Studies using specific biomarkers for human exposure assessment to exogenous and endogenous chemical agents.

The extent of formation of carcinogen adducts with DNA and protein may be used to assess the biologically effective dose of these carcinogens in the tissue under study. In normal human tissues, such carcinogen adducts arise in part from exposures to exogenous genotoxic compounds, although it has been shown that endogenously formed carcinogens also make a significant contribution to the observed DNA and protein damage. Although, highly sensitive analytical methods, such as immunoassay, 32P-postlabelling and mass spectrometry have been developed and successfully applied to measure carcinogen adducts, further methodological advances are making these methods more amenable to molecular epidemiological studies. Thus, the use of immunoslot blot assays allows a higher sample throughput for adduct quantification. Liquid chromatographic separations of adducts, either for their radiochemical detection following 32P-postlabelling or for their determination by mass spectrometry, improves the specificity and applicability of these techniques. In this review, the sensitivities and specificities of the analytical methods used for adduct detection are compared and the limitations of these methods described.     

History and perspectives of bioanalytical methods for chemical warfare agent detection

This paper provides a short historical overview of the development of bioanalytical methods for chemical warfare (CW) agents and their biological markers of exposure, with a more detailed overview of methods for organophosphorus nerve agents. Bioanalytical methods for unchanged CW agents are used primarily for toxicokinetic/toxicodynamic studies. An important aspect of nerve agent toxicokinetics is the different biological activity and detoxification pathways for enantiomers. CW agents have a relatively short lifetime in the human body, and are hydrolysed, metabolised, or adducted to nucleophilic sites on macromolecules such as proteins and DNA. These provide biological markers of exposure. In the past two decades, metabolites, protein adducts of nerve agents, vesicants and phosgene, and DNA adducts of sulfur and nitrogen mustards, have been identified and characterized. Sensitive analytical methods have been developed for their detection, based mainly on mass spectrometry combined with gas or liquid chromatography. Biological markers for sarin, VX and sulfur mustard have been validated in cases of accidental and deliberate human exposures. The concern for terrorist use of CW agents has stimulated the development of higher throughput analytical methods in support of homeland security.     

Direct observation of the alkylation products of deoxyguanosine and DNA by fast atom bombardment mass spectrometry.

By the methods of fast atom bombardment (FAB) mass spectrometry, thin-layer chromatography and ultraviolet absorption spectroscopy adducts have been studied which are formed by an antitumour alkylating drug thiotepa both in a model system, containing only deoxyguanosine (dGuo), and in DNA. Analysis of the model reaction mixture (dGuo + thiotepa) by FAB mass spectrometry permitted observation of adducts dGuo thiotepa, 2dGuo thiotepa, and also the products of their further modification in solution, which occurs by hydrolysis of the glycosidic bond and also by opening of the imidazole ring. In the case of DNA FAB mass spectrometry made it possible to characterize adducts of thiotepa with guanosine (Gua) and adenosine (Ade) without their preliminary purification. The site of alkylation of Gua in both dGuo and DNA is N7, and that of Ade in DNA is N3. The application of the results to the study of the molecular mechanism of the antitumour action of thiotepa is discussed.     

Applications of mass spectrometry for quantitation of DNA adducts

DNA adducts are formed when electrophilic molecules or free radicals attack DNA. 32P-postlabeling has been the most commonly used assay for quantitation of DNA adducts due mainly to its excellent sensitivity that allows quantitation at concentrations as low as approximately 1 adduct per 10(9) normal bases. Such methods, however, do not have the specificity desired for accurate and reliable quantitation, and are prone to produce false positives and artifacts. In the last decade, mass spectrometry in combination with liquid and gas chromatography has presented itself as a good alternative to these techniques since it can satisfy the need for specificity and reliability through the use of stable isotope-labeled internal standards and highly specific detection modes such as selected reaction monitoring and high-resolution mass spectrometry. In this article, the contribution of mass spectrometry to the quantitation of DNA adducts is reviewed with special emphasis on unique applications of mass spectrometry in the area of DNA adduct quantitation and recent applications with improvements in sensitivity.     

Separation and detection methods for covalent drug-protein adducts

Covalent binding of reactive metabolites of drugs to proteins has been a predominant hypothesis for the mechanism of toxicity caused by numerous drugs. The development of efficient and sensitive analytical methods for the separation, identification, quantification of drug-protein adducts have important clinical and toxicological implications. In the last few decades, continuous progress in analytical methodology has been achieved with substantial increase in the number of new, more specific and more sensitive methods for drug-protein adducts. The methods used for drug-protein adduct studies include those for separation and for subsequent detection and identification. Various chromatographic (e.g., affinity chromatography, ion-exchange chromatography, and high-performance liquid chromatography) and electrophoretic techniques [e.g., sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional SDS-PAGE, and capillary electrophoresis], used alone or in combination, offer an opportunity to purify proteins adducted by reactive drug metabolites. Conventionally, mass spectrometric (MS), nuclear magnetic resonance, and immunological and radioisotope methods are used to detect and identify protein targets for reactive drug metabolites. However, these methods are labor-intensive, and have provided very limited sequence information on the target proteins adducted, and thus the identities of the protein targets are usually unknown. Moreover, the antibody-based methods are limited by the availability, quality, and specificity of antibodies to protein adducts, which greatly hindered the identification of specific protein targets of drugs and their clinical applications. Recently, the use of powerful MS technologies (e.g., matrix-assisted laser desorption/ionization time-of-flight) together with analytical proteomics have enabled one to separate, identify unknown protein adducts, and establish the sequence context of specific adducts by offering the opportunity to search for adducts in proteomes containing a large number of proteins with protein adducts and unmodified proteins. The present review highlights the separation and detection technologies for drug-protein adducts, with an emphasis on methodology, advantages and limitations to these techniques. Furthermore, a brief discussion of the application of these techniques to individual drugs and their target proteins will be outlined.     

Analysis of DNA and protein adducts of benzo[a]pyrene in human tissues using structure-specific methods

We review studies which investigate the presence, using structure-specific analytical methods, of DNA or protein adducts of the carcinogen benzo[a]pyrene (BaP) in human tissues. The analytical methods include high performance liquid chromatography with fluorescence detection and gas chromatography-mass spectrometry. Although, for DNA detection these methods are somewhat less sensitive than non-specific techniques such as 32P-postlabeling and immunoassay, they have the distinct advantage of providing reliable structural information. In order to achieve adequate sensitivity, these methods often require the use of fairly large amounts of DNA (>100 microg) or protein (50-100mg). Most studies reviewed here measured tetraols released from DNA or protein by hydrolysis of adducts derived from (7R,8S)-dihydroxy-(9S,10R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), a major ultimate carcinogen of BaP. BPDE-DNA adducts were detected in 39% of 705 samples analyzed. BPDE-protein adducts were found in 59% of 772 samples. There was no single exposure situation that led to an overwhelming presence of detectable adducts. For example, BPDE-DNA adducts were detected in 45% of smokers, 33% of former smokers, 52% of non-smokers, 39% of occupationally exposed individuals, and 34% of environmentally exposed people. Adduct levels were influenced by polymorphisms in carcinogen metabolizing genes such as GSTM1, the presence of which was frequently protective. The relatively high occurrence of non-detectable adducts may result from low levels of BaP exposure and host factors such as genetic polymorphisms. Our analysis demonstrates that the presence of BaP adducts in human tissues cannot be assumed, even in situations where exposure to BaP is relatively high.     

Hydroxyl-specific fluorescence labeling of ABP-deoxyguanosine, PhIP-deoxyguanosine, and AFB1-formamidopyrimidine with BODIPY-FL

Detection and analysis of DNA adducts resulting from endogenous or exogenous exposures to carcinogens are essential not only for quantifying biologically effective doses but also for establishing relationships between exposure and cancer risk. We have developed and validated a procedure of high sensitivity and specificity based on fluorescence labeling of DNA adducts combined with high-performance liquid chromatography-laser-induced fluorescence detection. The fluorescent dye 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY FL) was used to label the deoxynucleoside adducts N-(2'-deoxyguanosine-8-yl)-4-aminobiphenyl and N-(2'-deoxyguanosine-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and the base adduct aflatoxin B(1)-formamidopyrimidine by acylation. The labeling reaction was carried out on adducts at 1pmol to 30nmol concentrations at 25 degrees C for 4h in dichloromethane with 200- to 5000-fold excess of BODIPY FL. BODIPY FL and its activating agents 1,3-dicyclohexylcarbodiimide and 4-dimethylaminopyridine were used at a molar ratio of 1:2:2. Under these conditions, all of the above adducts were quantitatively converted to bis-labeled products, as confirmed by mass spectrometry. Sites of derivatization of adduct deoxynucleosides were established primarily by nuclear magnetic resonance and by collision-induced dissociation mass spectrometric analysis, which indicated that the bis-BODIPY groups were located predominantely on the 3'- and 5'-hydroxyl groups of the deoxyribose ring.     

Use of DNA adducts to identify human health risk from exposure to hazardous environmental pollutants: the increasing role of mass spectrometry in assessing biologically effective doses of genotoxic carcinogens

The carcinogens to which humans are exposed are normally in the form of complex mixtures, and much effort has gone into determining the nature of the most significant carcinogenic components in these mixtures and their mechanisms of action. Essential to achieving this aim in exposed populations is the use of biomarkers, which can characterize the chemical nature of the carcinogens involved and identify key biological effects that result from the exposure. DNA adducts are particularly appropriate as biomarkers in the case of genotoxic carcinogens as they indicate the biologically effective dose of the genotoxin in the target tissue under study. This review considers in particular the use of mass spectrometry (MS), which is having an increasing role in the determination of DNA adducts. Compared to other existing DNA damage detection methods, such as 32P-postlabeling, HPLC-fluorescence or electrochemical detection, immunoassay-based techniques and modified Comet assays, MS provides improved structural characterization of adducts. Greater selectivity in the analyses is achieved by the use of tandem MS with selected reaction monitoring or constant neutral loss of ions. Use of capillary/nano liquid chromatography and micro/nano electrospray ionization improves the analytical sensitivity and higher throughput may be obtained by the use of online-column switching. The application of microfluidics technology offers exciting new possibilities for interfacing sample preparation to the mass spectrometer. Despite these improvements in the use of MS for adduct detection, the main current requirement is to validate these methods both analytically and in molecular epidemiology studies. More knowledge of the stability of stored samples is required. Development of sensitive mass spectrometric DNA adductomic screening systems, and of long-term biomarkers (e.g., phosphotriester adducts that are not repaired efficiently) seems important areas for the future assessment of the effects of human exposure to environmental genotoxins, together with studies of dose-response relationships at low doses.     

Characterization of adducts formed in reactions of acrolein with thymidine and calf thymus DNA

Acrolein, an important industrial chemical and environmental contaminant, has been shown to interact with nucleic acids in vitro and in vivo. In this study, we examined the reactivity of acrolein towards thymidine and calf-thymus double- and single-stranded DNA in aqueous buffered solutions. LC-MS Analyses of the reaction mixture of acrolein with thymidine showed the formation of five structurally different adducts. The structures of the products were determined on the basis of mass spectrometry, UV absorbance, and (1)H- and (13)C-NMR spectroscopy. The adducts were identified as 3-(3-oxopropyl)thymidine (dT1), 3-[(tetrahydro-2,4-dihydroxypyran-3-yl)methyl]thymidine (dT2), 2-(hydroxymethyl)-5-(thymidin-3-yl)pent-2-enal (dT3), 3-hydroxy-2-methylidene-5-(thymidin-3-yl)pentanal (dT4), and 2-[(thymidin-3-yl)methyl]penta-2,4-dienal (dT5). The adducts dT2-dT5 were formed in reaction of dT1 with acrolein. In the reaction of acrolein with calf-thymus DNA, dT1 was the only adduct detected in the DNA hydrolysate.     

Mass spectrometric analysis of a UV-cross-linked protein-DNA complex: tryptophans 54 and 88 of E. coli SSB cross-link to DNA

Protein-nucleic acid complexes are commonly studied by photochemical cross-linking. UV-induced cross-linking of protein to nucleic acid may be followed by structural analysis of the conjugated protein to localize the cross-linked amino acids and thereby identify the nucleic acid binding site. Mass spectrometry is becoming increasingly popular for characterization of purified peptide-nucleic acid heteroconjugates derived from UV cross-linked protein-nucleic acid complexes. The efficiency of mass spectrometry-based methods is, however, hampered by the contrasting physico-chemical properties of nucleic acid and peptide entities present in such heteroconjugates. Sample preparation of the peptide-nucleic acid heteroconjugates is, therefore, a crucial step in any mass spectrometry-based analytical procedure. This study demonstrates the performance of four different MS-based strategies to characterize E. coli single-stranded DNA binding protein (SSB) that was UV-cross-linked to a 5-iodouracil containing DNA oligomer. Two methods were optimized to circumvent the need for standard liquid chromatography and gel electrophoresis, thereby dramatically increasing the overall sensitivity of the analysis. Enzymatic degradation of protein and oligonucleotide was combined with miniaturized sample preparation methods for enrichment and desalting of cross-linked peptide-nucleic acid heteroconjugates from complex mixtures prior to mass spectrometric analysis. Detailed characterization of the peptidic component of two different peptide-DNA heteroconjugates was accomplished by matrix-assisted laser desorption/ionization mass spectrometry and allowed assignment of tryptophan-54 and tryptophan-88 as candidate cross-linked residues. Sequencing of those peptide-DNA heteroconjugates by nanoelectrospray quadrupole time-of-flight tandem mass spectrometry identified tryptophan-54 and tryptophan-88 as the sites of cross-linking. Although the UV-cross-linking yield of the protein-DNA complex did not exceed 15%, less than 100 pmole of SSB protein was required for detailed structural analysis by mass spectrometry.     

Characterization of the lysyl adducts formed from prostaglandin H2 via the levuglandin pathway.

Prostaglandin H(2) has been demonstrated to rearrange to gamma-ketoaldehyde prostanoids termed levuglandins E(2) and D(2). As gamma-dicarbonyl molecules, the levuglandins react readily with amines. We sought to characterize the adducts formed by synthetic levuglandin E(2) and prostaglandin H(2)-derived levuglandins with lysine. Using liquid chromatography/electrospray mass spectrometry, we found that the reaction predominantly produces lysyl-levuglandin Schiff base adducts that readily dehydrate to form lysyl-anhydrolevuglandin Schiff base adducts. These adducts were characterized by examination of their mass spectra, by analysis of the products of their reaction with sodium cyanide, sodium borohydride, and methoxylamine and by the mass spectra derived from collision-induced dissociation in tandem mass spectrometry. The Schiff base adducts also are formed on peptide-bound lysyl residues. In addition, synthetic levuglandin E(2) and prostaglandin H(2)-derived levuglandins produced pyrrole-derived lactam and hydroxylactam adducts upon reaction with lysine as determined by tandem mass spectrometry. A marked time dependence in the formation of these adducts was observed: Schiff base adducts formed very rapidly and robustly, whereas the lactam and hydroxylactam adducts formed more slowly but accumulated throughout the time of the experiment. These findings provide a basis for investigating protein modification induced by oxygenation of arachidonic acid by the cyclooxygenases.     

Imbalanced estrogen metabolism in the brain: possible relevance to the etiology of Parkinson’s disease

Damage to DNA by dopamine quinone and/or catechol estrogen quinones may play a significant role in the initiation of Parkinson’s disease (PD). Depurinating estrogen–DNA adducts are shed from cells and excreted in urine. The aim of this study was to discover whether higher levels of estrogen–DNA adducts are associated with PD. Forty estrogen metabolites, conjugates, and DNA adducts were analyzed in urine samples from 20 PD cases and 40 matched controls by using ultra performance liquid chromatography/tandem mass spectrometry. The levels of adducts in cases versus controls (P?<?0.005) suggest that unbalanced estrogen metabolism could play a causal role in the initiation of PD.     

Conformations of adducts formed between the genotoxic benzo[a]pyrene-7,8-dione and nucleosides studied by density functional theory

Benzo[a]pyrene (BP) is a widely distributed environmental pollutant that is metabolized by mammalian cells to a variety of genotoxic and carcinogenic intermediates that form covalent adducts with cellular DNA. One such pathway involves the metabolic activation of BP by members of the aldo-keto-reductase (AKR) family of enzymes to the highly reactive ortho-quinone, benzo[a]pyrene-7,8-dione (BPQ). This compound has been reported to react with the 2'-deoxynucleosides, dA and dG, under physiological conditions. Four BPQ-dG adducts and two -dA adducts were identified by mass spectrometry and NMR methods [Balu et al. (2004) Chem. Res. Toxicol. 17, 827-838]. However, the detailed conformations and absolute configurations around the linkage site have not been resolved. In order to determine the full conformations of these purine adducts, we carried out quantum mechanical geometry optimization using density functional theory. In the case of the BPQ-guanine adducts, six possible structures, each of which consists of two isomers, were identified. However, in the case of the adenine adducts, only four isomers were identified. The results suggest that stereoisomeric adduct pairs are expected to adopt opposite orientations with respect to the 5'-->3' direction of the modified DNA strands. The stereochemistry-dependent variations in adduct orientation may produce different biological effects, as has been observed in the case of DNA adducts derived from other metabolites of polycyclic aromatic hydrocarbons.     

Analysis of tamoxifen-DNA adducts in endometrial explants by MS and 32P-postlabeling

The nonsteroidal antiestrogen tamoxifen increases the risk of endometrial cancer; however, the mechanism for the induction of these tumors is not known. Recently, Sharma et al. [Biochem. Biophys. Res. Commun. 307 (2003) 157], using high performance liquid chromatography (HPLC) with online postcolumn photochemical activation and fluorescence detection, reported the presence of (E)-alpha-(deoxyguanosin- N2-yl)tamoxifen in DNA from human endometrial explants incubated with tamoxifen. Inasmuch as the methodology used by these investigators does not allow unambiguous characterization of tamoxifen-DNA adducts, we have used two additional techniques (HPLC coupled with electrospray ionization tandem mass spectrometry and 32P-postlabeling analyses) to assay for the presence of tamoxifen-DNA adducts in the human endometrial explant DNA. Tamoxifen-DNA adducts were not detected by either method.     

Fast Atom Bombardment and Tandem Mass Spectrometry for the Identification of Nucleoside Adducts with Phenyl Glycidyl Ether

Abstract

The present study deals with the use of fast atom bombardment (FAB) in combination with constant neutral loss (CNL) scanning, high resolution mass spectrometry and tandem mass spectrometry (MS-MS) with collisionally activated decomposition (CAD), as complementary methods for the identification and structural analysis of phenyl glycidyl ether-nucleoside adducts. Selective detection of the parent ions of the modified nucleosides at the 1–10 ng level has been achieved by suitably designed CNL scans. The elemental composition of the adducts has been determined by accurate mass measurements. CAD-MS has been carried out on the [M + H]⁺ and [M - H]^? ions to derive structural data on the size and nature of the base, sugar and alkyl substituent. In some cases, information on the alkylation site has been obtained, which is very useful for distinguishing isomeric adducts.     

Speciation of oxaliplatin adducts with DNA nucleotides

This paper describes a set of fast and selective high performance liquid chromatography (HPLC) methods coupled to electro-spray ionisation linear ion trap mass spectrometry (ESI-MS), sector-field inductively coupled plasma mass spectrometry (SF-ICP-MS) and UV detection for in vitro studies of the bifunctional adducts of oxaliplatin with mono-nucleotides, di-nucleotides and cellular DNA. The stationary phases and the optimised conditions used for each separation are discussed. Interaction of oxaliplatin with A and G mono-nucleotides resulted in the formation of five bifunctional platinum diaminocyclohexane (DACHPt) adducts. These were two isomers of the A-DACHPt-A and A-DACHPt-G adducts, and one G-DACHPt-G adduct, as confirmed by MS/MS spectra obtained by collision induced dissociation. These adducts were also characterised by UV absorption data and SF-ICP-MS elemental (195)Pt and (31)P signals. Further, interaction of oxaliplatin with AG and GG di-nucleotides resulted in the formation of three adducts: DACHPt-GG and two isomers of the DACHPt-AG adduct, as confirmed by ESI-MS and the complementary data obtained by UV and SF-ICP-MS. Finally, a very sensitive LC-ICP-MS method for the quantification of oxaliplatin GG intra-strand adducts (DACHPt-GG) was developed and used for monitoring the in vitro formation and repair of these adducts in human colorectal cancer cells. The method detection limit was 0.14 ppb Pt which was equivalent to 0.22 Pt adduct per 10(6) nucleotides based on a 10 μg DNA sample. This detection limit makes this method suitable for in vivo assessment of DACHPt-GG adducts in patients undergoing oxaliplatin chemotherapy.     

Mass spectrometric analysis of haemoglobin adducts formed by methyl bromide in vitro

An analytical procedure is described for the identification of the adducts formed by interaction of methyl bromide and haemoglobin. The reaction products of in vitro incubation of haemoglobin with methyl bromide have been characterised by electrospray mass spectrometry and gas chromatography-mass spectrometry. A prominent reactivity of several potential nucleophilic sites of haemoglobin was observed. Analogous results were recorded on blood samples of workers exposed to methyl bromide. The results obtained represent the basis for the complete structural characterisation of the modified haemoglobin and demonstrate the usefulness of the proposed analytical approach for the evaluation of alkylation degree and the identification of modified amino acids in proteins.     

Measurement of deaminated cytosine adducts in DNA using a novel hybrid thymine DNA glycosylase

The hydrolytic deamination of cytosine and 5-methylcytosine drives many of the transition mutations observed in human cancer. The deamination-induced mutagenic intermediates include either uracil or thymine adducts mispaired with guanine. While a substantial array of methods exist to measure other types of DNA adducts, the cytosine deamination adducts pose unusual analytical problems, and adequate methods to measure them have not yet been developed. We describe here a novel hybrid thymine DNA glycosylase (TDG) that is comprised of a 29-amino acid sequence from human TDG linked to the catalytic domain of a thymine glycosylase found in an archaeal thermophilic bacterium. Using defined-sequence oligonucleotides, we show that hybrid TDG has robust mispair-selective activity against deaminated U:G and T:G mispairs. We have further developed a method for separating glycosylase-released free bases from oligonucleotides and DNA followed by GC–MS/MS quantification. Using this approach, we have measured for the first time the levels of total uracil, U:G, and T:G pairs in calf thymus DNA. The method presented here will allow the measurement of the formation, persistence, and repair of a biologically important class of deaminated cytosine adducts.     

Biomonitoring of exposure to aromatic amines: haemoglobin adducts in humans

Haemoglobin (Hb) adducts from aromatic amines (AAs) are well established biomarkers of exposure. Tobacco smoking and occupational exposure are major sources of AA Hb adducts. The origin of background levels in non-smokers and non-occupationally exposed humans are largely unknown. Here we examine the determination of AA Hb adducts, focussing on the analytical strategies for Hb isolation, removal of unbound AAs from Hb solutions, hydrolysis of the Hb bound AAs, extraction, preconcentration, clean-up and derivatisation of the free amines for determination by gas chromatography-mass spectrometry. Finally, a detailed summary of available results on the determination of AA Hb adducts is given.     

Mass spectrometric determination of early and advanced glycation in biology

Protein glycation in biological systems occurs predominantly on lysine, arginine and N-terminal residues of proteins. Major quantitative glycation adducts are found at mean extents of modification of 1–5 mol percent of proteins. These are glucose-derived fructosamine on lysine and N-terminal residues of proteins, methylglyoxal-derived hydroimidazolone on arginine residues and N^ε-carboxymethyl-lysine residues mainly formed by the oxidative degradation of fructosamine. Total glycation adducts of different types are quantified by stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring mode. Metabolism of glycated proteins is followed by LC-MS/MS of glycation free adducts as minor components of the amino acid metabolome. Glycated proteins and sites of modification within them – amino acid residues modified by the glycating agent moiety - are identified and quantified by label-free and stable isotope labelling with amino acids in cell culture (SILAC) high resolution mass spectrometry. Sites of glycation by glucose and methylglyoxal in selected proteins are listed. Key issues in applying proteomics techniques to analysis of glycated proteins are: (i) avoiding compromise of analysis by formation, loss and relocation of glycation adducts in pre-analytic processing; (ii) specificity of immunoaffinity enrichment procedures, (iii) maximizing protein sequence coverage in mass spectrometric analysis for detection of glycation sites, and (iv) development of bioinformatics tools for prediction of protein glycation sites. Protein glycation studies have important applications in biology, ageing and translational medicine – particularly on studies of obesity, diabetes, cardiovascular disease, renal failure, neurological disorders and cancer. Mass spectrometric analysis of glycated proteins has yet to find widespread use clinically. Future use in health screening, disease diagnosis and therapeutic monitoring, and drug and functional food development is expected. A protocol for high resolution mass spectrometry proteomics of glycated proteins is given.