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Home arrow Health arrow Analysis of Protein Post-Translational Modifications by Mass Spectrometry
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Mass Spectrometry Behavior of Glycated Peptides

The mass increment indicating the detection of FL and other fructosamine- modified peptides is +162 Da. Glycation of intact proteins and large peptide chains has been detected by electrospray positive ion mass spectrometry and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Roberts and coworkers detected and quantified fructosamine- modified a- and p-chains of hemoglobin by deconvolution of multiply charged ion series [84], shown in later studies by peptide mapping to reflect fructosa- mine formation at sites a-K61, P-V1, and P-K66 [28]. Increase in molecular mass of human serum albumin (HSA) glycated by glucose prepared in vitro was measured by MALDI-TOF. This revealed that preparations of glycated HSA had a large increase in mass due to the high extent of glycation, dissimilar from the low increase in mass of glycated HSA in plasma samples in vivo. For example, HSA from human plasma had a mean mass increment of +243 Da, whereas model glucose-modified albumin prepared in vitro had a mean mass increment of +6780 Da [85]. This suggested that the albumin prepared with very high extent of glycation was a poor model for the albumin with minimal extent of glycation found in vivo.

For mass spectrometric analysis of glycated peptides, collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD) fragmentation of fructosamine-containing peptides produced characteristic fragment ions of the precursor fructosamines (M+162): by dehydration to an oxonium ion (M+144), further dehydration to a pyrylium ion (M+108), and dehydration and formaldehyde loss to an immonium ion (M+78) [42, 86-88] (Figure 8.2a). Pyrylium and furylium ions are detected in y ion series providing for fructosamine location [42]. In electron transfer dissociation (ETD) fragmentation, abundant and almost complete series of c- and z-type ions were observed, which greatly facilitated the peptide sequencing and fructosamine site location [89].

The FL degradation product and AGE CML were detected at the same sites as fructosamine residues in serum albumin, hemoglobin, and ribonuclease A [42, 90, 91].

MG-derived hydroimidazolone and dihydroxyimidazolidine may be detected in peptides glycated by MG and tryptic peptides of proteins glycated in vivo. They have mass increments on arginine residues of +72 Da and +54, respectively. A further minor MG-derived and stable AGE, .Ae-(1-carboxyethyl)lysine (CEL), may be detected as +72 Da on lysine residues [11, 12, 71, 74]. High collision energy fragmentation may dehydrate dihydroxyimidazolidine to hydro- imidazolone, and so discrimination is provided by detection of the peptide molecular ion [71]. In analysis of MG-modified lipoproteins, no advantage of ETD over CID in the detection of hydroimidazolone and dihydroxyimidazoli- dine has been found [11, 12]. Fragmentation of peptides modified by MG-H1 and related isomers gave complete series of b and y ions with mass increment of 54 Da relative to those of unmodified peptide and no neutral losses [11, 12, 71, 74]. A MG-H1-related fragment ion of m/z = 166.1 can be observed in the low mass region of the MS/MS spectra, with proposed immonium ion structure (Figure 8.2b). A similar fragment ion of m/z = 152.1 can be observed for

Fragmentation of fructosamine and hydroimidazolone glycation adducts

Figure 8.2 Fragmentation of fructosamine and hydroimidazolone glycation adducts. (a) Fragmentation of fructosyl-lysine by CID leading to the formation of oxonium, pyrylium, furylium, and immonium ions. (b) and (c) Fragmentation of hydroimidazolones formed by MG and glyoxal to immonium ions in CID and HCD [42, 86-88].

glyoxal-modified peptides [92] (Figure 8.2c). Hydroimidazolone and dihydrox- yimidazolidine residues are chemically labile AGEs, and conditions of preanalytic processing for proteomics analysis may influence mass spectrometric analysis outcomes. Tryptic digestion methods with prolonged periods of samples incubated at high pH and/or temperature leads to reversal of hydroimida- zolone to dihydroxyimidazolidine and deglycation. Alternatively, high pH and temperature may also stimulate dicarbonyl formation [93]. In earlier studies, using N-hydroxysuccinimidyl active ester derivatization of MG-H1 in chromatographic analysis, we found that incubation of MG-H1 in the presence of [15N2]arginine at pH 8.8 for 10 min at 55 °C led to migration of the MG moiety from MG-H1 to [15N2]arginine [19]. Hence, use of high pH and temperature in preanalytic processing may induce migration of the MG moiety between arginine residues and, potentially, also between proteins. Conventional tryptic digestion techniques require modification to minimize the increase of pH and avoid sample heating for peptide mapping and proteomics analysis of MG-modified proteins and related PTMs.

Trypsinization cleavage after lysine and arginine residues is impaired by gly- cation by glucose and MG, and glycated peptide with missed cleavage at the glycation site is detected [74, 75, 94]. In some cases, cleavage after dicarbonyl glycation of arginine was observed [92].

 
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