylammonium

CID

[39, 41]

Symmetric > asymmetric dimethyl- arginine

71.0604

Side-chain dimethyl- carbodiimidium

CID

[39, 41]

Symmetric

dimethyl-

arginine

31.0417

Neutral loss of monomethylamine

CID

[38-40, 42]

70.0525

Neutral loss of dimethyl- carbodiimidium

CID or ETD

Asymmetric

dimethyl-

arginine

45.0573

Neutral loss of dimethylamine

CID or ETD

[38-40, 42]

Notes: Hung et al., 2007 also provide additional resources by listing the accurate masses for modified y1 and dipeptide a and b ions to aid sequence assignment of acetylated and methylated peptides [43]. Zhang et al. provide information on the mechanisms of IM and NL fragment generated from acetylated and methylated peptides [30]. Gehrig et al. also provide mechanistic information on fragmentation of methylated peptides [38].

on the presence and absence of diagnostic neutral losses and IM ions aids software-based PTM assignment and localization [32, 33]. High mass accuracy measurement (<2 ppm) aids sequence and PTM analysis assignment since the chemical composition for the majority of detected MS2 fragment ions can be unambiguously assigned [33]. For lower resolution instruments, the use of heavy isotope-labeled synthetic peptide standards and retention time information enables confident assignment of acetylation and methylation sites [34].

For acetylation, it is the IM-NH3 (m/z, 126.091), rather than the IM (m/z, 143.1179), that is diagnostic [35]. The 143.1179 ion is not unique: isobaric masses can also result from GlyLeu-, LeuGly-, GlyIsoleu-, and IsoleuGly- containing peptides by the formation of either a2 or internal fragment. A specific feature of the Kac diagnostic ion is that the ion intensity is higher for peptides with N-terminal lysine relative to internal lysine ions [35]. In terms of fragmentation, the presence of Kac promotes selective cleavage of Lys-Xxx amide bond to generate more information of the peptide backbone sequence relative to the nonacetylated form. This is due to the presence of b(n)+ ions as the most abundant primary product ions [36]. The site of acetylation can be additionally inferred by the 170 Da mass differences between y and y + 1 ion or corresponding b ions (Figure 4.1). Note that high mass accuracy and resolution discriminate lysine acetylation (Д170.1056) from lysine trimethylation (Д170.1420) [21].

Peptides containing different types of methylations produce specific, diagnostic ions and neutral loss fragments [37-41, 44]. The diagnostic mass for monomethyl-lysine immonium-specific ion at m/z 98.1 can be used to discriminate

Representative spectrum of an acetylated peptide analyzed by TOF MS

Figure 4.1 Representative spectrum of an acetylated peptide analyzed by TOF MS. Peptide sequence CASIQKacFGER, precursor m/z 619.3, with the acetyl lysine diagnostic IM-NH3 (126.1) and mass difference of 170 Da, which together confirm the presence of acetyl lysine.

monomethyl-lysine from monomethyl-arginine [30, 37]. The symmetric and asymmetric forms of dimethyl-arginine can be discriminated by different neutral loss ions following CID [38, 40, 41] and ETD [28, 42]. Dimethyl- and trimethyl-lysine-containing peptides undergo specific neutral losses from the precursor, MH+-45 and MH+-59, respectively [30, 37, 44]. Neutral loss is only observed in ion series containing the modified lysines, information which enables site assignment of the PTM. The m/z values of monopeptide and dipeptide ions of y, a, and b types provide confirmatory data for N- and C-terminal end amino acids [43]. For example, trimethylation can be assigned to the N-termi- nus of the peptide when the b2-59 ion is detected [30].

A comparative analysis of CID and ETD revealed that ETD was superior for the analysis of protein methylation, particularly since CID spectra are complex and neutral loss-derived product ions can be at low abundance relative to ions resulting from -H2O losses [45]. Lysine-methylated peptides do not produce significant losses during ETD fragmentation, but despite this ETD has proved particularly useful for the analysis of monomethyl- and dimethyl-lysine, since they form multiply charged peptides [42]. Methylated peptides are often present at substoichiometric levels, and the sensitivity of detection can be enhanced by the use of targeted inclusion lists of theoretical m/z values for methylated peptides for selection and fragmentation - an approach of potential value to other PTMs [45].

Stable isotope labeling of methylated peptides in vivo with (13CD3)-methionine enables high-confidence detection of protein methylation sites by MS [39]. This technique, heavy methyl SILAC is a variant of Stable isotope labeling with amino acids in cell culture (SILAC); resulting in a mass difference of 4Da/methyl group, relative to the unlabeled (light) form of the peptide. Following mixing of heavy and light samples, the presence of a 1:1 methyl heavy:light precursor pair in the MS1 scan corroborates the assignment of the fragmentation spectrum to a methylated peptide. The mass difference within the SILAC pairs enables assignment of the number of methyl groups per identified peptide. While of proven utility in the identification of methylated peptides, the method has the drawback that heavy methyl SILAC also generates L-methionine or L-methionine-13CD3 SILAC pairs. A refinement of the method overcomes this limitation by the substitution of l- methionine with isomethionine (L-methionine-13C4). This “iMethyl-SILAC” approach results in near-isobaric methionine peptide pairs, thus iMethyl-SILAC pairs are specific to methylated peptides for improved confidence in the identification of methylated peptides: iMethyl SILAC led to a sixfold reduction in false discovery rate when compared with label-free identification of methylation sites [46].

 
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