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Protein Adductomics: Methodologies for Untargeted Screening of Adducts to Serum Albumin and Hemoglobin in Human Blood Samples
Protein Adductomics: Methodologies for Untargeted Screening of Adducts to Serum Albumin and...
Carlsson, Henrik;Rappaport, Stephen M.;Törnqvist, Margareta
Review Protein Adductomics: Methodologies for Untargeted Screening of Adducts to Serum Albumin and Hemoglobin in Human Blood Samples 1 , 1 2 Henrik Carlsson * , Stephen M. Rappaport and Margareta Törnqvist Division of Environmental Health Sciences, School of Public Health, University of California, Berkeley, CA 94720, USA; firstname.lastname@example.org Department of Environmental Science and Analytical Chemistry, Stockholm University, SE-106 91 Stockholm, Sweden; Margareta.Tornqvist@aces.su.se * Correspondence: email@example.com Received: 29 January 2019; Accepted: 27 February 2019; Published: 8 March 2019 Abstract: The reaction products of electrophiles in vivo can be measured as adducts to the abundant proteins, hemoglobin (Hb), and human serum albumin (HSA), in human blood samples. During the last decade, methods for untargeted screening of such adducts, called “adductomics”, have used liquid chromatography-mass spectrometry to detect large numbers of previously unknown Hb and HSA adducts. This review presents methodologies that were developed and used in our laboratories for Hb and HSA adductomics, respectively. We discuss critical aspects regarding choice of target protein, sample preparation, mass spectrometry, data evaluation, and strategies for identiﬁcation of detected unknown adducts. With this review we give an overview of these two methodologies used for protein adductomics and the precursor electrophiles that have been elucidated from the adducts. Keywords: proteins; protein adducts; electrophiles; adductomics; mass spectrometry; hemoglobin; human serum albumin 1. Introduction Electrophilic compounds occurring in vivo are generally formed by metabolism of molecules arising from endogenous (e.g., oxidative stress) and exogenous (e.g., diet and drugs) sources. Since these reactive species can modify nucleophilic sites of DNA and functional proteins, they can have toxicological implications. The inherent reactivity of electrophiles makes them difﬁcult to measure in vivo due to their short lifetimes, which has initiated analytical developments to measure their adducts to biomacromolecules. Thus, since the 1970s researchers have studied adducts of reactive electrophiles with abundant blood proteins, to obtain information about cumulative exposures to these reactive species over the mean residence times of particular proteins [1–3]. Protein adducts are covalent modiﬁcations resulting from reactions between electrophiles and nucleophilic sites in proteins, such as the N-terminus or the amino acid side chains containing sulfhydryl or amine functionalities. Since the early days of adduct measurements, mass spectrometry (MS) has been the analytical detection technique of choice for the adduct measurements because it provides both qualitative and quantitative information about the modiﬁed proteins. Measurements were targeted on adducts from expected reactive metabolites of toxic chemicals with a given protein. Important examples include hemoglobin (Hb) adducts from ethylene oxide [4,5], aromatic amines [6,7], and acrylamide [8,9] in occupational exposed workers and in smokers. Well-known examples of adducts measured to human serum albumin (HSA) are adducts of benzene and styrene in factory workers [10,11]. Many studies also showed a background adduct level in non-exposed control persons, as exempliﬁed with the Hb adducts from acrylamide, shown to be associated with consumption of cooked foods , and HSA adducts of High-Throughput 2019, 8, 6; doi:10.3390/ht8010006 www.mdpi.com/journal/highthroughput High-Throughput 2019, 8, 6 2 of 18 aﬂatoxin B1 from consumption of mycotoxin-contaminated foods [13,14]. These early studies clearly showed that humans have a continuous background exposure to electrophiles, both from endogenous and exogenous sources. During the last decade there has been a transition to untargeted approaches, “adductomics”, for analysis of the totality of adducts bound to a particular nucleophilic site  with the aim to contribute to the characterization of the human exposome . The adductomics concept was ﬁrst described by Kanaly et al. in 2006, who reported the simultaneous LC-MS detection of numerous DNA adducts in human tissues . A few years later, similar LC-MS approaches were described for protein adductomics by Li et al.  focusing on Cys34 adducts of HSA  and by Carlsson et al.  using N-terminal valine adducts of Hb . These latter two groups, at the University of California, Berkeley and Stockholm University, have continued to pursue adductomics of HSA and Hb, respectively. This review compares the two methodologies and lists the electrophilic species that have been characterized. For an updated and comprehensive overview of DNA adductomics the recent review by Villalta and Balbo is recommended . 2. Choice of Target Proteins in Blood Most electrophiles occurring in vivo are generated from enzymatic biotransformations of precursor molecules as well as from reactive oxygen and carbonyl species. Reactive functional groups make electrophiles capable of reacting with nucleophilic loci of proteins, notably, oxygen radicals, epoxides (adducts formed via nucleophilic substitution), activated double bonds of , -unsaturated carbonyl compounds (adducts formed via Michael addition), and aldehydes (adducts are Schiff bases formed via carbinolamine intermediates) [22,23]. Important nucleophilic sites for adduct formation in proteins are the sulfhydryl group of cysteine and amine functionalities of histidine, lysine, and N-terminal amino acids . Factors that affect the extent of adduct formation are the nucleophilicity and pKa of the nucleophile and steric factors that inﬂuence the access of the reactive electrophile to the nucleophilic site. Nucleophilic sites that are deprotonated at physiological pH (7.4) are favored for adduction because they have free electron pairs that can participate in the formation of covalent bonds. Because the chemistry of adduct formation differs across nucleophilic sites, for example Schiff bases are formed by reactions of aldehydes with amines and the formation of disulﬁde adducts require free sulfhydryl groups, it would be optimal to include several nucleophilic sites in characterizing a protein adductome. Important properties of Hb and HSA are summarized in Table 1. Regarding HSA, Cys34 has been the focus of all published studies on HSA adductomics thus far. This cysteine accounts for the antioxidant and detoxifying properties of albumin in the interstitial space and, therefore, is highly conserved in all mammalian species [24,25]. Because Cys34 has an unusually low pK of 6.55, compared to 8.0–8.55 for most protein thiols [25,26], the sulfhydryl group is predominantly in the deprotonated thiolate form (S ) at physiological pH (7.4). Since thiolate is an unusually strong nucleophile, many small electrophiles react preferentially at this site and makes Cys34 an exceptional nucleophilic locus for adductomics . With a few exceptions the in vivo formation of adducts involves only a very small fraction of the total amount of protein. For example, levels of typical Hb adducts of N-terminal valine (30–150 pmol/g Hb) correspond to 5–25 adducted Hb chains per 10 Hb chains in a human blood sample, whereas HSA adduct levels to Cys34 of 0.5–50 nmol/g HSA correspond to 3 modiﬁed HSA molecules per 3 5 10 –10 HSA molecules. Furthermore, since blood is the only tissue that is routinely available in studies of human health, adduct measurements have thus far focused on the two most abundant proteins in blood, i.e., Hb and HSA. The concentrations of Hb and HSA in human blood are approximately 120–160 mg/mL and 35–55 mg/mL, respectively, which enable adductomic analysis using between 5 (HSA) and 250 L (Hb) of blood per subject. In contrast, the concentration of DNA in human blood is about 30 g/mL, and DNA adductomics requires about 10 mL blood  per subject. High-Throughput 2019, 8, 6 3 of 18 Table 1. Properties of hemoglobin and human serum albumin. Property Human Hemoglobin Human Serum Albumin Tetramer, 64 kDa. Composed of Molecular weight two - and two -subunits (in 67 kDa adult Hb), both weighing 16 kDa Transport of e.g., hormones and Main functions Oxygen transport fatty acids, maintains oncotic pressure, antioxidant. Localization Red blood cells Blood plasma Conc (mg/mL) 120–160 (in blood) 35–55 (in serum) Turnover rate (days) ~120 (lifespan of red blood cells) ~20 (half-life in serum) Another important factor is the life-time of the protein in the human body, which provides a time window for integration of chronic exposures to reactive species [23,28]. Human serum albumin has a half-life of approximately 20 days  and Hb has a life-time (the same as the erythrocytes) of approximately 120 days . Chemically stable adducts of these proteins accumulate during chronic exposure and such adducts of HSA and Hb reach steady-state levels corresponding to the exposure during about one and two months, respectively [23,28]. This “signal-averaging” reduces variability due to short-term variation in exposure to reactive species and provides more stable measures of long-term exposure for epidemiologic investigations . In contrast, DNA adducts are more variable measures of exposure because of complex kinetics and turnover rates due to enzymatic repair of adducts, and result in low levels of accumulation [23,31]. The adductomics of Hb has focused exclusively on adducts to the N-terminal Val, which has a pK of approximately 7.8 (-chain) and 6.8 ( -chain) ; Hb is a tetramer composed of two - and two -chains, both with Val as the N-terminal amino acid. Another nucleophilic amino acid in Hb of potential interest for adductomic studies is -Cys93, which is a known adduction site for aromatic amines [7,33]. An important difference between HSA and Hb is the localization of the two proteins; HSA is, except in blood, also found in the interstitial ﬂuid whereas Hb is only found in red blood cells (RBCs). Their chemical environments are therefore partly different, which could give effects on the adductomes observed. In RBCs, the concentration of glutathione (GSH) is high compared to plasma concentrations (about 250 times higher in RBCs ), meaning that GSH has a far more important role as an electrophile scavenger in RBCs, giving some protection of Hb from incoming electrophiles. In plasma, Cys34 of HSA has a similar function. The sulfhydryl of Cys34 is a stronger and larger nucleophile than the amino group of N-terminal Hb Val, also with a lower pK , meaning that higher adduct levels are to be expected to this nucleophilic site. This is conﬁrmed when comparing adduct levels from a few electrophilic precursors in the published adductomics studies, with Hb adduct levels typically in the pmol/g Hb range and HSA adduct levels in the nmol/g HSA range. In fact, the human in vivo reaction rate between benzene oxide and HSA Cys34 was 30 times greater than that for Hb -Cys93 . This also means that much lower sample volumes may be used for analysis of HSA adducts compared to Hb adducts. 3. Sample Preparation In this section we describe and compare the sample preparation procedures for HSA and Hb adductomics [36,37]. Critical differences between the two methodologies are summarized in Table 2. The method for HSA adductomics uses only 5 L of plasma/serum. The ﬁrst step in the sample preparation involves precipitation of non-HSA proteins with 60% methanol. Following centrifugation, the protein content of the supernatant is approximately 70% HSA (of total protein content) . A portion of the supernatant is diluted with buffer and digested with trypsin, using a pressurized system (Barozyme HT48 from Pressure Biosciences Inc., South Easton, MA, USA) that facilitates High-Throughput 2019, 8, x FOR PEER REVIEW 4 of 17 content) . A portion of the supernatant is diluted with buffer and digested with trypsin, using a pressurized system (Barozyme HT48 from Pressure Biosciences Inc., South Easton, MA, USA) that facilitates complete digestion in 30 minutes, without prior reduction of the intramolecular HSA disulfides. The Cys34 residue is located on the third largest tryptic peptide, designated T3 with 21 34 41 sequence ALVLIAFAQYLQQC PFEDHVK with MW 2432.2562 Da (Figure 1). After digestion, formic acid is added to the sample to prevent further degradation. Prior to LC-MS analysis, a portion High-Throughput 2019, 8, 6 4 of 18 of the digest is diluted with 2% ACN/0.2% formic acid, and an isotopically labelled T3 peptide is added as an internal standard to monitor retention time drift and mass accuracy. Quantitation of adducts is performed relative to a housekeeping (HK) peptide, which is an HSA peptide of sequence complete digestion in 30 min, without prior reduction of the intramolecular HSA disulﬁdes. 41 50 LVNEVTEFAK with MW 1148.6077 Da. The peak area ratio (PAR = adduct peak area/HK peptide The Cys34 residue is located on the third largest tryptic peptide, designated T3 with sequence pe 21 ak area) has been sh34 own to be a41 reliable measure of adduct concentration over a wide dynamic ALVLIAFAQYLQQC PFEDHVK with MW 2432.2562 Da (Figure 1). After digestion, formic acid is range . Recently the method has been adapted for analysis of HSA extracted from dried blood added to the sample to prevent further degradation. Prior to LC-MS analysis, a portion of the digest is spots (DBS), which require additional steps prior to digestion . The small amount of protein diluted with 2% ACN/0.2% formic acid, and an isotopically labelled T3 peptide is added as an internal needed for HSA adductomics, as well as the possibility to use DBS for the analysis, is an attractive standard to monitor retention time drift and mass accuracy. Quantitation of adducts is performed advantage of this methodology. The fast sample preparation allows for processin41 g and MS analysi 50 s relative to a housekeeping (HK) peptide, which is an HSA peptide of sequence LVNEVTEFAK of about 12 samples per day (with duplicate injections). with MW 1148.6077 Da. The peak area ratio (PAR = adduct peak area/HK peptide peak area) has been shown to be a reliable measure of adduct concentration over a wide dynamic range . Recently the Table 2. Critical aspects of the methodologies for Hb and HSA adductomics. method has been adapted for analysis of HSA extracted from dried blood spots (DBS), which require additional steps prior to digestion . The small amount of protein needed for HSA adductomics, Methodolo as well as the possibility to use DBS for the analysis, is an attractive advantage of this methodology. gical Hb adductomics HSA adductomics The fast sample preparation allows for processing and MS analysis of about 12 samples per day aspect (with duplicate injections). Nucleophil N-terminal Val Cys34 ic site Table 2. Critical aspects of the methodologies for Hb and HSA adductomics. Sample 250 µ L whole blood/lysate 5 µ L plasma/serum volume Methodological Aspect Hb Adductomics HSA Adductomics Enrichmen Detachment of adducts using the reagent Precipitation of non-HSA Nucleophilic site N-terminal Val Cys34 t of fluorescein isothiocyanate, followed by clean-up proteins, followed by tryptic Sample volume 250 L whole blood/lysate 5 L plasma/serum adducts using solid phase extraction digestion Detachment of adducts using the reagent Fluorescein thiohydantoin derivatives of Tryptic peptide containing the Precipitation of non-HSA proteins, Enrichment of adducts ﬂuorescein isothiocyanate, followed by Analytes followed by tryptic digestion adducted Val adducted Cys34 residue clean-up using solid phase extraction MW of >502 Da (starting at the MW of the methyl >2432 Da (analyzed as triply Fluorescein thiohydantoin derivatives of Tryptic peptide containing the Analytes analytes adduct) charged positive ions) adducted Val adducted Cys34 residue Nano-liquid chromatography - >502 Da (starting at the MW of the methyl >2432 Da (analyzed as triply Analytical Liquid chromatography - mass spectrometry MW of analytes high-resolution mass adduct) charged positive ions) method (LC-MS)/high-resolution MS (LC-HRMS) spectrometry (nLC-HRMS) Liquid chromatography-mass Nano-liquid Type of LC Analytical method spectrometry (LC-MS)/high-resolution MS chromatography-high-resolution Reversed phase, C18, 120 µ L/min Monolithic, 750 nL/min (LC-HRMS) mass spectrometry (nLC-HRMS) column Type Type oof f LC column Reversed phase, C18, 120 L/min Monolithic, 750 nL/min MS: Triple quadropole in multiple reaction MS and HRMS: Orbitrap in data MS: Triple quadropole in multiple reaction monitoring mode; HRMS: Orbitrap in data Type of MS and MS HRMS: Orbitrap in data MS monitoring mode; HRMS: Orbitrap in data dependent acquisition mode method dependent acquisition mode independent acquisition mode independent acquisition mode method Figure 1. Following tryptic digestion, Cys34 is located on the third largest tryptic peptide, designated T3. Figure 1. Following tryptic digestion, Cys34 is located on the third largest tryptic peptide, designated T3. The methodology for Hb adductomics is based on a modiﬁed Edman procedure in which adducted N-terminal amino acids are speciﬁcally detached using the reagent ﬂuorescein isothiocyanate (FITC) (Figure 2). In the 1980s, during the development of the ﬁrst successful method for analysis of N-terminal Hb adducts it was found that the detachment of adducted N-terminal Val with isothiocyanate reagents was favored over non-adducted Val, providing a basis for selective enrichment of adducts [39–41]. This methodology was then developed for LC-MS analysis using FITC as the reagent [42,43]. To perform Hb adductomics 5 mg of FITC (dissolved in dimethyl sulfoxide) is added to 250 L of whole blood or lysate (both work equally well for the methodology) and mixed at 37 C overnight . This reaction, an example of a modiﬁed Edman degradation, generates High-Throughput 2019, 8, x FOR PEER REVIEW 5 of 17 The methodology for Hb adductomics is based on a modified Edman procedure in which adducted N-terminal amino acids are specifically detached using the reagent fluorescein isothiocyanate (FITC) (Figure 2). In the 1980s, during the development of the first successful method for analysis of N-terminal Hb adducts it was found that the detachment of adducted N-terminal Val High-Thr wioughput th isoth 2019 iocya , 8,n 6ate reagents was favored over non-adducted Val, providing a basis for selective 5 of 18 enrichment of adducts [39–41]. This methodology was then developed for LC-MS analysis using FITC as the reagent [42,43]. To perform Hb adductomics 5 mg of FITC (dissolved in dimethyl sulfoxide) is detached adduct derivatives, in the form of ﬂuorescein thiohydantoins (FTHs) (Figure 2). To purify the added to 250 µ L of whole blood or lysate (both work equally well for the methodology) and mixed FTH a derivatives, t 37°C overni the ght pr [4oteins 4]. This ar re eaﬁrst ction,pr an ecipitated example o with f a m acetonitrile odified Edm(ACN) an degra and datio then n, genera centrifuged. tes detached adduct derivatives, in the form of fluorescein thiohydantoins (FTHs) (Figure 2). To purify At this step, stable isotope-substituted internal standards corresponding to one or several FTH the FTH derivatives, the proteins are first precipitated with acetonitrile (ACN) and then centrifuged. derivatives of known adducts are added. The FTH derivatives have a carboxylic acid functional At this step, stable isotope-substituted internal standards corresponding to one or several FTH group (originating from the ﬂuorescein part of the molecules), which may be protonated/deprotonated derivatives of known adducts are added. The FTH derivatives have a carboxylic acid functional with acids/bases. This property is utilized in the work-up of the FTHs. Following precipitation and group (originating from the fluorescein part of the molecules), which may be centrifugation, the FTH-containing ACN supernatant is alkalized (deprotonation of the carboxylic protonated/deprotonated with acids/bases. This property is utilized in the work-up of the FTHs. acid) with ammonium hydroxide then added to a mixed-mode anion exchange solid phase extraction Following precipitation and centrifugation, the FTH-containing ACN supernatant is alkalized (SPE) column. The deprotonated anionic FTHs are retained on the column, which are then washed (deprotonation of the carboxylic acid) with ammonium hydroxide then added to a mixed-mode anion with ACN, H O, and cyanoacetic acid in H O, in that order. To elute the FTHs a solution of cyanoacetic exchange 2 solid phase extraction (SPE) co 2lumn. The deprotonated anionic FTHs are retained on the acid in colACN umn, wh is used, ich arewhich then wbr ash eaks ed wi both th ACN, the H hydr 2O, a ophobic nd cyanoand acetianionic c acid in interactions H2O, in that ord with er. To the elcolumn. ute the FTHs a solution of cyanoacetic acid in ACN is used, which breaks both the hydrophobic and The eluted samples are evaporated to dryness using air and then re-dissolved in H O/ACN (6:4), anionic interactions with the column. The eluted samples are evaporated to dryness using air and preparing the samples for LC-MS analysis. Although the sample processing for Hb adductomics is then re-dissolved in H2O/ACN (6:4), preparing the samples for LC-MS analysis. Although the sample more time-consuming than the method for HSA adductomics, with the over-night derivatization being processing for Hb adductomics is more time-consuming than the method for HSA adductomics, with the limiting step, the method is fast compared to earlier methods for Hb adduct analysis [40,45] and is the over-night derivatization being the limiting step, the method is fast compared to earlier methods applicable for semi-high throughput analysis [44,46]. for Hb adduct analysis [40,45] and is applicable for semi-high throughput analysis [44,46]. Figure Fig 2. ure Adducted 2. AdductN-terminal ed N-termina amino l amino acids acids ar are e detached detached usi using ng ththe e rea reagent gent fluo ﬂuor rescei escein n isoth isothiocyanate iocyanate (FITC) generating adduct derivatives in the form of fluorescein thiohydantoins (FTHs). (FITC) generating adduct derivatives in the form of ﬂuorescein thiohydantoins (FTHs). 4. Mass 4. Ma Spectrometry ss Spectrometry The method for HSA adductomics uses nanoflow LC and nanoelectrospray-ionization and the The method for HSA adductomics uses nanoﬂow LC and nanoelectrospray-ionization and the mass spectrometry is performed with a Hybrid Ion Trap Orbitrap high-resolution mass spectrometer mass spectrometry is performed with a Hybrid Ion Trap Orbitrap high-resolution mass spectrometer (HRMS) (ThermoFisher Scientific, MA, USA) . One microliter of each sample digest is injected (HRMS) (ThermoFisher Scientiﬁc, MA, USA) . One microliter of each sample digest is injected into into a monolithic nanoflow column that has excellent properties for separation of hydrophobic a monolithic nanoﬂow column that has excellent properties for separation of hydrophobic peptides peptides and proteins. The analysis requires about 40 min per injection, and each sample is injected and proteins. The analysis requires about 40 min per injection, and each sample is injected in duplicate in duplicate followed by blank and wash injections, making the total time per sample about 2 h. The followed by blank and wash injections, making the total time per sample about 2 h. The ionization is ionization is done in positive-ion mode and the mass spectrometer is operated in the data-dependent done in positive-ion mode and the mass spectrometer is operated in the data-dependent acquisition acquisition (DDA) mode, which selects the most abundant ions for fragmentation (MS2) using the (DDA) mode, which selects the most abundant ions for fragmentation (MS2) using the ion trap. ion trap. The combination of high resolution MS1 data from the Orbitrap analysis and ion trap MS2 data, enables the classification of unknown compounds as adducts and determination of adduct The combination of high resolution MS1 data from the Orbitrap analysis and ion trap MS2 data, masses with high accuracy. enables the classiﬁcation of unknown compounds as adducts and determination of adduct masses with high accuracy. Hemoglobin adductomics has almost exclusively been performed with triple quadrupole mass spectrometers which require a different strategy for untargeted screening of adducts compared to high resolution mass spectrometers. Similar to early methods for DNA adductomics and HSA adductomics [17,18], the screening of N-terminal Hb adducts using triple quadrupole MS has been done in the multiple reaction monitoring (MRM) mode . From the analysis of FTH derivatives of known N-terminal Hb adducts it was observed that all such analytes exhibit similar fragmentation pathways when performing MS/MS, resulting in at least three common fragments (Figure 3). This observation is the underlying principle for MRM screening of FTH derivatives of unknown N-terminal Hb adducts, High-Throughput 2019, 8, x FOR PEER REVIEW 6 of 17 Hemoglobin adductomics has almost exclusively been performed with triple quadrupole mass spectrometers which require a different strategy for untargeted screening of adducts compared to high resolution mass spectrometers. Similar to early methods for DNA adductomics and HSA adductomics [17,18], the screening of N-terminal Hb adducts using triple quadrupole MS has been done in the multiple reaction monitoring (MRM) mode . From the analysis of FTH derivatives of known N-terminal Hb adducts it was observed that all such analytes exhibit similar fragmentation pathways when performing MS/MS, resulting in at least three common fragments (Figure 3). This observation is the underlying principle for MRM screening of FTH derivatives of unknown N- High-Throughput 2019, 8, 6 6 of 18 terminal Hb adducts, in which sequential lists of MRM transitions are set up for the m/z range of interest with four diagnostic fragments, corresponding to the most common fragmentation pathways, being screened for each incremental m/z unit. Although the most sensitive alternative on a triple in which sequential lists of MRM transitions are set up for the m/z range of interest with four quadrupole platform, the analysis of large numbers of MRM transitions is relatively slow. To acquire diagnostic fragments, corresponding to the most common fragmentation pathways, being screened for a sufficient number of data points for quantitative analysis each sample needs to be injected several each incremental m/z unit. Although the most sensitive alternative on a triple quadrupole platform, times, covering different increments of the studied m/z range with each injection. In the first the analysis of large numbers of MRM transitions is relatively slow. To acquire a sufﬁcient number of application of Hb adductomics each sample was injected 12 times to cover a range of 135 m/z, data points for quantitative analysis each sample needs to be injected several times, covering different demonstrating the time-consuming aspects of this methodology . Recently, the methodology has increments of the studied m/z range with each injection. In the ﬁrst application of Hb adductomics been adapted for HRMS using a Hybrid Quadrupole Orbitrap MS (Thermo Scientific, MA, USA) . each sample was injected 12 times to cover a range of 135 m/z, demonstrating the time-consuming On this platform the MS analysis was performed in the data independent acquisition (DIA) mode. In aspects of this methodology . Recently, the methodology has been adapted for HRMS using a this mode, all ions within a specified m/z range are fragmented, with collective fragmentation patterns for all ions within that range recorded. This is an advantageous approach when screening Hybrid Quadrupole Orbitrap MS (Thermo Scientiﬁc, MA, USA) . On this platform the MS analysis for low-level compounds, such as adducts, in complex biological samples compared to DDA for was performed in the data independent acquisition (DIA) mode. In this mode, all ions within a which only abundant ions will be fragmented, thereby risking missing interesting compounds. In a speciﬁed m/z range are fragmented, with collective fragmentation patterns for all ions within that blood sample derivatized with FITC the concentration of reagent byproducts is high (5 mg FITC is range recorded. This is an advantageous approach when screening for low-level compounds, such as used for each 250 µ L of blood; see Sample Preparation above), making DIA a better suited alternative adducts, in complex biological samples compared to DDA for which only abundant ions will be to DDA to maximize detection of potential adducts. A disadvantage with DIA is that the fragmented, thereby risking missing interesting compounds. In a blood sample derivatized with measurements are slower than for DDA, meaning that the samples need to be injected several times FITC the concentration of reagent byproducts is high (5 mg FITC is used for each 250 L of blood; to cover large m/z ranges. When screening the m/z range 500–700 m/z for Hb adducts each sample see Sample Preparation above), making DIA a better suited alternative to DDA to maximize detection was injected four times, with each injection covering 50 m/z . To confirm possible adducts of potential adducts. A disadvantage with DIA is that the measurements are slower than for DDA, observed in the DIA mode, additional follow-up measurements were performed in the parallel meaning reactithat on m the onito samples ring moneed de, into wh be ich i injected ons of spec several ific m times /z (un to it r cover esoluti lar onge ) are m/z frag ranges. mented.When screening For Hb adductomics conventional LC-MS has been performed using a flow rate of 0.12 mL/min the m/z range 500–700 m/z for Hb adducts each sample was injected four times, with each injection and electrospray ionization in the positive mode . A volume of 10–20 µ L of the sample is injected covering 50 m/z . To conﬁrm possible adducts observed in the DIA mode, additional follow-up (depending on type of instrumentation and purpose of experiment) on a reversed phase C18 column measurements were performed in the parallel reaction monitoring mode, in which ions of speciﬁc m/z with the dimensions 2.1 mm × 150 mm and a gradient is applied to separate sample components. The (unit resolution) are fragmented. chromatographic program is 35 minutes per injection. Figure Fig3. ure Fluor 3. Fluo escein rescein thiohydantoin thiohydantoin (FTH) (FTH) de derivatives rivatives of of NN-terminal -terminal HbHb addadducts ucts exhib exhibit it simila similar r fragmentation pathways when performing tandem mass spectrometry (MS/MS). fragmentation pathways when performing tandem mass spectrometry (MS/MS). 5. Data Evaluation For Hb adductomics conventional LC-MS has been performed using a ﬂow rate of 0.12 mL/min and electrospray ionization in the positive mode . A volume of 10–20 L of the sample is injected Untargeted screening MS experiments generate huge and complex sets of data, that are both (depending difficult on antype d timof e-co instr nsum umentation ing to evaand luate. purpose In comof pari experiment) son with meta onba olro eversed mics anphase d proteo C18 mics column , adductomics is a small field of research and there are no commercial software solutions for the with the dimensions 2.1 mm 150 mm and a gradient is applied to separate sample components. evaluation of adductomic data. The chromatographic program is 35 min per injection. For HSA adductomics, unknown species detected as triply charged ions in the HRMS are + 2+ classified as Cys34 HSA adducts if they exhibit a sufficient number of b - and y -fragments that are 5. Data Evaluation Untargeted screening MS experiments generate huge and complex sets of data, that are both difﬁcult and time-consuming to evaluate. In comparison with metabolomics and proteomics, adductomics is a small ﬁeld of research and there are no commercial software solutions for the evaluation of adductomic data. For HSA adductomics, unknown species detected as triply charged ions in the HRMS are classiﬁed + 2+ as Cys34 HSA adducts if they exhibit a sufﬁcient number of b - and y -fragments that are characteristic of the T3 peptide (m/z = 811.7594). This process is described in detail by Grigoryan et al. 2016 . To facilitate this time-consuming task, in-house software, written in the programming language R, has been developed for processing MS2 spectra according to the established rules for adduct selection . The internal standard that is added in the ﬁnal step of sample preparation (isotopically labeled T3 modiﬁed with iodoacetamide) is used to monitor variations in retention times and mass High-Throughput 2019, 8, 6 7 of 18 accuracy throughout the analysis. The resulting processed spectra are inspected manually to control for false positives. For quantiﬁcation of detected adducts, selected ion chromatograms generated using the monoisotopic masses (MIMs) of the adducted T3 peptides are used. Peak area ratios relative to the HK peptide facilitate relative quantitation and statistical analyses and can provide approximate concentrations in pmol adduct/mg HSA . The added masses of the adducts are calculated by subtracting the MIM of the thiolate form of the unmodiﬁed T3 peptide from the MIM of the adducted T3 peptides. The calculated added masses are used to propose elemental compositions of adducts and to suggest likely structures and electrophilic precursors. When evaluating data from Hb adductomics, adduct candidates are selected based on the observation of at least two of the four screened diagnostic fragments (see Mass Spectrometry Analysis above and Figure 3). Compounds considered as possible adducts are further studied in product ion scan mode and their fragmentation patterns compared with those of known adduct analytes. The selection of adducts is done manually. The evaluation of MRM data is time-consuming but rather straightforward, which is not the case for DIA data for which the evaluation requires extensive manual inspection as well as follow-up experiments to select adducts with certainty. To determine the added mass of an adduct, the m/z of the FTH derivative of unmodiﬁed Val is subtracted from the m/z of the FTH derivative of the Val adduct. Adduct levels of Hb adducts are estimated using a semiquantitative approach, assuming that all FTH derivatives of Val adducts have similar responses in the MS analysis. Internal standard calibration has been done using the stable-isotope internal standard corresponding to the FTH derivative of the Val adduct of acrylamide (AA), which is added to all samples during the sample preparation immediately after the FITC derivatization. A calibration curve of a reference standard of AA-Val-FTH and the corresponding internal standard is used for this purpose. The average of the integrated peak areas for the detected diagnostic transitions for each adduct is used for the determinations. The adduct levels are then adjusted for the Hb concentrations in the blood samples, measured separately using a spectrophotometric device. Adduct levels are reported in the unit pmol/g Hb. The assumption that all FTH derivatives of Val adducts have similar responses in the MS analysis is a simpliﬁcation but a reasonable approximation for the low molecular weight adducts observed. The supposed similar responses are supported by the comparable responses for the different known adducts that have been studied as FTH derivatives in more detail [48–52]. 6. Identiﬁcation of Adducts Following untargeted detection of adducts the next challenge is their identiﬁcation. This section describes how previously unknown adducts have been identiﬁed following HSA and Hb adductomics. With HSA adductomics about 75 adducts (or HSA variants) have been reported thus far with added masses ranging from 46 to 510 Da [36,53–55] (negative added masses refer to truncations and deletions). Most of these adducts have been annotated to presumed structures and 11 have been identiﬁed through comparisons with reference standards [36,55,56]. The HSA adducts detected in the four published adductomics studies are presented in Table 3. Annotations include Cys34 oxidation products, truncations and deletions, and disulﬁdes of low-molecular-weight thiols. Many of the HSA modiﬁcations detected through adductomics result from oxidation of Cys34 to produce adducts with one, two or three oxygens. Because the one-oxygen addition product, i.e. the sulfenic acid (Cys34-SOH) is unstable, it reacts with the adjacent amino acid Gln33 to produce a cyclic sulﬁnamide, which is detected . Formation of the Cys34 sulfenic acid, also leads to disulﬁde formation with small circulating thiols, which represent the largest class of adducts detected with HSA adductomics thus far. With Hb adductomics 25 different N-terminal Val adducts have been detected, with added masses ranging from 15 to 198 Da, with 13 of them identiﬁed thus far [19,50,52,57]. These Hb adducts are presented in Table 4. High-Throughput 2019, 8, 6 8 of 18 Table 3. HSA adducts detected on the T3 peptide that contain Cys34. Ret. Time MIM Observed Added Mass Suggested Elemental Composition of Adduct Annotation (min) (m/z, +3) (Da) Added Mass (to Cys34S ) a,b,d 27.5 796.4309 45.9913 CH S Cys34!Gly 796.43 a,b,d 800.43 28.4 800.4317 33.9873 SH Cys34!Dehydroalanine a,b,d,e 27.2 805.7632 17.9965 SH , +O Cys34!Oxoalanine 805.76 a,b,c,d 808.73 28.3 808.7306 9.0923 Not Cys34 adduct a,c 810.45 30.5 810.4536 3.9280 Not Cys34 adduct a,b,c,d f 811.43 30.4 811.4254 2431.2480 +C H N O S T3 Dimer 114 172 27 30 a,b,c,d 811.76_1 27.9 811.7608 1.0072 +H T3 Labile adduct a,b,c,d,e f 811.76_2 28.6 811.7609 1.0097 +H Unmodiﬁed T3 a,b,c,d,e f 27.8 816.4200 13.9766 H , +O 816.42 S-Monooxidation a,b,c,d,e 816.43 29.1 816.4321 15.0233 +CH Methylation (not at Cys34) a,c,d 820.09 28.8 820.0920 25.9995 +CN S-Cyanylation a,b,c,d,e f 822.42 27.7 822.4236 32.9946 +HO S-Dioxidation 823.39 27.2 823.3971 35.9173 Not Cys34 adduct 825.76 26.7 825.7644 43.0188 +C H O S-Acetylation 2 3 26.7 826.0990 44.0226 +C H O Unknown (likely S-addition of an aldehyde) 826.10 2 4 826.44 27.9 826.4358 45.0332 +C H O Ethylene oxide adduct 2 5 b,d 827.09 28.8 827.0901 46.9960 +CH S S-Methylthiolation 827.09 30.1 827.0946 47.0022 +HO + CH Cys34 sulﬁnic acid plus methylation (not Cys34) 2 2 27.5 827.0958 47.0129 +CH O S-(O)-O-CH3 827.1 3 2 a,b,c,d,e f 827.76 28.0 827.7550 48.9889 +HO S-Trioxidation 829.13 27.4 829.1264 53.1052 Not Cys34 adduct a,b 829.40 28.6 829.3972 53.9177 Not Cys34 adduct 829.44 28.9 829.4369 54.0366 +C H N Acrylonitrile adduct 3 4 26.5 830.4059 56.9433 Unknown 830.41 830.44 27.3 830.4359 57.0333 +C H O Unknown (likely S-addition of an aldehyde) 3 5 830.77 27.5 830.7685 58.0313 +C H NO Methylisocyanate adduct 2 4 27.8 833.0813 64.9696 +HO S S-Addition of SO 833.08 2 2 a, d f 835.11 28.2 835.1079 71.0494 +C H O S-Addition of crotonaldehyde 4 7 d,e 837.10 27.4 837.1041 77.0380 +C H S-Phenylation 6 5 d,e f 27.7 839.7797 85.0647 +C H O 839.78 S-Addition of tiglic aldehyde 5 9 High-Throughput 2019, 8, 6 9 of 18 Table 3. Cont. Ret. Time MIM Observed Added Mass Suggested Elemental Composition of Adduct Annotation (min) (m/z, +3) (Da) Added Mass (to Cys34S ) S-Addition of pyruvate or malonate b,c 28.5 841.0987 89.0183 +C H O 841.10 3 5 3 semialdehyde 841.43 27.8 841.4251 90.0013 +C H NOS S-Mercaptoacetamide 2 4 a,b,c,d 841.75 28.6 841.7529 90.9827 +C H O S S-Addition of mercaptoacetic acid 2 3 2 a,b,c,d 845.42 27.7 845.4249 101.9987 +C H NOS S-Addition of Cys (-H O) 3 4 2 28.6 845.7528 102.9842 +C H O S S-Cys (possibly NH ! OH, H O) 845.75 3 3 2 2 2 847.10 26.7 847.0963 107.0145 +C H O S S-Methylethyl-sulfonylation 3 7 2 a,b,c 30.2 847.1082 107.0481 +C H O S-Addition of benzaldehyde or quinone methide 847.11 7 7 a,b 847.77 27.3 847.7664 109.0251 Cys34 Adduct with unknown annotation a,b,d 849.07 28.0 849.0698 112.9353 +HO S S-Addition of S O H 3 2 2 3 a,b,d 27.9 850.0975 116.0182 +C H NOS S-Addition of hCys (-H O) 850.10 4 6 2 a,b,c,d f 851.43 26.9 851.4290 120.0109 +C H NO S S-Addition of Cys 3 6 2 a,b,c,d 27.8 851.7572 120.9973 +C H O S S-Addition of Cys (NH !OH) 851.76 3 5 3 2 a,b 853.78 27.7 853.7839 127.0776 Cys34 Adduct with unknown annotation 854.44 25.9 854.4428 129.0539 +C H O S-Addition of BDE 6 9 3 855.44 29.0 855.4373 132.0378 +C H NO Oxindole 8 6 a,b,c,d f 856.10_1 27.0 856.1003 134.0250 +C H NO S S-Addition of hCys 4 8 2 a,b,c,d f 27.3 856.1001 134.0244 +C H NO S 856.10_2 S-Addition of hCys 4 8 2 856.43 27.2 856.4287 135.0117 +C H O S S-Addition of hCys (NH !OH) 4 7 3 2 857.09 29.0 857.0876 136.9812 Unknown a,b,d 857.1 27.4 857.1000 137.0257 Unknown 859.41 25.5 859.4085 143.9513 Unknown b,c 860.77 29.3 860.7717 148.0342 +C H NO S + CH S-hCys, plus methylation (not Cys34) 4 8 2 2 a,d 26.3 862.0881 151.9901 Not Cys34 adduct 862.09 a,b,d 864.08 26.2 864.0772 157.9574 Not Cys34 adduct a,b,c,d 27.5 864.4319 159.0198 +C H N O S S-Addition of CysGly (-H O) 864.43 5 7 2 2 2 a,b,c 865.43 28.3 865.4314 162.0176 +C H NO S S-(N-acetyl)Cys 5 8 3 866.76 27.0 866.7572 165.9973 +C H NO S S-S-hCys trisulﬁde 4 8 2 2 a,b,c,d f 26.5 870.4360 177.0319 +C H N O S 870.44 S-Addition of CysGly 5 9 2 3 872.7309 26.7 872.7309 183.9187 Not Cys34 adduct a,b,d 870.44 27.2 873.4310 186.0187 Unknown b,c 27.5 875.1062 191.0409 +C H N O S + CH S-CysGly, plus methylation (not Cys34) 875.11 5 9 2 3 2 875.42 27.7 875.4231 191.9950 Cys34 Adduct with unknown annotation b,c 894.13 26.1 894.1270 248.1029 Unknown High-Throughput 2019, 8, 6 10 of 18 Table 3. Cont. Ret. Time MIM Observed Added Mass Suggested Elemental Composition of Adduct Annotation (min) (m/z, +3) (Da) Added Mass (to Cys34S ) a,b,c,d f 27.0 894.4426 249.0516 +C H N O S 894.44 S-Addition of GluCys 8 13 2 5 899.11 29.3 899.1140 263.0612 Unknown a,b,c,d f 913.45 26.9 913.4494 306.0722 +C H N O S S-Addition of GSH 10 16 3 6 918.12 29.2 918.1222 320.0852 Unknown 927.14 27.1 927.1408 347.1412 Unknown 928.78 32.2 928.7842 352.0712 Unknown b,c,d 25.9 931.8215 361.1881 Unknown 931.82 941.16 25.3 941.1570 389.1967 Unclear modiﬁcation site b,c,d 965.49 25.8 965.4920 462.1994 Unknown c c 970.16 28.1 970.1643 476.2112 Unknown b,d 974.51 25.1 974.5068 489.2460 Unknown b,c 976.82 28.0 976.8205 496.1840 Unknown 25.3 981.4956 510.2126 Cys34 adduct with unknown annotation 981.50 a b c d Notes: GSH, glutathione; hCys, homocysteine; MIM, monoisotopic mass. Detected by Grigoryan et al. 2016 . Detected by Lu et al. 2017 . Detected by Liu et al. 2018 . e f Detected by Grigoryan et al. 2018 . Adduct was detected in samples that were reduced with tris(2-carboxyethyl)phosphine (TCEP) prior to digestion; reduces all disulﬁde bonds. Annotation conﬁrmed with a synthetic standard. High-Throughput 2019, 8, 6 11 of 18 Table 4. N-terminal Hb adducts detected using adductomics to date. Identity/Precursor Elemental Composition [M+H] m/z rt (min) Added Mass (Da) to Val-NH b, c Methylation 16.6 15 CH 503.1 b, c 517.1 Ethylation 17.9 29 C H 2 5 519.1 Unknown 12.3 31 Unknown 519.1 Unknown 15.7 31 Unknown b, c Ethylene oxide 14.1 45 C H O 533.1 2 5 b d 542.1 Acrylonitrile 18.0 54 C H N 3 4 b, c 547.1 Carboxy-methylation/Glyoxal 14.2 59 C H O 2 3 2 b, c Methyl vinyl ketone 16.6 71 C H O 559.1 4 7 b, c 560.1 Acrylamide 12.9 72 C H NO 3 6 b, c Acrylic acid/Carboxyethylation 14.5 73 C H O 561.1 3 5 2 561.1 Methylglyoxal 12.6 73 C H O 3 5 2 563.1 Glycidol 12.4 75 C H O 3 7 2 b, c 573.1 Ethyl vinyl ketone 18.1 85 C H O 5 9 576.1 Glycidamide 12.2 88 C H NO 3 6 2 b, c Unknown 13.0 89 Unknown 577.1 b, c 593.1 Unknown 11.3 105 Unknown 595.1 Unknown 15.1 107 Unknown b,c 4-Hydroxybenzyl 17.00 107 C H O 595.1 7 7 b,c 615.1 1-Octen-3-one 22.2 127 C H O 8 15 b,c 617.1 Unknown 14.8 129 Unknown b,c 625.1 Unknown 13.9 137 Unknown b,c 631.1 Unknown 15.2 143 Unknown 651.1 Unknown 11.2 163 Unknown 659.1 Unknown 16.4 171 Unknown 686.1 Unknown 11.2 198 Unknown a b c Notes: The names of identiﬁed adducts or known/probable precursors are in bold (conﬁrmed with reference adducts). Detected by Carlsson et al. 2014 . Detected by Carlsson et al. 2017 . Acrylonitrile adducts are typically only observed in samples from smokers. High-Throughput 2019, 8, 6 12 of 18 The results of an adductomic experiment will be a list of adducts detected, with their molecular weights, estimated concentrations, and retention times. This is the only information available to formulate hypotheses about adduct identities. The added mass may be calculated as described above, by subtracting the m/z of an unmodiﬁed precursor molecule, from the adduct m/z. The accuracy of the calculated added mass will depend on the type of mass spectrometer used, with high resolution instruments providing more accurate data. Based on the added mass the elemental composition of the adduct may be suggested. A practical tool to help with this task is the Molecular Formula ﬁnder, provided by ChemCalc (http://www.chemcalc.org/mf_ﬁnder) . With a likely composition of the adduct, the adduct structure or corresponding precursor electrophiles can be suggested. As described above, electrophiles that form adducts commonly have reactive functional groups, such as activated double bonds, aldehydes, and epoxides. Such features should be considered when considering possible precursors, as well as the chemistry of the speciﬁc nucleophilic sites and limitations of the methodology used. One limitation with the present method for Hb adductomics is when the N-terminal nitrogen atom is blocked for the reaction with the isothiocyanate reagent, as when it is tertiary or when it is substituted with acyl groups. This exclude ring-closed adducts, e.g., the adduct from diepoxybutane , from being detected. Adducts from isocyanates, carbamoylated N-terminal valines, could though be detached as hydantoins from Hb after acidiﬁcation in a procedure similar to the normal Edman procedure, as has been shown in several studies (e.g., for methylisocyanate  and dimethylformamide ). Of the identiﬁed or annotated adducts observed to Hb and HSA there has been very little overlap thus far (when analyzing blood samples from smokers, acrylonitrile, and ethylene oxide adducts were observed both to Hb and HSA [19,36], and the recently identiﬁed 4-hydroxybenzyl Hb adduct may correspond to the unknown HSA adduct with the same annotated composition C H O, added mass 107.0481 Da ). This emphasizes the need of several nucleophilic 7 7 targets when exploring the adductome. With adducts of low mass there may be few possible structures and precursors, facilitating the adduct identiﬁcation. However, as MWs exceed about 70 Da the numbers of possibilities increase rapidly, and annotation becomes difﬁcult. To facilitate hypotheses about possible annotations specialized databases can be used. The simplest of these databases is “Search for Species Data by Molecular Weight” provided by the National Institute of Standards and Technologies (https: //webbook.nist.gov/chemistry/mw-ser) , which list species based on the input value. Although there is no speciﬁc database for adductomics, the UNIMOD database that lists protein modiﬁcations may be useful (www.unimod.org) . Databases for metabolomics may also contain valuable information. Among these there are large databases such as the human metabolome database (http://www.hmdb.ca/metabolites) , but also more speciﬁc databases such as the Toxic Exposome Database (T3DB) (http://www.t3db.ca)  and the Exposome-Explorer Database (http://exposome- explorer.iarc.fr)  can be relevant for adductomics . Having established one or more candidate annotations for a given adduct, the next step is to perform literature searches, consider possible metabolic activations of possible precursors, working backwards to determine whether suspected adduct precursors have been previously described. Both Hb and HSA adductomics are performed with gradient-elution reversed-phase chromatography that separates compounds based on lipophilicity. Thus, the retention times of the analytes correlate with their lipophilicities, and this information can also be useful in suggesting adduct identities. For FTH derivatives of Val adducts the theoretical Log P values (calculated using ChemDraw from CambridgeSoft, Cambridge, MA, USA) correlate with their retention times (with a coefﬁcient of determination, R = 0.88) . By drawing the hypothetical adduct derivatives in ChemDraw and comparing the theoretical Log Ps with the observed retention times of the investigated unknown adducts, the hypotheses may be further evaluated prior to initiating the costly and time-consuming identiﬁcation. For the FTH derivatives with relatively low MWs generally ranging from about 500–700 Da, this is a very viable strategy while the situation for the larger T3 adducts (MWs > 2432 Da) is more complex. For such large molecules the retention times are not as affected by small modiﬁcations High-Throughput 2019, 8, 6 13 of 18 and cannot be expected to be as reproducible as for reverse phase separations of small molecules. Although the chromatographic program includes a 30 min gradient (from 2–45% ACN), the majority of T3 adducts elute during a 5 min period, indicating the small differences in retention even for large modiﬁcations such as glutathione (added mass of 306 Da) . To conﬁrm adduct identities, reference adducts should be synthesized and compared with the unknown adducts of interest. Generation of reference adducts can simply involve adding the proposed precursor electrophiles to either plasma or whole blood/lysate. The concentrations of the electrophiles and the reaction times necessary to yield sufﬁcient concentrations of the adducts vary depending on the reactivity and stability of the electrophiles. Detailed experiments for generating reference adducts have previously been published by Grigoryan et al. [36,55,56] for HSA adducts, and Carlsson et al. [19,57] for Hb adducts. Following incubations with electrophiles the samples should be processed and analyzed according to the respective methods. The synthetic adducts should then be compared with the unidentiﬁed adducts observed concurrently in untreated human samples to minimize methodological and instrumental variations. The comparison should be done with regard to m/z of the precursor ions, fragmentation patterns (MS2) as well as retention times. The identiﬁcation process is summarized in the form of a ﬂowchart in Figure 4. High-Throughput 2019, 8, x FOR PEER REVIEW 12 of 17 Subtract m/z of Unknown adduct Adduct mass unmodified peptide MS1, MS2, retention time, Suggested elemental from adduct m/z concentration. composition Search for matching compounds. Databases and literature. Reference adduct Generate reference Proposed structure Compare MS1, MS2 and adduct Proposed electrophilic retention time with unknown precursor adduct Figure Figure4. 4. Flowchart Flowchart depicting depicting the the pr process ocess of of identifying identifyingunknown unknownadducts adducts using usingadductome adductome d data. ata. The small FTH derivatives of Hb-Val adducts are advantageous for identiﬁcation purposes, The small FTH derivatives of Hb-Val adducts are advantageous for identification purposes, with with highly reproducible retention times and distinct MS2 spectra in which the relative abundances highly reproducible retention times and distinct MS2 spectra in which the relative abundances of of fragments may be used to match spectra for unknown and reference adducts with high fragments may be used to match spectra for unknown and reference adducts with high agreement agreement [50,57]. For example, the electrophiles methylglyoxal and acrylic acid form Hb adducts [50,57]. For example, the electrophiles methylglyoxal and acrylic acid form Hb adducts with the same with the same elemental composition, assumed to be 1-carboxyethyl-Val, and 2-carboxyethyl-Val elemental composition, assumed to be 1-carboxyethyl-Val, and 2-carboxyethyl-Val respectively, but respectively, but the retention times and MS2 spectra of their FTH derivatives are signiﬁcantly different the retention times and MS2 spectra of their FTH derivatives are significantly different and allows and allows for unambiguous identiﬁcation . This simpler pathway of generating reference adducts for unambiguous identification . This simpler pathway of generating reference adducts does not does not mean that a fully characterized (with NMR) reference compound is not often desired, mean that a fully characterized (with NMR) reference compound is not often desired, and sometimes and sometimes required for unequivocal identiﬁcation. required for unequivocal identification. The identiﬁcation of adducts is complicated and time-consuming, and potentially expensive. The identification of adducts is complicated and time-consuming, and potentially expensive. In In many cases there will be structural isomers that will complicate structural identiﬁcation. For some many cases there will be structural isomers that will complicate structural identification. For some small adducts, such as addition of one, two or three oxygens to Cys34, accurate mass is sufﬁcient to small adducts, such as addition of one, two or three oxygens to Cys34, accurate mass is sufficient to conﬁrm the identity. However, in most cases the adducts and often precursor molecules will require confirm the identity. However, in most cases the adducts and often precursor molecules will require regular organic synthesis work. A recent example, representing a chemically complex generation regular organic synthesis work. A recent example, representing a chemically complex generation of of a reference adduct is the identiﬁcation of 4-hydroxybenzyl adducts to N-terminal Val in Hb . a reference adduct is the identification of 4-hydroxybenzyl adducts to N-terminal Val in Hb . Two Two precursors were found to generate the adduct; 4-quinone methide and 4-hydroxybenzaldehyde. precursors were found to generate the adduct; 4-quinone methide and 4-hydroxybenzaldehyde. The The quinone methide is not commercially available and not chemically stable. A precursor with quinone methide is not commercially available and not chemically stable. A precursor with protective protective groups was therefore synthesized in-lab, and activated prior to the reaction with Val. groups was therefore synthesized in-lab, and activated prior to the reaction with Val. This reflect the This reﬂect the often complex process to generate a reference adduct. often complex process to generate a reference adduct. 7. Conclusions The concept of protein adductomics was introduced nearly a decade ago. Since then the methodologies have been refined for the current generation of instrumentation and adapted to showcase the strengths and possibilities of adductomics. Many previously unknown adducts have been detected, and many of them have been identified. When comparing the present methodologies for HSA and Hb adductomics, the HSA method is more versatile and suited for high-throughput studies. By analyzing tryptic digests of HSA this method is truly untargeted and although adducts to Cys34 have been the primary focus to date, adducts to other nucleophilic sites in HSA (e.g., Lys199) may be detected simultaneously in a single LC-MS run. Other advantages of HSA adductomics is the low sample volumes needed (5 µ L) and the streamlined selection of adducts using the in-house software. The method for Hb adductomics is limited to N-terminal Hb adducts and require relatively large sample volumes (250 µ L), and will benefit from lower detection levels with more sensitive MS instrumentation than used in the published adductomics work. This methodology, however, benefits from the relatively small molecules resulting from the derivatization of N-terminal adducts, that are advantageous in the identification of unknown adducts. The FTH derivatives separate well using reversed phase chromatography, with highly reproducible retention times, and give distinct MS2 spectra. The minimal overlap of the adducts so far detected and identified with the different methods highlights the need to analyze several nucleophilic sites to explore the full adductome. In the small field of adductomics the two methods described in this review are the two best suited methods for High-Throughput 2019, 8, 6 14 of 18 7. Conclusions The concept of protein adductomics was introduced nearly a decade ago. Since then the methodologies have been reﬁned for the current generation of instrumentation and adapted to showcase the strengths and possibilities of adductomics. Many previously unknown adducts have been detected, and many of them have been identiﬁed. When comparing the present methodologies for HSA and Hb adductomics, the HSA method is more versatile and suited for high-throughput studies. By analyzing tryptic digests of HSA this method is truly untargeted and although adducts to Cys34 have been the primary focus to date, adducts to other nucleophilic sites in HSA (e.g., Lys199) may be detected simultaneously in a single LC-MS run. Other advantages of HSA adductomics is the low sample volumes needed (5 L) and the streamlined selection of adducts using the in-house software. The method for Hb adductomics is limited to N-terminal Hb adducts and require relatively large sample volumes (250 L), and will beneﬁt from lower detection levels with more sensitive MS instrumentation than used in the published adductomics work. This methodology, however, beneﬁts from the relatively small molecules resulting from the derivatization of N-terminal adducts, that are advantageous in the identiﬁcation of unknown adducts. The FTH derivatives separate well using reversed phase chromatography, with highly reproducible retention times, and give distinct MS2 spectra. The minimal overlap of the adducts so far detected and identiﬁed with the different methods highlights the need to analyze several nucleophilic sites to explore the full adductome. In the small ﬁeld of adductomics the two methods described in this review are the two best suited methods for analyzing large number of samples, even though the numbers of samples that can be analyzed during a certain period are limited compared to methods used, for example, for metabolomics. The constant improvement of MS instrumentation will beneﬁt adductomics by decreasing detection limits and improving mass accuracies. The most challenging aspect of adductomics is, however, the quantiﬁcation of unknown adducts. Hemoglobin adductomics rely on a more traditional approach with internal standard calibration and report estimated adducts levels assuming similar responses for all adducts. Human serum albumin adductomics employs an innovative housekeeping peptide to normalize peak areas for the amounts of HSA in the digests. Both approaches acknowledge the impossible task of giving accurate quantitative estimates of unknowns, and for the purposes of adductomics they work well, meaning that the relative concentrations of a set of adducts in a population of samples can be obtained for preliminary evaluations. If follow-up studies heighten interest in these adducts, then they can be identiﬁed and standardized for subsequent targeted analyses. Another challenging aspect of adductomics is formation of artifacts from storing and processing of samples. In the case of HSA adductomics the digestion procedure could potentially expose the nucleophilic sites in the protein for electrophiles present in the samples. For Hb adductomics the derivatization procedure could potentially form byproducts or chemically affect adducts. To control for such processes, various control experiments may be performed (cf. Carlsson 2014 ), but it is not likely that artefact formation for certain adducts, for instance during storage , may be fully controlled. Thankfully, it can be assumed that the extent of such artifact formation should be the same for all samples processed in a given study, e.g., from disease cases and concurrent controls, thereby reducing the likelihood of false positives in the statistical analysis. As a complement to the HSA adductomics method we are currently investigating the analysis of intact HSA isoforms in plasma samples, using a time-of-ﬂight MS. When analyzing the intact protein, the sample preparation prior to the MS analysis is minimal, as is the risk of introducing artifacts. Cysteine 34 of HSA is easily oxidized and the analysis of the intact protein provides valuable insights about the state of HSA in samples, prior to digestion. By comparing the two approaches, using both bottom-up and top-down proteomic data of HSA in plasma samples, a more complete view of HSA and the various modiﬁed forms is gained. An important goal of adductomics is to provide a deeper understanding of the underlying chemistry describing how proteins are modiﬁed by reactive compounds in vivo. With the methods described in this review we have increased our understanding of this fascinating aspect of biological chemistry. High-Throughput 2019, 8, 6 15 of 18 Author Contributions: H.C. planned and wrote the manuscript and prepared ﬁgures and tables. S.M.R. and M.T. assisted with the writing and gave expert advice regarding the topic. Funding: This research was supported by two grants from the US National Institutes of Health to S.M.R. (grant R33CA191159 from the National Cancer Institute and grant P42ES004705 from the National Institute for Environmental Health Sciences) and a grant from the Swedish Research Council to M.T. 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This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
Multidisciplinary Digital Publishing Institute
Protein Adductomics: Methodologies for Untargeted Screening of Adducts to Serum Albumin and Hemoglobin in Human Blood Samples
Rappaport, Stephen M.
, Volume 8 (1) –
Mar 8, 2019
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