Use of Narrow Mass-Window, High-Resolution Extracted Product Ion

Mar 27, 2013 - Life and Health Sciences, Aston University, Aston Triangle, Birmingham, B4 7ET, United Kingdom. •S Supporting Information. ABSTRACT: ...
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Use of Narrow Mass-Window, High-Resolution Extracted Product Ion Chromatograms for the Sensitive and Selective Identification of Protein Modifications Corinne M. Spickett,‡ Ana Reis,‡ and Andrew R. Pitt*,‡ Life and Health Sciences, Aston University, Aston Triangle, Birmingham, B4 7ET, United Kingdom S Supporting Information *

ABSTRACT: Protein modifications, including oxidative modifications, glycosylations, and oxidized lipid−protein adducts, are becoming increasingly important as biomarkers and in understanding disease etiology. There has been a great deal of interest in mapping these on Apo B100 from low density lipoprotein (LDL). We have used extracted ion chromatograms of product ions generated using a very narrow mass window from high-resolution tandem mass spectrometric data collected on a rapid scanning quadrupole time-of-flight (QTOF) instrument, to selectively and sensitively detect modified peptides and identify the site and nature of a number of protein modifications in parallel. We have demonstrated the utility of this method by characterizing for the first time oxidized phospholipid adducts to LDL and human serum albumin and for the detection of glycosylation and kynurenin formation from the oxidation of tryptophan residues in LDL.

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negative LDL ex vivo.3 Tyrosine nitration has also been detected ex vivo on albumin of LA-apoA-I(−/−) mice.18 The MS methods used are sensitive and powerful, but they rely on the precise site and nature of the modification already being identified.9 An alternative approach is the use of scanning routines such as precursor ion or neutral loss; these are more generic and enable the identification of any protein or peptide containing a specific modification.19−22 We tested this approach previously for the identification of hydroxylation and chlorination of tyrosine and tryptophan using the amino acid immonium ion as the reporter, but using conventional precursor ion scanning routines, some false positives were observed, prompting the development of a more specific MS3 fragmentation method.23 Similar problems with the use of the nitrotyrosine immonium ion at m/z 181.1 have been reported, together with the suggestion that the problem might be circumvented by accurate mass analysis.24 Similar approaches have been used to identify protein carbonyls using a dinitrophenyhdrazine (DNPH) chemical labeling approach and a reporter ion for the dinitrophenylhydrazone (DNP) product.13 For the analysis of adducts between electrophilic lipid peroxidation products and proteins, again there are numerous reports of analyses of specific fatty acid-derived aldehyde adducts reacted with individual proteins in vitro.14,22,25−27 Similar problems of false positive identifications are encountered, and similar solutions have been described. 26−28 Hydroxynonenal (HNE) adducts of ApoB-100 have been

xidative damage to biomolecules is a common factor in inflammatory disease and offers the potential insights into disease mechanisms and diagnostic markers. Oxidative modifications to low and high density lipoproteins (LDL and HDL) have been much studied and are considered to be valuable reporters of systemic inflammatory status, as well as contributing to disease pathology.1,2 Apolipoprotein B100 (ApoB-100), the protein in LDL, can display a variety of oxidative and nonoxidative post-translational modifications (oxPTMs and PTMs), including glycosylation, glycation, oxidation, and adduct formation, which vary or play a role in disease conditions.3−6 The glycosylation pattern of lipoproteins has been reported to change in disease, and ApoB-100 may also be modified by advanced glycation endproducts (AGEs).6,7 Adduct formation can arise through reactions of cysteine, histidine, and lysine residues with electrophilic products of phospholipid peroxidation.8−10 All these modifications can affect lipoprotein recognition by receptors and immunogenicity,1,2,10,11 so it is extremely important to be able to elucidate such structural modifications in order to understand their role in pathology. Many studies have detected and identified specific modifications of proteins and lipoproteins, especially in vitro,12−15 but few studies have adopted a global approach to the analysis of PTMs. The most informative methodology for analyzing PTMs is mass spectrometry, which has the potential for precise localization and identification of modifications.16 For example, following oxidation of LDL or HDL in vitro with myeloperoxidase-derived oxidants, tyrosine chlorination was detected by mass spectrometry (MS),17 while nitrations of tyrosine and tryptophan, as well as cysteine trioxidation and phenylalanine hydroxylation, have been observed in electro© 2013 American Chemical Society

Received: January 27, 2013 Accepted: March 27, 2013 Published: March 27, 2013 4621

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detected using enzyme linked immunosorbent assay (ELISA)29 and by mass spectrometry,14 but data on other lipid modification are scarce. Very little work has been done on the formation of intact phospholipid adducts with proteins of any kind, despite the fact that more than a decade ago adducts of oxidized phospholipids (oxPLs) with LDL were reported on the basis of detection with monoclonal antibodies.30,31 This reflects the difficulty of analysis of such adducts with MS, owing to differences in ionizability, m/z, fragmentation susceptibility, and polarity compared to unmodified peptides. Improved MS technologies are therefore also required to detect oxidized phospholipid adducts to proteins. A significant proportion of the false positive rate in neutral loss and precursor ion scanning is due to the limited resolution of the second quadrupole, giving a minimum detection window of around 0.3−0.5 Da. This is particularly problematic when the reporter ion is small (70% sequence 4623

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with peptides (region labeled iii). There were also some signals in this region that were found to be false positives as they appeared to be late eluting peptides without adducts, but they could be relatively easily identified as they had very low (20 000 MS/MS spectra in a standard 1 h LC run, it is possible to mine very deeply into samples. This could be further improved if sufficient quality MS/MS data could be extracted from methods that do not select a precursor ion in Q1, such as the MSE34,35 and SWATH44 approaches. Additionally, low intensity signals are unlikely to give sufficient quality MS/MS data to allow unambiguous identification of the structure. Limitations of tryptic digestion can be overcome with orthogonal digestion strategies if necessary. The high data content of analyses on modern MS instruments means that significant sequence coverage can be obtained even for very large proteins, as demonstrated by the >70% sequence coverage obtained for Apo B100, and the sensitivity of the method demonstrated in the identification for the first time of intact oxidized phospholipid adducts of this protein; any limitations are significantly outweighed by the reduction in false positives and the sensitivity of the method and the fact that it is label free.

aldehyde derived from the oxidation of palmitoyl-arachidonoylphosphatidylcholine (PAPC) with the peptide LVELTHQYK*LK is shown in Supplementary Figure S-3, Supporting Information. The fatty acid chain can be identified as palmitoyl from the peak at m/z 313 Da, and the mass of the oxidized chain can be inferred from this information; the mass of the peptide can be calculated from the m/z of the precursor, and the sequence derived from the spectrum. This demonstrates that the methodology can be used very effectively to identify modifications from clinical samples. Some of the modifications identified in this study are shown in Supplementary Table S-1, Supporting Information; these are illustrative, and we have not undertaken an exhaustive analysis. High-resolution XIC extraction methodology was tested further by analysis of glycosylation sites on ApoB100. A number of well-known reporter ions have been used in precursor ion scanning for the detection of glycosylation, such as m/z 204.09 (HexNAc+) for general glycosylation and m/z 274.1 ([NeuAc − H2O]+) for sialic acid-containing glycans.33,42 An example of the data generated from precursor ion scanning for m/z 274.1 on a 5500 QTrap is shown in Figure 4a. Comparison of this data with XICs generated with a ±0.25 Da and a ±0.0025 Da window for m/z 274.0905 is shown in Figure 4b,c, respectively, and demonstrates again how the highresolution XIC can be used to effectively target sialic acid containing glycans and minimize false positives. A number of intense fragment ions are present in the m/z 274.1 region of the MS/MS spectra (Figure 4d) that would significantly interfere with the detection of the sialic acid fragment at lower resolution. The interfering ions may come from short peptide fragments, some of which are identified in Figure 4e. All the MS/MS data analyzed so far that show a significant intensity reporter at m/z 274.0905 ± 0.0025 (positive peaks in Figure 4c) appear to contain peptides modified with sialic acidcontaining glycans (confirmed by data in Supplementary Figure S-4, Supporting Information). Although the m/z 204.1 fragment has previously been used for the detection of HexNAc glycosylated peptides, interestingly, even selecting m/z 204.0872 with a ±0.005 Da window still generated a high number of false positives. However, the MS/MS spectra of true positive glycopeptides identified using the narrow window XIC of m/z 204.0872 were all found to contain certain other fragment ions (at m/z 138.0512, 168.0648, and 186.0750 Da) that gave better selectivity for glycosylation than m/z 204.1 and, therefore, have better potential as reporter product ions. The best results were obtained with m/z 138.0512, which demonstrated a good combination of sensitivity and selectivity, giving a minimal level of false positive identifications; using an XIC of 138.0512 ± 0.0025, all the peptides characterized contained glycosylated chains. The general nature of this approach was demonstrated by reanalysis of the LDL data for the identification of peptides containing an oxidatively modified amino acid. Kynureninemodified peptides formed by mono-oxidation of tryptophan can be identified using the immonium ion at m/z 174.1.43 The combined MS/MS spectra at 174.1 Da shows the presence of a number of interfering fragment ions (Supplementary Figure S5a, Supporting Information), but generation of an XIC at m/z 174.055 ± 0.0025 (Supplementary Figure S-5b, Supporting Information) selectively identified kynurenin containing peptides, as demonstrated by the MS/MS sequencing of an example low intensity peptide ion at 791.3 Da (Supplementary Figure S-5c, Supporting Information). The presence of a strong



CONCLUSIONS The use of narrow XIC window chromatograms of product ions generated from high-resolution, high acquisition rate MS/ MS data significantly reduces the false positive rates especially when more than one reporter ion is used in tandem and provides high quality MS/MS data for the identification of the modified peptides. This label-free approach can be used for the parallel detection of a number of modifications and has been used to identify for the first time intact oxidized phospholipid adducts on a protein. It can be applied to a wide range of modifications, including glycosylation and oxidative modifications of LDL.



ASSOCIATED CONTENT

S Supporting Information *

Supplementary Figures S-1 to S-5. This material is available free of charge via the Internet at http://pubs.acs.org.



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions ‡

C.M.S., A.R., and A.R.P. contributed equally to the paper. All authors have given approval to the final version of the manuscript. 4626

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Notes

(25) Wakita, C.; Honda, K.; Shibata, T.; Akagawa, M.; Uchida, K. Free Radical Biol. Med. 2011, 51, 1−4. (26) Carini, M.; Aldini, G.; Facino, R. M. Mass Spectrom. Rev. 2004, 23, 281−305. (27) Rauniyar, N.; Prokai, L. Proteomics 2009, 9, 5188−5193. (28) Orioli, M.; Aldini, G.; Benfatto, M. C.; Facino, R. M.; Carini, M. Anal. Chem. 2007, 79, 9174−9184. (29) Uchida, K.; Toyokuni, S.; Nishikawa, K.; Kawakishi, S.; Oda, H.; Hiai, H.; Stadtman, E. R. Biochemistry 1994, 33, 12487−12494. (30) Bird, D. A.; Gillotte, K. L.; Horkko, S.; Friedman, P.; Dennis, E. A.; Witztum, J. L.; Steinberg, D. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 6347−6352. (31) Boullier, A.; Gillotte, K. L.; Horkko, S.; Green, S. R.; Friedmann, P.; Dennis, E. A.; Witztum, J. L.; Steinberg, D.; Quehenberger, O. J. Biol. Chem. 2000, 275, 9163−9169. (32) Steen, H.; Mann, M. Anal. Chem. 2002, 74, 6230−6236. (33) Jebanathirajah, J.; Steen, H.; Roepstorff, P. J. Am. Soc. Mass Spectrom. 2003, 14, 777−784. (34) Niggeweg, R.; Kocher, T.; Gentzel, M.; Buscaino, A.; Taipale, M.; Akhtar, A.; Wilm, M. Proteomics 2006, 6, 41−53. (35) Silva, J. C.; Denny, R.; Dorschel, C. A.; Gorenstein, M.; Kass, I. J.; Li, G. Z.; McKenna, T.; Nold, M. J.; Richardson, K.; Young, P.; Geromanos, S. Anal. Chem. 2005, 77, 2187−2200. (36) Cristobal, A.; Hennrich, M. L.; Giansanti, P.; Goerdayal, S. S.; Heck, A. J. R.; Mohammed, S. Analyst 2012, 137, 3541−3548. (37) Andrews, G. L.; Simons, B. L.; Young, J. B.; Hawkridge, A. M.; Muddiman, D. C. Anal. Chem. 2011, 83, 5442−5446. (38) Jerlich, A.; Pitt, A. R.; Schaur, R. J.; Spickett, C. M. Free Radical Biol. Med. 2000, 28, 673−682. (39) Yue, H.; Jansen, S. A.; Strauss, K. I.; Borenstein, M. R.; Barbe, M. F.; Rossi, L. J.; Murphy, E. J. Pharm. Biomed. Anal. 2007, 43, 1122− 1134. (40) Shevchenko, A.; Tomas, H.; Havlis, J.; Olsen, J. V.; Mann, M. Nat. Protoc. 2006, 1, 2856−2860. (41) Reis, A.; Domingues, P.; Ferrer-Correia, A. J.; Domingues, M. R. J. Am. Soc. Mass Spectrom. 2006, 17, 657−660. (42) Harazono, A.; Kawasaki, N.; Kawanishi, T.; Hayakawa, T. Glycobiology 2005, 15, 447−462. (43) Todorovski, T.; Fedorova, M.; Hoffmann, R. J. Mass Spectrom. 2011, 46, 1030−1038. (44) Gillet, L. C.; Navarro, P.; Tate, S.; Rost, H.; Selevsek, N.; Reiter, L.; Bonner, R.; Aebersold, R. Mol. Cell. Proteomics 2012, 11, DOI: 10.1074/mcp.O111.016717.

The authors declare no competing financial interest.



ACKNOWLEDGMENTS Funding was provided by the Engineering and Physical Sciences Research Council (EPSRC), UK; The European Union FP7 PEOPLE Programme. We gratefully acknowledge funding for a Marie-Curie Intra-European Fellowship (FP7PEOPLE-2009-IEF Project ID 255076 “ATHERO_MASS”) and from the Engineering and Physical Sciences Research Council (EPSRC grant EP/I017887/1). We would also like to thank Aston University and ABSciex for the provision of the mass spectrometers used in this study and Professor David Webb, University of Edinburgh, for providing clinical plasma samples.



REFERENCES

(1) Berliner, J. A.; Heinecke, J. W. Free Radical Biol. Med. 1996, 20, 707−727. (2) Malle, E.; Marsche, G.; Panzenboeck, U.; Sattler, W. Arch. Biochem. Biophys. 2006, 445, 245−255. (3) Hamilton, R. T.; Asatryan, L.; Nilsen, J. T.; Isas, J. M.; Gallaher, T. K.; Sawamura, T.; Hsiai, T. K. Arch. Biochem. Biophys. 2008, 479, 1−14. (4) Obama, T.; Kato, R.; Masuda, Y.; Takahashi, K.; Aiuchi, T.; Itabe, H. Proteomics 2007, 7, 2132−2141. (5) Ravandi, A.; Kuksis, A.; Shaikh, N. A. Arterioscler., Thromb., Vasc. Biol. 2000, 20, 467−477. (6) Tertov, V. V.; Kaplun, V. V.; Sobenin, I. A.; Orekhov, A. N. Atherosclerosis 1998, 138, 183−195. (7) Garner, B.; Harvey, D. J.; Royle, L.; Frischmann, M.; Nigon, F.; Chapman, M. J.; Rudd, P. M. Glycobiology 2001, 11, 791−802. (8) Annangudi, S. P.; Deng, Y. J.; Gu, X. R.; Zhang, W. J.; Crabb, J. W.; Salomon, R. G. Chem. Res. Toxicol. 2008, 21, 1384−1395. (9) Chakraborty, S.; Cai, Y.; Tarr, M. A. Anal. Biochem. 2010, 404, 109−117. (10) Negre-Salvayre, A.; Vieira, O.; Escargueil-Blanc, I.; Salvayre, R. Mol. Aspects Med. 2003, 24, 251−261. (11) Vindis, C.; Escargueil-Blanc, I.; Uchida, K.; Elbaz, M.; Salvayre, R.; Negre-Salvayre, A. Redox Rep. 2007, 12, 96−100. (12) Aldini, G.; Regazzoni, L.; Orioli, M.; Rimoldi, I.; Facino, R. M.; Carini, M. J. Mass Spectrom. 2008, 43, 1470−1481. (13) Bollineni, R. C.; Fedorova, M.; Hoffmann, R. J. Proteomics 2011, 74, 2351−2359. (14) Bolgar, M. S.; Yang, C. Y.; Gaskell, S. J. J. Biol. Chem. 1996, 271, 27999−28001. (15) Maeshima, T.; Honda, K.; Chikazawa, M.; Shibata, T.; Kawai, Y.; Akagawa, M.; Uchida, K. Chem. Res. Toxicol. 2012, 25, 1384−1392. (16) Spickett, C. M.; Pitt, A. R. Amino Acids 2012, 42, 5−21. (17) Bergt, C.; Fu, X. Y.; Huq, N. P.; Kao, J.; Heinecke, J. W. J. Biol. Chem. 2004, 279, 7856−7866. (18) Parastatidis, I.; Thomson, L.; Fries, D. M.; Moore, R. E.; Tohyama, J.; Fu, X.; Hazen, S. L.; Heijnen, H. F.; Dennehy, M. K.; Liebler, D. C.; Rader, D. J.; Ischiropoulos, H. Circ. Res. 2007, 101, 368−376. (19) Steen, H.; Kuster, B.; Fernandez, M.; Pandey, A.; Mann, M. Anal. Chem. 2001, 73, 1440−1448. (20) Annan, R. S.; Carr, S. A. J. Protein Chem. 1997, 16, 391−402. (21) Petersson, A. S.; Steen, H.; Kalume, D. E.; Caidahl, K.; Roepstorff, P. J. Mass Spectrom. 2001, 36, 616−625. (22) Fenaille, F.; Tabet, J. C.; Guy, P. A. Rapid Commun. Mass Spectrom. 2004, 18, 67−76. (23) Mouls, L.; Silajdzic, E.; Haroune, N.; Spickett, C. M.; Pitt, A. R. Proteomics 2009, 9, 1617−1631. (24) Abello, N.; Kerstjens, H. A. M.; Postma, D. S.; Bischoff, R. J. Proteome Res. 2009, 8, 3222−3238. 4627

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