MS, with or without pre-analysis peptide fractionation, can be used to decipher the residues on proteins where oxidative modifications caused by peroxynitrite, singlet oxygen or electrophilic lipids have occurred. Peroxynitrite nitrates tyrosine and tryptophan residues on the surface of actin. Singlet oxygen, formed by the interaction of UVA light with tryptophan, can oxidize neighbouring cysteine, histidine, methionine, tyrosine and tryptophan residues. Dose–response inactivation by 4HNE (4-hydroxynonenal) of hBAT (human bile acid CoA:amino acid N-acyltransferase) and CKBB (cytosolic brain isoform of creatine kinase) is associated with site-specific modifications. FT-ICR (Fourier-transform ion cyclotron resonance)–MS using nanoLC (nano-liquid chromatography)–ESI (electrospray ionization)–MS or direct-infusion ESI–MS with gas-phase fractionation identified 14 4HNE adducts on hBAT and 17 on CKBB respectively. At 4HNE concentrations in the physiological range, one member of the catalytic triad of hBAT (His362) was modified; for CKBB, although all four residues in the active site that were modifiable by 4HNE were ultimately modified, only one, Cys283, occurred at physiological concentrations of 4HNE. These results suggest that future in vivo studies should carefully assess the critical sites that are modified rather than using antibodies that do not distinguish between different modified sites.
- high-resolution mass spectrometry
- 4-hydroxynonenal (4HNE)
- oxidative stress
- post-translational modification
- protein oxidation
PTMs (post-translational modifications) of proteins occur extensively throughout their lifetime in a cell. For certain proteins, rather than the addition of a modifying group such as a phosphate, the PTM may be the removal of an N-terminal amino acid (e.g. methionine) or a longer N-terminal peptide, or the conversion of a C-terminal glycine into a C-terminal amidate. Similarly, some proteins are translated as a single polypeptide but are then digested by specific proteases to release individual and very bioactive proteins. Examples include chromogranin A in the brain  and the polypeptide containing the capsid protein in HIV . Another large group of PTMs are those formed enzymatically, such as phosphorylation [3,4], N-glycosylation , O-glycosylation , lysine N-methylation  and lysine N-acetylation . These proteins are also then subject to modification by enzymes that remove the PTMs, i.e. phosphatases, glycosidases , lysine N-demethylases  and acyl hydrolases .
The remaining group of protein PTMs result from chemically reactive species generated during different levels and types of oxidative stress which may be exacerbated by inflammatory conditions in infectious and chronic diseases (Table 1). Activation of neutrophils and other monocytes leads to the generation of a respiratory burst and the formation of superoxide anion radical (O2•−) . This oxidant species can undergo two main reactions: the first is a chemical reaction with another radical, nitric oxide (NO•), to form peroxynitrite (ONO2−) and the second, a catalytic one, with superoxide dismutase to form oxygen and hydrogen peroxide [13,14]. Peroxynitrite reacts with tyrosine and tryptophan residues to form nitrotyrosine  and nitrotryptophan [16–19]. It may also act as an oxidizing agent and modify cysteine residues. Hydrogen peroxide is also an oxidizing agent and under certain circumstances converts protein cysteine thiol (sulfhydryl) groups to sulfenic (SOH), sulfinic (SO2H) and sulfonic (SO3H) acids [20–22]. It may also be converted by neutrophil myeloperoxidase into hypochlorous acid (HOCl) that in turn reacts with nitrite to form nitryl chloride; we have shown previously that this increases the chlorination of a tyrosine-like residue in polyphenols [23,24]. Interestingly, conversion of peroxynitrite into nitryl chloride blocks nitration of tyrosine groups . Another oxidizing species is singlet oxygen (1O2) which is generated following the impact of UV light with tryptophan residues  and during the respiratory burst in neutrophils ; the former occurs in the lens of the eye and may be the basis of cataract formation. Singlet oxygen has a very short lifetime in a solution containing a protein and reacts with cysteine, histidine, methionine, tyrosine and tryptophan residues [28,29]. Singlet oxygen also reacts with polyunsaturated lipids to generate electrophilic lipid products such as malondialdehyde and 4HNE (4-hydroxynonenal) . These electrophilic lipids react with lysine, arginine and N-terminal amino acids to form Schiff bases, as well as with cysteine, lysine and histidine groups to form Michael adducts . Since Michael adducts are formed by the reaction of the unsaturated bond with the amino group, the aldehyde group remains unreacted. The resulting PTM is an example of a protein-associated carbonyl.
Biology of oxidative stress
It has long been appreciated that oxidative stress is part of the aetiology of many chronic diseases, including cardiovascular disease, diabetes, arthritis, autoimmune disease and many neurodegenerative diseases. Oxidation of LDL (low-density lipoprotein) is well recognized as a biomarker of cardiovascular disease. Its failure to be metabolized leads to its accumulation in foam cells. However, there are many other proteins that are also undergoing oxidative PTMs. Proteins containing carbonyl groups can be reacted with 2,4-dinitrophenylhydrazine to form 2,4-dinitrophenylhydrazones . These proteins can be separated by two-dimensional gel electrophoresis and detected by Western blotting with an anti-2,4-dinitrophenylhydrazone antibody and visualized by a secondary antibody coupled to a fluorescent probe . We have used a similar reagent (biotin hydrazide) to visualize protein carbonyls in livers of normal and ApoE−/− (apolipoprotein E-knockout) mice . Nitration can be monitored as well by Western blotting with anti-nitrotyrosine antibodies ; similarly, there are anti-4HNE antibodies to detect proteins where 4HNE adducts have formed .
The antibody-based Western blot procedures mentioned above contain two implicit assumptions. The first is that the antibody detects all modified groups of a given type, e.g. all the nitrotyrosine residues or all the 4HNE-amino acid adducts. Since the antibody is raised against specific nitrotyrosine-containing peptides, it cannot be guaranteed that it reacts equally with each one, given that the neighbouring amino acid residues are so different. Similarly, one has to ask the question: do anti-4HNE antibodies distinguish between Schiff bases and Michael adducts?
The effect of oxidative PTMs
Aside from the structural issues, a much deeper second assumption awaits us: that an oxidative modification is deleterious to the function of the protein. This seems, at first thought, a reasonable assumption; however, since oxidative stress is an unavoidable consequence of living in an oxygen-rich atmosphere, intuitively it seems that low levels of oxidation could have no effect or could even have benefit, subtly altering a protein's properties. Consistent with this latter thought process, in vitro data have shown that mild oxidation lowers kinetic barriers for HDL (high-density lipoprotein) remodelling, thereby improving its ability to take up cholesterol .
Rationale for the use of MS in studying oxidative stress
MS approaches to the study of proteins have developed at an amazing speed over the last 20 years. ESI (electrospray ionization)  and MALDI (matrix-assisted laser-desorption ionization)  are soft-ionization procedures that enable intact peptides and proteins to go into the gas phase. Any oxidant-induced changes in the chemistry of a protein (or a peptide derived from it) will be accompanied by changes in its mass. By using proteases to break the protein into smaller peptide pieces, the amino acid residue(s) where the oxidative modification has occurred can be determined. The changes in mass for peptides containing specific modifications are shown in Table 1.
Models of oxidative stress
Two models of oxidative stress for PTM analysis by MS are described here: the first is the reaction of the electrophilic lipid 4HNE with hBAT (human bile acid CoA:amino acid N-acyltransferase) and with CKBB (cytosolic brain isoform of creatine kinase). The second is the reaction of singlet oxygen with αB-crystallin as a product of UV light exposure. Site-specific PTM analysis was performed on a LTQ (linear quadrupole ion trap)–FT-ICR (Fourier-transform ion cyclotron resonance) hybrid mass spectrometer (LTQ FT; Thermo Fisher Scientific) with chip-based direct-infusion ESI and/or nanoLC (nano-liquid chromatography)–ESI.
Oxidative modifications of recombinant human CKBB and αB-crystallin were analysed by direct infusion with a fully automated monolithic silicon microchip-based electrospray interface, the TriVersa NanoMate (Advion) with gas-phase mass selection . Before MS analysis, 10 μM recombinant CKBB (Sigma–Aldrich) was incubated with increasing amounts of 4HNE (5, 10, 30, 100, 500 or 3000 μM) for 2 h at 37°C, as described previously . Excess 4HNE was removed by treatment with 1 mM histidine. Recombinant human αB-crystallin, expressed in Escherichia coli and purified using ion-exchange and gel filtration as described previously , was exposed to 50 mJ/cm2 of UVA light (320–400 nm) over a period of 2 h. The samples were immersed in an ice-water bath to maintain the temperature at approx. 5°C. The modified protein was digested overnight with sequencing grade trypsin or chymotrypsin in 25 mM NH4HCO3 buffer (pH 8.0) (at 37°C for trypsin and at room temperature for chymotrypsin). Aliquots (10 μl) of digested CKBB and αB-crystallin were loaded on to C18 ZipTip columns (Millipore). Peptides were eluted with 15 μl of a 4:1 acetonitrile/water solution in 0.1% methanoic (formic) acid and then diluted 1:1 with 0.1% methanoic acid. The NanoMate was set to load 5 μl of sample (8 pmol) which was electrosprayed by applying a 1.9 kV spray voltage and a 0.3 psi (1 psi=6.9 kPa) nitrogen head pressure to the sample tip to obtain a constant spray for 20–30 min. The capillary temperature, capillary voltage and tube lens voltage were set to 150°C, 20 V and 100 V respectively. The LTQ FT mass spectrometer was operated in a ‘top three’ data-dependent acquisition mode with gas-phase mass selection. The mass spectrometer was set to perform full FT-ICR–MS scans for the determination of the three most abundant precursor ions with the use of Xcalibur software (Thermo Fisher Scientific). For each of the three most abundant ions, a FT-ICR–MS SIM (selected-ion monitoring) scan was performed followed by MS/MS (tandem MS) in the LTQ ion trap. The cycle time for the full scan followed by successive FT-ICR SIM and LTQ–MS/MS scans for the three most abundant ions was approx. 2.1 s. Gas-phase mass selection, also termed gas-phase fractionation , was performed in the ion trap with four mass window selections: a 3 min FT-ICR–MS scan (m/z 225–500), a 7 min FT-ICR–MS scan (m/z 450–800), a 3 min FT-ICR–MS scan (m/z 750–1200) and a 1 min FT-ICR–MS scan (m/z 1150–2000) for a total method acquisition time of 14 min. The resolution was set to 100000 for full FT-ICR–MS scans and to 50000 for FT-ICR–MS SIM scans. Dynamic exclusion was enabled after a repeat count of three for the duration of the method. This method was validated against the nanoLC–ESI method described above as well as by nanoLC–ESI–QTOF (hybrid quadrupole orthogonal time-of-flight)–MS/MS as described for 4HNE and cytochrome c by Isom et al. .
Recombinant hBAT was purified as described previously . A 1.6 μM solution was reacted with 8, 16, 32, 64 or 128 μM 4HNE at 4°C for 1 h and excess 4HNE was removed by adjusting the reaction mixture to 1 mM histidine . The modified proteins were digested overnight with trypsin and chymotrypsin as described above. The samples were analysed by nanoLC (Eksigent) on a 15 cm×75 μm i.d. (internal diameter) reverse-phase C18 column with a linear gradient of 5–95% acetonitrile in 0.1% methanoic acid at a flow rate of 200 nl·min−1. Eluted tryptic and chymotryptic peptides were electrosprayed at 2 kV. Peptide fragmentation was induced by CID (collision-induced dissociation) in the ion trap, and fragment ions were also analysed in the ion trap. The LTQ FT mass spectrometer was operated in a ‘top three’ data-dependent acquisition mode. The mass spectrometer was set to switch between a full FT-ICR–MS scan (m/z 200–2000) followed by successive FT-ICR–MS SIM scans and LTQ–MS/MS scans of the three most abundant precursor ions in the full FT-ICR–MS scan as determined by the Xcalibur software. Dynamic exclusion was enabled after a repeat count of three for a period of 90 s.
LTQ–FT–MS/MS data, from both nanoLC–ESI and direct-infusion ESI runs, were searched against custom FASTA sequence databases containing the protein of interest as well as nine unrelated human proteins, as a negative control, with the TurboSEQUEST algorithm within Bioworks 3.2 (Thermo Fisher Scientific). Monoisotopic precursor and fragment ion masses were searched with a mass tolerance of 2 p.p.m. and 1 Da respectively. For identification of 4HNE-modified peptides, the TurboSEQUEST searches were amended to search for the mass additions of 156.1150, 138.1045 and 120.0939 for Michael, Schiff base and 2-pentylpyrrole adducts respectively. For identification of oxidized αB-crystallin peptides, the TurboSEQUEST searches were amended to search for the mass additions of 15.9949, 31.9898 and 47.9844. Additionally, FT-ICR mass spectra were extracted from each sample dataset for manual identification of modifications based on high mass accuracy. The modified peptides were manually validated by their absence in the unmodified FT-ICR mass spectra; a mass accuracy cut-off of 2 p.p.m. was used.
Detecting peptide modifications
4HNE forms multiple adducts on both CKBB (Table 2) and hBAT (Table 3) [33,39]. In both cases, the principal sites of adduct formation are histidine residues, as Michael adducts. Even the lysine adducts on hBAT are mostly Michael adducts, as are the cysteine groups on CKBB. It appears that 4HNE forms more Michael adducts (+m/z 156.1150) than Schiff bases (+m/z 138.1045), although this may be a reflection of the instability of the latter. In one case, an apparent Schiff base was formed on the histidine residue in the hBAT tryptic peptide A335H*AEQAIGQLKR346 as shown in the ECD (electron capture dissociation) mass spectrum (Figure 1). However, this 138.1045 change in mass of the peptide may be a result of in-source dehydration, as noted previously .
In the case of CKBB, modifications of all four 4HNE-modifiable residues in the active site were detected. Modification of the active-site Cys283 was identified at all concentrations of 4HNE where inactivation of enzymatic activity was observed (Table 2 and Figure 2). At 5 μM 4HNE, there was no significant inhibition of CKBB activity; nonetheless, there was modification of a non-active-site cysteine residue (Cys254). This demonstrates the importance of MS in PTM analysis, as some modifications are not deleterious in effect. In addition, in order to detect the Cys254 4HNE modification, it was necessary to use nanoLC–ESI–FT-ICR–MS to obtain sufficient sensitivity. This modification was not detected using direct-infusion ESI and gas-phase fractionation. As observed for His336 on hBAT (Figure 1), several of the histidine residues (His7, His219, His234 and His296) on CKBB formed 4HNE Michael adducts (+156.1150) as well as potential dehydrated Michael adducts (+138.1045). A similar pair of 4HNE adducts were observed for Cys254 (Table 2).
Besides oxidation by electrophilic lipids such as 4HNE, proteins are also prone to oxidation by UV light-induced oxidants such as singlet oxygen and hydrogen peroxide. The crystallin proteins in the lens of the eye do not undergo any turnover from birth to death . Since they are exposed to UVA light (320–400 nm) throughout life and to increasing amounts of UVB light (280–320 nm) with age, the oxidative changes can be significant and lead to a loss of function of the chaperone activities of these proteins . Analysis of tryptic peptides revealed that recombinant human αB-crystallin exposed to 50 mJ/cm2 of UVA light for 2 h is oxidized on methionine and tryptophan residues in the N-terminal region (Met1 and Trp9) and the region responsible for chaperone function (Met68 and Trp60) . Interestingly, several isobaric forms of the tryptic peptides were observed. For M1DIAIHHPWIR11 and A57PSWFDTGLSEMR69, there were two mono-oxygenated isomers, three di-oxygenated isomers and two tri-oxygenated isomers. The tandem mass spectrum of the triply oxygenated A57PSWFDTGLSEMR69 peptide is shown in Figure 3. The y2–y7 fragment ions all indicate that Met68 is modified by a single oxygen atom, whereas the jump in mass between b3 and b4 (218 Da) for Trp60 is due to two oxygen atoms (186+2×16).
HNE modifications of hBAT were analysed by nanoLC–ESI–FT-ICR due to sample limitations. Functionally, its enzyme activity was completely inhibited at 32 μM (Figure 2). A total of 14 modifications were observed at the different concentrations of 4HNE (Table 3). Seven of these modifications (His62, His271, His280, Lys329, Lys334, His336 and His362) were detected at the lowest concentration tested (8 μM; 1:5 molar ratio of hBAT/4HNE). Only one (His362) was a member of the catalytic triad (Cys235-Asp328-His362) . Because of their proximity to Asp328, the modifications at Lys329, Lys334 and His336 may have also contributed to changes in hBAT activity.
Oxidative PTM of proteins is widespread. In normal tissues, proteins often exist in multiple electrophoretically resolvable forms, many of which are oxidized states. It remains to be determined whether all oxidative modifications are necessarily deleterious to the function of the protein. In a recent study, mild oxidation of HDL with hydrogen peroxide increased its ability to abstract cholesterol from macrophages . In contrast, oxidation with HOCl resulted in a greater oxidant stress and loss of function . Thus whether a particular oxidative modification is deleterious may be dependent on the specific protein, and the dose, as well as the exact chemical nature of the modification.
The versatility, high mass accuracy and high mass-resolving capabilities of modern FT-ICR–MS instruments are well suited to the identification and characterization of oxidative PTMs. If an investigator has access to purified recombinant protein, direct-infusion automated nanoelectrospray is a very convenient way to examine the effects of a wide range of concentrations of the oxidative agent. In the present study with human CKBB, 8 pmol of protein was consumed in analysis performed at six different concentrations of 4HNE. In the direct-infusion method, the mass range is divided into four parts; the very high mass resolution means that even peptides that have the same nominal mass can be resolved. Our direct-infusion method made use of gas phase rather than chromatographic fractionation and is accordingly much faster. Furthermore, since each nozzle of the electrospray chip is only used once, there is no possibility of carryover that can be a difficulty in LC analytical approaches. It is also possible to carry out real-time dynamic exclusion of previously observed peptides in order to examine low-intensity ones. However, when the amount of protein is restricted, or to detect modifications occurring at a very low level, nanoLC–ESI–FT-ICR is recommended (Table 3). In the nanoLC approach, the sample is concentrated by the LC, elutes in a smaller volume and hence has better signal-to-noise than direct infusion. The limitation of nanoLC is the longer time that it takes for each analysis (90 min per LC run compared with 15 min per infusion for each concentration). NanoLC–ESI–QTOF was less sensitive than either FT-ICR technique (Table 2); however, newer instruments in this category may not be as limited.
Finally, although MS can be very effective in studying oxidative PTMs, it should be borne in mind that it cannot, in a MS/MS experiment, establish with certainty which isomers of tryptophan residues are formed; however, an ion-trap method based on MSn may be suitable, since it would enable further fragmentation of isobaric MS/MS daughter ions.
The structural analysis of protein oxidative modifications will continue to be enhanced by future refinement of MS instrumentation. The most meaningful studies will result from a combination of high-resolution MS and functional studies, where the oxidative modifications are correlated with functional effects. Besides HDL , there may be other proteins whose functions are enhanced by oxidative modifications. It will be important to identify others in this category to balance the presumption that all modifications are deleterious.
Support for the UAB Center for Nutrient–Gene Interaction in Cancer Prevention was provided by a grant-in-aid (U54 CA100949, S.B., Principal Investigator) from the National Cancer Institute. Support for research on botanicals and dietary supplements and their effects on protein oxidation at the Purdue University–University of Alabama at Birmingham Botanical Center for Age-Related Disease was provided by a grant (P50 AT00477, Connie M. Weaver, Principal Investigator) from the National Center for Complementary and Alternative Medicine and the NIH (National Institutes of Health) Office of Dietary Supplements. S.M.E. and H.K. were supported by funds from the U.S. Agency for International Development, Kikkoman Corporation, and the Cranberry Institute to H.K. Support for the research on bile acid amino acid amidation was provided by a grant (R01 DK46390, S.B., Principal Investigator) from the National Institute for Diabetes, Digestive and Kidney Diseases. Support for research on UV light-induced damage to lens crystallins was provided by a grant from the Alabama EyeSight Foundation. Funds for the purchase of the mass spectrometers were provided by the National Center for Research Resources (S10 RR17261, S.B., Principal Investigator). Additional support for the operation of the UAB Biomedical FT-ICR laboratory was provided by the Supporters of the UAB Comprehensive Cancer Center.
Bioanalysis in Oxidative Stress: A Biochemical Society Focused Meeting held at the University of Exeter, U.K., 2–3 April 2008. Organized and Edited by John Moody (Plymouth, U.K.) and Paul Winyard (Peninsula Medical School, Exeter, U.K.).
Abbreviations: CKBB, cytosolic brain isoform of creatine kinase; ECD, electron capture dissociation; ESI, electrospray ionization; FT-ICR, Fourier-transform ion cyclotron resonance; hBAT, human bile acid CoA:amino acid N-acyltransferase; HDL, high-density lipoprotein; 4HNE, 4-hydroxynonenal; LC, liquid chromatography; LTQ, linear quadrupole ion trap; MS/MS, tandem MS; PTM, post-translational modification; QTOF, quantitative time-of-flight; SIM, selected-ion monitoring
- © The Authors Journal compilation © 2008 Biochemical Society