A Metal-coded Affinity Tag Approach to Quantitative Proteomics

Glycoprotein structure determination and quantification by MS requires efficient isolation of glycopeptides from a proteolytic digest of complex protein mixtures. Here we describe that the use of acids as ion-pairing reagents in normal-phase chromatography (IP-NPLC) considerably increases the hydrophobicity differences between non-glycopeptides and glycopeptides, thereby resulting in the reproducible isolation of N-linked high mannose type and sialylated glycopeptides from the tryptic digest of a ribonuclease B and fetuin mixture. The elution order of non-glycopeptides relative to glycopeptides in IP-NPLC is predictable by their hydrophobicity values calculated using the Wimley-White water/octanol hydrophobicity scale. O-linked glycopeptides can be efficiently isolated from fetuin tryptic digests using IP-NPLC when N-glycans are first removed with PNGase. IP-NPLC recovers close to 100% of bacterial N-linked glycopeptides modified with non-sialylated heptasaccharides from tryptic digests of periplasmic protein extracts from Campylobacter jejuni 11168 and its pglD mutant. Label-free nano-flow reversed-phase LC-MS is used for quantification of differentially expressed glycopeptides from the C. jejuni wild-type and pglD mutant followed by identification of these glycoproteins using multiple stage tandem MS. This method further confirms the acetyltransferase activity of PglD and demonstrates for the first time that heptasaccharides containing monoacetylated bacillosamine are transferred to proteins in both the wild-type and mutant strains. We believe that IP-NPLC will be a useful tool for quantitative glycoproteomics.

No consensus has been reached on the proper time to add stable-isotope labeled (SIL) peptides in protein cleavage isotope dilution mass spectrometry workflows. While quantifying 24 monolignol pathway enzymes in the xylem tissue of Populus trichocarpa, we compared the protein concentrations obtained when adding the SIL standard peptides concurrently with the enzyme or after quenching of the digestion (i.e. postdigestion) and observed discrepancies for nearly all tryptic peptides investigated. In some cases, greater than 30-fold differences were observed. To explain these differences and potentially correct for them, we developed a mathematical model based on pseudo-first-order kinetics to account for the dynamic production and decay (e.g. degradation and precipitation) of the native peptide targets in conjunction with the decay of the SIL peptide standards. A time course study of the digests confirmed the results predicted by the proposed model and revealed that the discrepancy between concurrent and postdigestion introduction of the SIL standards was related to differential decay experienced by the SIL peptide and the native peptide in each method. Given these results, we propose concurrent introduction of the SIL peptide is most appropriate, though not free from bias. Mathematical modeling of this method reveals that overestimation of protein quantities would still result when rapid peptide decay occurs and that this bias would be further exaggerated by slow proteolysis. We derive a simple equation to estimate the bias for each peptide based on the relative rates of production and decay. According to this equation, nearly half of the peptides evaluated here were estimated to have quantitative errors greater than 10% and in a few cases over 100%. We conclude that the instability of peptides can often significantly bias the protein quantities measured in protein cleavage isotope dilution mass spectrometry-based assays and suggest peptide stability be made a priority when selecting peptides to use for quantification.

Homolytic decomposition of PUFA 2The abbreviations used are: PUFA, polyunsaturated fatty acid; DDE, trans, trans-2,4-decadienal; EDE, 4,5-epoxy-(2E)-decenal; HNE, 4-hydroxy-(2E)-nonenal; HPNE, 4-hydroperoxy-(2E)-nonenal; MDA, malondialdehyde; ONE, 4-oxo-(2E)-nonenal; DODE, 9,12-dioxo-(10E)-dodecenoic acid; MS, mass spectrometry; ROS, reactive oxygen species; COX, cyclooxygenase; LOX, lipoxygenase; GST, glutathione S-transferase; LC-MRM-MS, liquid chromatography-multiple reaction monitoring-mass spectrometry; LA, linoleic acid; HPODE, hydroperoxy-(9Z),(11E)-octadecadienoic acid; AA, arachidonic acid; HPETE, hydroperoxyeicosatetraenoic acid; HETE, hydroxyeicosatetraenoic acid; APCI, atmospheric pressure chemical ionization; MS/MS, tandem MS; ESI, electrospray ionization; dGuo, 2′-deoxyguanosine; ∈DNA, etheno-DNA; H∈, heptanone-etheno; dAdo, 2′-deoxyadenosine; dCyd, 2′-deoxycytidine; C∈, carboxynonanone-etheno; GC, gas chromatography. 2The abbreviations used are: PUFA, polyunsaturated fatty acid; DDE, trans, trans-2,4-decadienal; EDE, 4,5-epoxy-(2E)-decenal; HNE, 4-hydroxy-(2E)-nonenal; HPNE, 4-hydroperoxy-(2E)-nonenal; MDA, malondialdehyde; ONE, 4-oxo-(2E)-nonenal; DODE, 9,12-dioxo-(10E)-dodecenoic acid; MS, mass spectrometry; ROS, reactive oxygen species; COX, cyclooxygenase; LOX, lipoxygenase; GST, glutathione S-transferase; LC-MRM-MS, liquid chromatography-multiple reaction monitoring-mass spectrometry; LA, linoleic acid; HPODE, hydroperoxy-(9Z),(11E)-octadecadienoic acid; AA, arachidonic acid; HPETE, hydroperoxyeicosatetraenoic acid; HETE, hydroxyeicosatetraenoic acid; APCI, atmospheric pressure chemical ionization; MS/MS, tandem MS; ESI, electrospray ionization; dGuo, 2′-deoxyguanosine; ∈DNA, etheno-DNA; H∈, heptanone-etheno; dAdo, 2′-deoxyadenosine; dCyd, 2′-deoxycytidine; C∈, carboxynonanone-etheno; GC, gas chromatography.-derived lipid hydroperoxides results in formation of the α,β-unsaturated aldehydic bifunctional electrophiles DDE, EDE, HNE, HPNE, MDA (shown as β-hydroxyacrolein), ONE, DODE, and 5,8-dioxo-(10E)-octenoic acid (see Fig. 1) (1Blair I.A. Exp. Gerontol. 2001; 36: 1473-1481Crossref PubMed Scopus (169) Google Scholar, 2Lee S.H. Blair I.A. Trends Cardiovasc. Med. 2001; 11: 148-155Crossref PubMed Scopus (121) Google Scholar, 3Blair, I. A. (2005) in Encyclopedia of Mass Spectrometry (Caprioli, R. M., and Gross, M. L., eds) Vol. 3, pp. 283–307, Elsevier Ltd., OxfordGoogle Scholar). Intracellular formation of the bifunctional electrophiles can then result in the formation of GSH, protein, and DNA adducts (1Blair I.A. Exp. Gerontol. 2001; 36: 1473-1481Crossref PubMed Scopus (169) Google Scholar, 2Lee S.H. Blair I.A. Trends Cardiovasc. Med. 2001; 11: 148-155Crossref PubMed Scopus (121) Google Scholar, 3Blair, I. A. (2005) in Encyclopedia of Mass Spectrometry (Caprioli, R. M., and Gross, M. L., eds) Vol. 3, pp. 283–307, Elsevier Ltd., OxfordGoogle Scholar, 5Marnett L.J. Riggins J.N. West J.D. J. Clin. Investig. 2003; 111: 583-593Crossref PubMed Scopus (393) Google Scholar). The analysis of lipid hydroperoxide-derived DNA adducts can facilitate molecular epidemiology studies by providing insight into the amount of a genotoxin that has reached the DNA of the tissue under study (6Kensler T.W. Qian G.S. Chen J.G. Groopman J.D. Nat. Rev. Cancer. 2003; 3: 321-329Crossref PubMed Scopus (179) Google Scholar, 7Sharma R.A. Farmer P.B. Clin. Cancer Res. 2004; 10: 4901-4912Crossref PubMed Scopus (86) Google Scholar). DNA repair enzymes, such as those involved in base excision repair, are able to excise the DNA adducts so that they can potentially be excreted in the urine (7Sharma R.A. Farmer P.B. Clin. Cancer Res. 2004; 10: 4901-4912Crossref PubMed Scopus (86) Google Scholar). This suggests that non-invasive MS-based techniques could be used to monitor urinary DNA adducts arising from lipid hydroperoxide-mediated DNA damage. Unfortunately, to date, the analysis of urinary DNA adducts of lipid hydroperoxide-derived bifunctional electrophiles has not been particularly successful. ONE-derived DNA adducts, which can arise only from lipid peroxidation, have now been characterized in the tissues of mouse models (8Williams M.V. Lee S.H. Pollack M. Blair I.A. J. Biol. Chem. 2006; 281: 10127-10133Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Therefore, it might eventually be possible to detect these specific lipid hydroperoxide-derived DNA adducts after they have been excised from the DNA and excreted in the urine (see Fig. 2).FIGURE 2Formation of ∈DNA and H∈DNA adducts through homolytic decomposition of lipid hydroperoxides. BER, base excision repair.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Oxidative stress represents one of the major cellular responses to toxic insults, carcinogenic chemicals, and environmental agents such as viruses and mycotoxins (9Ames B.N. Shigenaga M.K. Hagen T.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7915-7922Crossref PubMed Scopus (5382) Google Scholar). During oxidative stress, there is a decrease in the amount of intracellular GSH and an increase in GSSG (10Zhu P. Oe T. Blair I.A. Rapid Commun. Mass Spectrom. 2008; 22: 432-440Crossref PubMed Scopus (62) Google Scholar) with a concomitant increase in ROS and reactive nitrogen species (9Ames B.N. Shigenaga M.K. Hagen T.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7915-7922Crossref PubMed Scopus (5382) Google Scholar) and an increase in the activity of COX-2 (11Lee S.H. Williams M.V. Dubois R.N. Blair I.A. J. Biol. Chem. 2005; 280: 28337-28346Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) and LOXs (10Zhu P. Oe T. Blair I.A. Rapid Commun. Mass Spectrom. 2008; 22: 432-440Crossref PubMed Scopus (62) Google Scholar), involved in lipid peroxidation (1Blair I.A. Exp. Gerontol. 2001; 36: 1473-1481Crossref PubMed Scopus (169) Google Scholar). ROS are generated constantly in vivo by a variety of endogenous processes, including normal mitochondrial aerobic respiration, phagocytosis of bacteria- or virus-containing cells, and peroxisome-mediated degradation of fatty acids (9Ames B.N. Shigenaga M.K. Hagen T.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7915-7922Crossref PubMed Scopus (5382) Google Scholar). Increased ROS production occurs in inflammation; during radiation; or during metabolism of hormones, drugs, and environmental toxins. This can overwhelm endogenous protective mechanisms and increase ROS-mediated damage to GSH, proteins, DNA, and lipids. The resulting ROS-derived lipid hydroperoxides can then undergo homolytic decomposition to various bifunctional electrophiles that form DNA adducts with different lipid modifications (1Blair I.A. Exp. Gerontol. 2001; 36: 1473-1481Crossref PubMed Scopus (169) Google Scholar, 2Lee S.H. Blair I.A. Trends Cardiovasc. Med. 2001; 11: 148-155Crossref PubMed Scopus (121) Google Scholar, 3Blair, I. A. (2005) in Encyclopedia of Mass Spectrometry (Caprioli, R. M., and Gross, M. L., eds) Vol. 3, pp. 283–307, Elsevier Ltd., OxfordGoogle Scholar;5Marnett L.J. Riggins J.N. West J.D. J. Clin. Investig. 2003; 111: 583-593Crossref PubMed Scopus (393) Google Scholar). Alternatively, the bifunctional electrophiles can arise from homolytic decomposition of COX- and LOX-derived lipid hydroperoxides (11Lee S.H. Williams M.V. Dubois R.N. Blair I.A. J. Biol. Chem. 2005; 280: 28337-28346Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 12Lee S.H. Rangiah K. Williams M.V. Wehr A.Y. Dubois R.N. Blair I.A. Chem. Res. Toxicol. 2007; 20: 1665-1675Crossref PubMed Scopus (33) Google Scholar, 13Jian W. Lee S.H. Arora J.S. Silva Elipe M.V. Blair I.A. Chem. Res. Toxicol. 2005; 18: 599-610Crossref PubMed Scopus (24) Google Scholar). Intracellular lipid hydroperoxides are detoxified by GSH peroxidase-mediated reduction to lipid alcohols, with GSH providing the reducing equivalents (4Blair I.A. Curr. Drug Metab. 2006; 7: 853-872Crossref PubMed Scopus (101) Google Scholar). Peroxidized phospholipids are detoxified by peroxiredoxin 6 to the corresponding phospholipid alcohols in a GSH-dependent manner (14Manevich Y. Fisher A.B. Free Radic. Biol. Med. 2005; 38: 1422-1432Crossref PubMed Scopus (322) Google Scholar). GSH also readily forms adducts with lipid hydroperoxide-derived bifunctional electrophiles, providing an additional protective pathway (4Blair I.A. Curr. Drug Metab. 2006; 7: 853-872Crossref PubMed Scopus (101) Google Scholar, 12Lee S.H. Rangiah K. Williams M.V. Wehr A.Y. Dubois R.N. Blair I.A. Chem. Res. Toxicol. 2007; 20: 1665-1675Crossref PubMed Scopus (33) Google Scholar, 15Jian W. Lee S.H. Mesaros C. Oe T. Silva Elipe M.V. Blair I.A. Chem. Res. Toxicol. 2007; 20: 1008-1018Crossref PubMed Scopus (44) Google Scholar). Formation of the GSH adducts is generally facilitated by GSTs, and the resulting adducts are exported from cells by ATP-binding and non-ATP-binding cassette transporters (10Zhu P. Oe T. Blair I.A. Rapid Commun. Mass Spectrom. 2008; 22: 432-440Crossref PubMed Scopus (62) Google Scholar). Total GSH and GSSG concentrations are modulated in cells by low extracellular concentrations of lipid hydroperoxide-derived bifunctional electrophiles such as HNE. This can increase GSH concentrations by up-regulation of γ-glutamylcysteine ligase, a key enzyme involved in GSH biosynthesis. In contrast, high concentrations of bifunctional electrophiles deplete GSH (4Blair I.A. Curr. Drug Metab. 2006; 7: 853-872Crossref PubMed Scopus (101) Google Scholar) through a direct GST-mediated reaction as well as by inhibition of enzymes involved in GSH biosynthesis. GSH/GSSG homeostasis plays an important role in maintaining cellular redox status, and changes of the half-cell reduction potential of the 2GSH/GSSG couple correlate with the biological status of the cell (10Zhu P. Oe T. Blair I.A. Rapid Commun. Mass Spectrom. 2008; 22: 432-440Crossref PubMed Scopus (62) Google Scholar). It is difficult to accurately quantify intracellular GSH and GSSG concentrations because of the ease with which free sulfhydryl groups are oxidized during the lysis of cells. To overcome this problem, a stable isotope dilution LC-MRM-MS method was developed using 4-fluoro-7-sulfamoylbenzofurazan derivatization of GSH (10Zhu P. Oe T. Blair I.A. Rapid Commun. Mass Spectrom. 2008; 22: 432-440Crossref PubMed Scopus (62) Google Scholar). Increased lipid peroxidation resulting from transfection of the human 15-LOX-1 gene into a mouse macrophage cell line was found to increase intracellular GSH biosynthesis. This resulted in a lower resting cellular redox potential and provided protection against exogenous lipid hydroperoxide-derived bifunctional electrophiles. Therefore, increased intracellular lipid peroxidation can induce a protective adaptive response (10Zhu P. Oe T. Blair I.A. Rapid Commun. Mass Spectrom. 2008; 22: 432-440Crossref PubMed Scopus (62) Google Scholar). ROS-mediated oxidation of esterified LA-containing lipids and free LA results in the formation of four HPODE isomers as enantiomeric pairs. They are subsequently reduced to the corresponding hydroxyoctadecadienoic acids. Similarly, ROS-mediated oxidation of AA-containing lipids and free AA results in the formation of a complex mixture of HPETEs that are reduced to racemic hydroxyeicosatetraenoic acids (HETEs), including (15S)- and (15R)-HETE (11Lee S.H. Williams M.V. Dubois R.N. Blair I.A. J. Biol. Chem. 2005; 280: 28337-28346Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 12Lee S.H. Rangiah K. Williams M.V. Wehr A.Y. Dubois R.N. Blair I.A. Chem. Res. Toxicol. 2007; 20: 1665-1675Crossref PubMed Scopus (33) Google Scholar, 16Brash A.R. J. Biol. Chem. 1999; 274: 23679-23682Abstract Full Text Full Text PDF PubMed Scopus (1137) Google Scholar). Lipid hydroperoxides can also be formed by the action of LOXs (9Ames B.N. Shigenaga M.K. Hagen T.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7915-7922Crossref PubMed Scopus (5382) Google Scholar) and COXs (17Porter N.A. Caldwell S.E. Mills K.A. Lipids. 1995; 30: 277-290Crossref PubMed Scopus (996) Google Scholar) on PUFAs. High sensitivity, stable isotope dilution, normal phase, chiral LC-electron capture APCI-MS/MS methodology (18Singh G. Gutierrez A. Xu K. Blair I.A. Anal. Chem. 2000; 72: 3007-3013Crossref PubMed Scopus (180) Google Scholar) makes it possible to separate and quantify all of the enantiomers and regioisomers of the PUFA alcohols derived from LA and AA (19Lee S.H. Williams M.V. Dubois R.N. Blair I.A. Rapid Commun. Mass Spectrom. 2003; 17: 2168-2176Crossref PubMed Scopus (156) Google Scholar, 20Lee S.H. Blair I.A. Methods Enzymol. 2007; 433: 159-174Crossref PubMed Scopus (34) Google Scholar). Eicosanoids and isoprostanes can be quantified at the same time. This method revealed that endogenous (15S)-HPETE (as measured by (15S)-HETE release) was formed in amounts that were similar to prostaglandin E2 in unstimulated rat intestinal epithelial cells stably expressing COX-2 (RIES cells) (21Lee S.H. Oe T. Blair I.A. Science. 2001; 292: 2083-2086Crossref PubMed Scopus (409) Google Scholar). When exogenous AA was added to the RIES cells, the profile of eicosanoids was found to be dependent upon the time of incubation as well as the AA concentration (12Lee S.H. Rangiah K. Williams M.V. Wehr A.Y. Dubois R.N. Blair I.A. Chem. Res. Toxicol. 2007; 20: 1665-1675Crossref PubMed Scopus (33) Google Scholar). Interestingly, COX-2-mediated biosynthesis of prostaglandin E2 and (15S)-HETE was found to increase linearly with the addition of increasing amounts of AA to the cells. From the intercepts on the y axes of the regression lines, it was possible to calculate the amount of each eicosanoid formed from endogenous AA in resting cells. The calculated values were very close to the experimentally determined values. It was also possible to estimate how much endogenous AA (0.56 μm) had been mobilized by phospholipases in the unstimulated RIES cells. The ability to determine how much of an eicosanoid metabolite is formed in the absence of exogenous AA stimulation is particularly useful when endogenous production of the metabolite is below the detection limit of the assay that is being utilized. Studies on DNA adducts resulting from lipid hydroperoxide-derived bifunctional electrophiles have relied heavily on the use of reversed-phase LC-ESI-MS methodology (3Blair, I. A. (2005) in Encyclopedia of Mass Spectrometry (Caprioli, R. M., and Gross, M. L., eds) Vol. 3, pp. 283–307, Elsevier Ltd., OxfordGoogle Scholar). However, many of the bifunctional electrophiles themselves are poorly ionized under ESI conditions. Aldehydic bifunctional electrophiles can be converted to oxime derivatives to improve ESI efficiency. However, this results in syn- and anti-oxime isomers together with extremely complex LC chromatograms (22Lee S.H. Blair I.A. Chem. Res. Toxicol. 2000; 13: 698-702Crossref PubMed Scopus (230) Google Scholar). Therefore, normal phase LC-APCI-MS was employed to quantify the lipid hydroperoxide-derived bifunctional electrophiles (21Lee S.H. Oe T. Blair I.A. Science. 2001; 292: 2083-2086Crossref PubMed Scopus (409) Google Scholar, 23Williams M.V. Lee S.H. Blair I.A. Rapid Commun. Mass Spectrom. 2005; 19: 849-858Crossref PubMed Scopus (30) Google Scholar, 24Lee S.H. Oe T. Arora J.S. Blair I.A. J. Mass Spectrom. 2005; 40: 661-668Crossref PubMed Scopus (26) Google Scholar). HNE was more readily ionized than the other bifunctional electrophiles, so its APCI response was almost an order of magnitude more intense compared with the other bifunctional electrophiles. HPNE, trans-EDE, and ONE were the major products identified from the Fe(II)-mediated homolytic decomposition of (13S)-HPODE (24Lee S.H. Oe T. Arora J.S. Blair I.A. J. Mass Spectrom. 2005; 40: 661-668Crossref PubMed Scopus (26) Google Scholar). The possible formation of ONE and HNE from HPNE was confirmed by treating HPNE with increasing concentrations of Fe(II). Vitamin C was more than twice as efficient at initiating the decomposition of 13-HPODE to bifunctional electrophiles compared with transition metal ions. It also induced the conversion of HPNE to ONE and HNE. Normal phase LC-APCI-MS methodology also showed that ONE was a major product from both Fe(II)-and vitamin C-mediated homolytic decomposition of (15S)-HPETE (23Williams M.V. Lee S.H. Blair I.A. Rapid Commun. Mass Spectrom. 2005; 19: 849-858Crossref PubMed Scopus (30) Google Scholar). A number of DNA adducts arising from lipid hydroperoxide-derived bifunctional electrophiles (Fig. 1) have been identified by LC-MS (3Blair, I. A. (2005) in Encyclopedia of Mass Spectrometry (Caprioli, R. M., and Gross, M. L., eds) Vol. 3, pp. 283–307, Elsevier Ltd., OxfordGoogle Scholar). The reaction between (13S)-HPODE and dGuo was shown to result in the formation of a single ∈DNA adduct, H∈dGuo (Fig. 2) (25Rindgen D. Nakajima M. Wehrli S. Xu K. Blair I.A. Chem. Res. Toxicol. 1999; 12: 1195-1204Crossref PubMed Scopus (136) Google Scholar). The initially formed ethano adducts arose from highly regioselective nucleophilic addition of N-2 of the dGuo to the C-1 aldehyde of ONE, followed by reaction of N-1 at C-2 of the resulting α,β-unsaturated ketone. H∈dAdo DNA adducts were also detected in the reaction between dAdo and 13-HPODE-derived ONE (Fig. 2) (26Rindgen D. Lee S.H. Nakajima M. Blair I.A. Chem. Res. Toxicol. 2000; 13: 846-852Crossref PubMed Scopus (70) Google Scholar, 27Lee S.H. Rindgen D. Bible Jr., R.H. Hajdu E. Blair I.A. Chem. Res. Toxicol. 2000; 13: 565-574Crossref PubMed Scopus (103) Google Scholar). Furthermore, the reaction between ONE and dCyd resulted in formation of H∈dCyd (Fig. 2) as a major product (28Pollack M. Oe T. Lee S.H. Silva Elipe M.V. Arison B.H. Blair I.A. Chem. Res. Toxicol. 2003; 16: 893-900Crossref PubMed Scopus (79) Google Scholar). The endogenous formation of unsubstituted ∈DNA adducts was suggested originally to arise from lipid hydroperoxide-mediated epoxidation of HNE to 2,3-epoxy-4-hydroxynonanal and subsequent reaction of this bifunctional electrophile with DNA bases (29Sodum R.S. Chung F.L. Cancer Res. 1991; 51: 137-143PubMed Google Scholar). Subsequently, it was suggested that this reaction was unlikely to occur in cells because of the relatively high pKa of the lipid hydroperoxides (30Douki T. Odin F. Caillat S. Favier A. Cadet J. Free Radic. Biol. Med. 2004; 37: 62-70Crossref PubMed Scopus (78) Google Scholar). Unsubstituted etheno adducts were also formed from the reaction of lipid hydroperoxide-derived DDE with dAdo or dGuo in the presence of peroxides (31Loureiro A.P. Di M.P. Gomes O.F. Medeiros M.H. Chem. Res. Toxicol. 2000; 13: 601-609Crossref PubMed Scopus (74) Google Scholar). This pathway of endogenous ∈DNA adduct formation would require intracellular epoxidation of DDE to occur more rapidly than its detoxification by GSTs and aldoketoreductases (32Jian W. Arora J.S. Oe T. Shuvaev V.V. Blair I.A. Free Radic. Biol. Med. 2005; 39: 1162-1176Crossref PubMed Scopus (46) Google Scholar). Unsubstituted etheno adducts were formed when DNA bases were treated with EDE and HPNE (Fig. 2) (33Lee S.H. Oe T. Blair I.A. Chem. Res. Toxicol. 2002; 15: 300-304Crossref PubMed Scopus (74) Google Scholar, 34Lee S.H. Arora J.A. Oe T. Blair I.A. Chem. Res. Toxicol. 2005; 18: 780-786Crossref PubMed Scopus (61) Google Scholar). However, HPNE was much more reactive to DNA bases compared with EDE. Based on this increased reactivity, it appears that HPNE is most likely the major lipid hydroperoxide-derived bifunctional electrophile responsible for the formation of unsubstituted ∈DNA adducts (Fig. 2) (34Lee S.H. Arora J.A. Oe T. Blair I.A. Chem. Res. Toxicol. 2005; 18: 780-786Crossref PubMed Scopus (61) Google Scholar). Previous studies have shown that environmental chemicals such as vinyl chloride, vinyl fluoride, and chloroethylene oxide form the same unsubstituted ∈DNA adducts (Fig. 2) (35Barbin A. Friesen M. O'Neill I.K. Croisy A. Bartsch H. Chem. Biol. Interact. 1986; 59: 43-54Crossref PubMed Scopus (13) Google Scholar, 36Swenberg, J. A., Bogdanffy, M. S., Ham, A., Holt, S., Kim, A., Morinello, E. J., Ranasinghe, A., Scheller, N., and Upton, P. B. (1999) in Exocyclic DNA Adducts in Mutagenesis and Carcinogenesis (Singer, B., and Bartsch, H., eds) Volume 150, pp. 29–43, IARC Science Publications, Scholar). Therefore, unsubstituted ∈DNA adducts not arise from lipid It has been that a of 13-HPODE would to the formation of a of K. Lipid Res. 2003; PubMed Scopus Google Scholar). is to rapidly form HPNE, which in is converted to ONE and HNE (23Williams M.V. Lee S.H. Blair I.A. Rapid Commun. Mass Spectrom. 2005; 19: 849-858Crossref PubMed Scopus (30) Google Scholar, 24Lee S.H. Oe T. Arora J.S. Blair I.A. J. Mass Spectrom. 2005; 40: 661-668Crossref PubMed Scopus (26) Google Scholar). This suggested that the (Fig. might also be formed from the homolytic decomposition of Subsequently, was and shown to be the 13-HPODE-derived bifunctional electrophile responsible for the formation of DNA adducts S.H. Silva Elipe M.V. Arora J.S. Blair I.A. Chem. Res. Toxicol. 2005; 18: PubMed Scopus Google Scholar). amounts of H∈dGuo and were formed from the homolytic decomposition of (13S)-HPODE in the presence of study showed that the bifunctional electrophile 5,8-dioxo-(10E)-octenoic acid was a major product arising from the homolytic decomposition of and that it formed DNA adducts W. Lee S.H. Arora J.S. Silva Elipe M.V. Blair I.A. Chem. Res. Toxicol. 2005; 18: 599-610Crossref PubMed Scopus (24) Google Scholar). have been the to methodology for the of and adducts formed from of lipid hydroperoxide-derived bifunctional electrophiles to DNA (1Blair I.A. Exp. Gerontol. 2001; 36: 1473-1481Crossref PubMed Scopus (169) Google Scholar, 2Lee S.H. Blair I.A. Trends Cardiovasc. Med. 2001; 11: 148-155Crossref PubMed Scopus (121) Google Scholar, 5Marnett L.J. Riggins J.N. West J.D. J. Clin. Investig. 2003; 111: 583-593Crossref PubMed Scopus (393) Google Scholar, M.V. Lee S.H. Pollack M. Blair I.A. J. Biol. Chem. 2006; 281: 10127-10133Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, S.H. Williams M.V. Dubois R.N. Blair I.A. J. Biol. Chem. 2005; 280: 28337-28346Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, T. Odin F. Caillat S. Favier A. Cadet J. Free Radic. Biol. Med. 2004; 37: 62-70Crossref PubMed Scopus (78) Google Scholar, M. J.D. Blair I.A. L.J. Science. PubMed Scopus Google Scholar, M. L.J. Blair I.A. Biol. Mass Spectrom. PubMed Scopus (65) Google Scholar, M. L.J. Blair I.A. J. Mass Spectrom. 1995; 30: Scopus Google Scholar, Blair I.A. L.J. 17: PubMed Scopus Google Scholar, M. Blair I.A. Chem. Res. Toxicol. 10: PubMed Scopus Google Scholar, M. Blair I.A. L.J. Chem. Res. Toxicol. 10: PubMed Scopus Google Scholar, A. J. P.B. F. J.A. Chem. Res. Toxicol. 1999; 12: PubMed Scopus Google Scholar, J. Chung F.L. Chem. Res. Toxicol. 1999; 12: PubMed Scopus (62) Google Scholar, Chem. Res. Toxicol. 2000; 13: PubMed Scopus (74) Google Scholar, Anal. Chem. 2001; PubMed Scopus Google Scholar, Chem. Res. Toxicol. 2002; 15: PubMed Scopus Google Scholar, M. L.J. J. Mass Spectrom. 2004; 39: PubMed Scopus Google Scholar, Chem. Res. Toxicol. 2004; 17: PubMed Scopus Google Scholar, K. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). MDA (Fig. 1) is one of the most of the lipid hydroperoxide-derived bifunctional electrophiles that DNA damage L.J. Riggins J.N. West J.D. J. Clin. Investig. 2003; 111: 583-593Crossref PubMed Scopus (393) Google Scholar, M. J.D. Blair I.A. L.J. Science. PubMed Scopus Google Scholar, M. L.J. Blair I.A. Biol. Mass Spectrom. PubMed Scopus (65) Google Scholar, M. L.J. Blair I.A. J. Mass Spectrom. 1995; 30: Scopus Google Scholar, Blair I.A. L.J. 17: PubMed Scopus Google Scholar, M. Blair I.A. L.J. Chem. Res. Toxicol. 10: PubMed Scopus Google Scholar, M. L.J. J. Mass Spectrom. 2004; 39: PubMed Scopus Google Scholar, L.J. 2000; PubMed Scopus Google Scholar). However, the adduct it forms with dGuo in vivo from base that are generated from ROS-mediated damage to the of DNA K. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). can also be formed from MDA during biosynthesis. is not a specific of lipid hydroperoxide-mediated DNA adduct it be a useful of endogenous DNA damage resulting from oxidative has been detected in the DNA of M. J.D. Blair I.A. L.J. Science. PubMed Scopus Google Scholar) and models M. L.J. Blair I.A. Biol. Mass Spectrom. PubMed Scopus (65) Google Scholar) as well as in human M. Blair I.A. L.J. Chem. Res. Toxicol. 10: PubMed Scopus Google Scholar). is excreted into rat urine as the corresponding metabolite H. L.J. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus (13) Google Scholar). Unfortunately, the endogenous of in human urine to be low to the amount of base and DNA damage M. L.J. J. Mass Spectrom. 2004; 39: PubMed Scopus Google Scholar). However, this important the that other lipid hydroperoxide-derived DNA adducts might undergo metabolism they are excreted in the isotope dilution capture chemical methodology was used to determine the of adducts in human DNA J. Chung F.L. Chem. Res. Toxicol. 1999; 12: PubMed Scopus (62) Google Scholar). A subsequent study the use of which it possible to the DNA adduct to the or to an Chem. Res. Toxicol. 2000; 13: PubMed Scopus (74) Google Scholar). this and specific it was shown that was in at much lower in human DNA than found by the The between the capture chemical and studies was suggested to result from formation of during the and derivatization This to the with high of lipid hydroperoxide-derived DNA adducts in human tissue COX-2 can AA into which intracellular reduction to (15S)-HETE (4Blair I.A. Curr. Drug Metab. 2006; 7: 853-872Crossref PubMed Scopus (101) Google Scholar, Y. Fisher A.B. Free Radic. Biol. Med. 2005; 38: 1422-1432Crossref PubMed Scopus (322) Google Scholar). In of oxidative stress, reducing are that (15S)-HPETE might to undergo homolytic decomposition to bifunctional electrophiles. COX-2 is to the which the potential of bifunctional electrophiles into the RIES cells, which stably it was possible to that endogenous production of (15S)-HPETE resulted in the formation of H∈dGuo adducts (11Lee S.H. Williams M.V. Dubois R.N. Blair I.A. J. Biol. Chem. 2005; 280: 28337-28346Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). from in studies (21Lee S.H. Oe T. Blair I.A. Science. 2001; 292: 2083-2086Crossref PubMed Scopus (409) Google Scholar, 23Williams M.V. Lee S.H. Blair I.A. Rapid Commun. Mass Spectrom. 2005; 19: 849-858Crossref PubMed Scopus (30) Google Scholar), there was a increase in endogenous H∈dGuo adduct formation when vitamin C was added to the RIES cells (11Lee S.H. Williams M.V. Dubois R.N. Blair I.A. J. Biol. Chem. 2005; 280: 28337-28346Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). H∈dGuo adduct formation and (15S)-HPETE biosynthesis were both by a specific COX-2 and by The role of COX-2 induced DNA damage in vivo was using the mouse of (8Williams M.V. Lee S.H. Pollack M. Blair I.A. J. Biol. Chem. 2006; 281: 10127-10133Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The in is to of This results in increased together with of Therefore, a useful to the of increased in vivo COX-2 DNA was from the of and and then in the presence of isotope dilution was to and In separate it was shown that the H∈DNA adducts were not generated as during and by (Fig. The use of DNA a method to an endogenous DNA adduct can arise during and of DNA or during LC-MS H∈dGuo was increased from normal bases in to normal bases in H∈dCyd was much This suggested that base excision repair H∈dAdo and H∈dCyd H∈dGuo in the DNA and that the excised adducts might be excreted in the Interestingly, adducts were which suggests that peroxidation of LA not to DNA damage that was to the In with this could the only when esterified (32Jian W. Arora J.S. Oe T. Shuvaev V.V. Blair I.A. Free Radic. Biol. Med. 2005; 39: 1162-1176Crossref PubMed Scopus (46) Google Scholar). The of DNA repair that have to adducts arising from endogenous has been in of biological to L.J. 2000; PubMed Scopus Google Scholar). DNA is very of the DNA damage results in (7Sharma R.A. Farmer P.B. Clin. Cancer Res. 2004; 10: 4901-4912Crossref PubMed Scopus (86) Google Scholar). This that DNA adducts, which are and in the can be used as of DNA damage. and which are formed by lipid hydroperoxide-mediated DNA might eventually be useful urinary (Fig. It is that in with high stable isotope dilution LC-MRM-MS be for the specific of urinary H∈DNA adducts Anal. Chem. 2001; PubMed Scopus Google Scholar, M. L.J. J. Mass Spectrom. 2004; 39: PubMed Scopus Google Scholar). the that H∈dCyd is highly in human cells M. Blair I.A. M. Chem. Res. Toxicol. 2006; 19: PubMed Scopus Google Scholar) suggests that the of urinary H∈DNA adducts might the of human that are at for through lipid DNA damage.

Cell surface glycosylation is an important element in defining the life of pathogenic bacteria. Tannerella forsythia is a Gram-negative, anaerobic periodontal pathogen inhabiting the subgingival plaque biofilms. It is completely covered by a two-dimensional crystalline surface layer (S-layer) composed of two glycoproteins. Although the S-layer has previously been shown to delay the bacterium's recognition by the innate immune system, we characterize here the S-layer protein O-glycosylation as a potential virulence factor. The T. forsythia S-layer glycan was elucidated by a combination of electrospray ionization-tandem mass spectrometry and nuclear magnetic resonance spectroscopy as an oligosaccharide with the structure 4-Me-β-ManpNAcCONH(2)-(1→3)-[Pse5Am7Gc-(2→4)-]-β-ManpNAcA-(1→4)-[4-Me-α-Galp-(1→2)-]-α-Fucp-(1→4)-[-α-Xylp-(1→3)-]-β-GlcpA-(1→3)-[-β-Digp-(1→2)-]-α-Galp, which is O-glycosidically linked to distinct serine and threonine residues within the three-amino acid motif (D)(S/T)(A/I/L/M/T/V) on either S-layer protein. This S-layer glycan obviously impacts the life style of T. forsythia because increased biofilm formation of an UDP-N-acetylmannosaminuronic acid dehydrogenase mutant can be correlated with the presence of truncated S-layer glycans. We found that several other proteins of T. forsythia are modified with that specific oligosaccharide. Proteomics identified two of them as being among previously classified antigenic outer membrane proteins that are up-regulated under biofilm conditions, in addition to two predicted antigenic lipoproteins. Theoretical analysis of the S-layer O-glycosylation of T. forsythia indicates the involvement of a 6.8-kb gene locus that is conserved among different bacteria from the Bacteroidetes phylum. Together, these findings reveal the presence of a protein O-glycosylation system in T. forsythia that is essential for creating a rich glycoproteome pinpointing a possible relevance for the virulence of this bacterium.

Most naturally occurring mammalian cancers and immortalized tissue culture cell lines share a common characteristic, the overexpression of full-length HMGA1 (high mobility group A1) proteins. The HMGA1 protooncogene codes for two closely related isoform proteins, HMGA1a and HMGA1b, and causes cancerous cellular transformation when overexpressed in either transgenic mice or "normal" cultured cell lines. Previous work has suggested that the in vivo types and patterns of the HMGA1 post-translational modifications (PTMs) differ between normal and malignant cells. The present study focuses on the important question of whether HMGA1a and HMGA1b proteins isolated from the same cell type have identical or different PTM patterns and also whether these isoform patterns differ between non-malignant and malignant cells. Two independent mass spectrometry methods were used to identify the types of PTMs found on specific amino acid residues on the endogenous HMGA1a and HMGA1b proteins isolated from a non-metastatic human mammary epithelial cell line, MCF-7, and a malignant metastatic cell line derived from MCF-7 cells that overexpressed the transgenic HMGA1a protein. Although some of the PTMs were the same on both the HMGA1a and HMGA1b proteins isolated from a given cell type, many other modifications were present on one but not the other isoform. Furthermore, we demonstrate that both HMGA1 isoforms are di-methylated on arginine and lysine residues. Most importantly, however, the PTM patterns on the endogenous HMGA1a and HMGA1b proteins isolated from non-metastatic and metastatic cells were consistently different, suggesting that the isoforms likely exhibit differences in their biological functions/activities in these cell types.

Regulatory T cells promote corneal endothelial cell survival following transplantation via interleukin-10.

A major question in G protein-coupled receptor signaling concerns the quaternary structure required for signal transduction. Do these transmembrane receptors function as monomers, dimers, or larger oligomers? We have investigated the oligomeric state of the model G protein-coupled receptor rhodopsin (Rho), which absorbs light and initiates a phototransduction-signaling cascade that forms the basis of vision. In this study, different forms of Rho were isolated using gel filtration techniques in mild detergents, including n-dodecyl-beta-D-maltoside, n-tetradecyl-beta-D-maltoside, and n-hexadecyl-beta-D-maltoside. The quaternary structure of isolated Rho was determined by transmission electron microscopy, demonstrating that in micelles containing n-dodecyl-beta-D-maltoside, Rho exists as a mixture of monomers and dimers whereas in n-tetradecyl-beta-D-maltoside and n-hexadecyl-beta-D-maltoside Rho forms higher ordered structures. Especially in n-hexadecyl-beta-D-maltoside, most of the particles are present in tightly packed rows of dimers. The oligomerization of Rho seems to be important for interaction with its cognate G protein, transducin. Although the activated Rho (Meta II) monomer or dimers are capable of activating the G protein, transducin, the activation process is much faster when Rho exists as organized dimers. Our studies provide direct comparisons between signaling properties of Meta II in different quaternary complexes.

Crystal structures of the xenobiotic metabolizing cytochrome P450 2B4 have demonstrated markedly different conformations in the presence of imidazole inhibitors or in the absence of ligand. However, knowledge of the plasticity of the enzyme in solution has remained scant. Thus, hydrogen-deuterium exchange mass spectrometry (DXMS) was utilized to probe the conformations of ligand-free P450 2B4 and the complex with 4-(4-chlorophenyl)imidazole (4-CPI) or 1-biphenyl-4-methyl-1H-imidazole (1-PBI). The results of DXMS indicate that the binding of 4-CPI slowed the hydrogen-deuterium exchange rate over the B'- and C-helices and portions of the F-G-helix cassette compared with P450 2B4 in the absence of ligands. In contrast, there was little difference between the ligand-free and 1-PBI-bound exchange sets. In addition, DXMS suggests that the ligand-free P450 2B4 is predominantly open in solution. Interestingly, a new high resolution structure of ligand-free P450 2B4 was obtained in a closed conformation very similar to the 4-CPI complex. Molecular dynamics simulations performed with the closed ligand-free structure as the starting point were used to probe the energetically accessible conformations of P450 2B4. The simulations were found to equilibrate to a conformation resembling the 1-PBI-bound P450 2B4 crystal structure. The results indicate that conformational changes observed in available crystal structures of the promiscuous xenobiotic metabolizing cytochrome P450 2B4 are consistent with its solution structural behavior.

We present the first large scale study characterizing both N- and O-linked glycosylation in a site-specific manner on hundreds of proteins. We demonstrate that a lectin-affinity fractionation step using wheat germ agglutinin enriches not only peptides carrying intracellular O-GlcNAc, but also those bearing ER/Golgi-derived N- and O-linked carbohydrate structures. Liquid chromatography-MS (LC/MS) analysis with high accuracy precursor mass measurements and high sensitivity ion trap electron-transfer dissociation (ETD) were utilized for structural characterization of glycopeptides. Our results reveal both the identity of the precise sites of glycosylation and information on the oligosaccharide structures possible on these proteins. We report a novel iterative approach that allowed us to interpret the ETD data set directly without making prior assumptions about the nature and distribution of oligosaccharides present in our glycopeptide mixture. Over 2500 unique N- and O-linked glycopeptides were identified on 453 proteins. The extent of microheterogeneity varied extensively, and up to 19 different oligosaccharides were attached at a given site. We describe the presence of the well-known mucin-type structures for O-glycosylation, an EGF-domain-specific fucosylation and a rare O-mannosylation on the transmembrane phosphatase Ptprz1. Finally, we identified three examples of O-glycosylation on tyrosine residues.

Human nasal epithelial cells derived from multiple subjects exhibit differential responses to H3N2 influenza virus infection in vitro

A new method for enrichment of phosphopeptides in complex mixtures derived by proteolytic digestion of biological samples has been developed. The method is based on calcium phosphate precipitation of the phosphopeptides prior to further enrichment with established affinity enrichment methods. Calcium phosphate precipitation combined with phosphopeptide enrichment using Fe(III) IMAC provided highly selective enrichment of phosphopeptides. Application of the method to a complex peptide sample derived from rice embryo resulted in more than 90% phosphopeptides in the enriched sample as determined by mass spectrometry. Introduction of a two-step IMAC enrichment procedure after calcium phosphate precipitation resulted in observation of an increased number of phosphopeptides.

The extracellular matrix (ECM) molecules play important roles in many biological and pathological processes. During tissue remodeling, the ECM molecules that are glycosylated are different from those of normal tissue owing to changes in the expression of many proteins that are responsible for glycan synthesis. Vitronectin (VN) is a major ECM molecule that recognizes integrin on hepatic stellate cells (HSCs). The present study attempted to elucidate how changes in VN glycans modulate the survival of HSCs, which play a critical role in liver regeneration. Plasma VN was purified from partially hepatectomized (PH) and sham-operated (SH) rats at 24 h after operation and non-operated (NO) rats. Adhesion of rat HSCs (rHSCs), together with phosphorylation of focal adhesion kinase, in PH-VN was decreased to one-half of that in NO- or SH-VN. Spreading of rHSCs on desialylated NO-VN was decreased to one-half of that of control VN, indicating the importance of sialylation of VN for activation of HSCs. Liquid chromatography/multiple-stage mass spectrometry analysis of Glu-C glycopeptides of each VN determined the site-specific glycosylation. In addition to the major biantennary complex-type N-glycans, hybrid-type N-glycans were site-specifically present at Asn(167). Highly sialylated O-glycans were found to be present in the Thr(110)-Thr(124) region. In PH-VN, the disialyl O-glycans and complex-type N-glycans were decreased while core-fucosylated N-glycans were increased. In addition, immunodetection after two-dimensional PAGE indicated the presence of hyper- and hyposialylated molecules in each VN and showed that hypersialylation was markedly attenuated in PH-VN. This study proposes that the alteration of VN glycosylation modulates the substrate adhesion to rat HSCs, which is responsible for matrix restructuring.

The effect of phosphorylation on the shape of tyrosine hydroxylase (TH) was studied directly using gel filtration and indirectly using electrospray ionization mass spectrometry. Phosphorylation of Ser(19) and Ser(40) produced a TH molecule with a more open conformation than the non-phosphorylated form. The conformational effect of Ser(19) phosphorylation is less pronounced than that of the Ser(40) phosphorylation. The effect of Ser(19) and Ser(40) phosphorylation appears to be additive. Binding of dopamine produced a more compact form when compared with the non-dopamine-bound TH. The interdependence of Ser(19) and Ser(40) phosphorylation was probed using electrospray ionization mass spectrometry. The rate constants for the phosphorylation of Ser(19) and Ser(40) were determined by electrospray ionization mass spectrometry using a consecutive reaction model. The rate constant for the phosphorylation of Ser(40) is approximately 2- to 3-fold higher if Ser(19) is already phosphorylated. These results suggest that phosphorylation of Ser(19) alters the conformation of tyrosine hydroxylase to allow increased accessibility of Ser(40) to kinases.

The Intestinal Metabolome: An Intersection Between Microbiota and Host

A shotgun lipidomics approach that allowed the analysis of eight lipid classes directly from crude extracts of the soil bacterium Sinorhizobium meliloti is presented. New MS-MS transitions are reported for the analysis of monomethylphosphatidylethanolamines, dimethylphosphatidylethanolamines, and three bacterial non-phosphorus-containing lipid classes [sulfoquinovosyldiacylglycerols, ornithines, and diacylglyceryl-(N,N,N-trimethyl)-homoserines]. Unique MS-MS transitions allowed the analysis of isomeric species from various lipid classes without chromatography. Analyses required small sample amounts and minimal preparation; thus, this methodology has excellent potential to be used as a screening tool for the analysis of large numbers of samples in functional genomics studies. FA distributions within lipid classes of S. meliloti are described for the first time, and the relative positions of fatty acyl substituents (sn-1, sn-2) in phospholipids are presented. FA distributions in diacylglyceryl-(N,N,N-trimethyl)-homoserines were identical to those of phospholipids, indicating a common biosynthetic origin for these lipids. The method was applied to the analysis of mutants deficient in the PhoB regulator protein. Increased lipid cyclopropanation was observed in PhoB-deficient mutants under P(i) starvation.