A Comprehensive Analytical Strategy To Identify Malondialdehyde

Subscriber access provided by University of Newcastle, Australia

Technical Note

A comprehensive analytical strategy to identify malondialdehyde-modified proteins and peptides Juliane Weißer, Claudia Ctortecka, Clara J. Busch, Shane R. Austin, Karin Nowikovsky, Koji Uchida, Christoph J. Binder, and Keiryn L. Bennett Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b05065 • Publication Date (Web): 01 Mar 2017 Downloaded from http://pubs.acs.org on March 2, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

A comprehensive analytical strategy to identify malondialdehydemodified proteins and peptides Juliane Weißer,1 Claudia Ctortecka,1 Clara J Busch,1,2,† Shane R Austin,1,3,§ Karin Nowikovsky,3 Koji Uchida,4 Christoph J Binder,1,2 and Keiryn L Bennett1* 1

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090, Vienna,

Austria 2

Department of Laboratory Medicine, Medical University of Vienna, 1090, Vienna, Austria

3

Department of Internal Medicine I, Medical University of Vienna, 1090, Vienna, Austria

4

Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan

†

present address: Max-Delbrück-Center for Molecular Medicine, 13092, Berlin, Germany

§

present address: University of the West Indies, Department of Biological and Chemical Sciences, PO Box 64, Bridgetown, Barbados

*Corresponding author: email: [email protected]

1 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 22

ABSTRACT: Mass spectrometric-based proteomics is a powerful tool to analyse post-translationally modified proteins. Carbonylation modifications that result from oxidative lipid breakdown are a class of posttranslational modifications that are poorly characterised with respect to protein targets and function. This is partly due to the lack of dedicated mass spectrometry-based technologies to facilitate the analysis of these modifications. Here, we present a comprehensive approach to identify malondialdehydemodified proteins and peptides. Malondialdehyde is amongst the most abundant of the lipid peroxidation products; and malondialdehyde-derived adducts on proteins have been implicated in cardiovascular diseases, neurodegenerative disorders and other clinical conditions. Our integrated approach targets three levels of the overall proteomic workflow: (i) sample preparation, by employing a targeted enrichment strategy; (ii) high-performance liquid chromatography, by using a gradient optimised for the separation of the modified peptides; and (iii) tandem mass spectrometry, by improving the spectral quality of very low-abundance peptides. By applying the optimised procedure to a whole cell lysate spiked with a low amount of malondialdehyde-modified proteins, we were able to identify up to 350 different modified peptides and localise the modification to a specific lysine residue. This methodology allows the comprehensive analysis of malondialdehyde-modified proteins.


Page 3 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Introduction Post-translational modifications (PTMs) often pose a significant challenge in mass spectrometric-based proteomics. The chromatographic, ionisation and fragmentation behaviour of modified proteins and peptides often differs from the unmodified version1. For confident identification and localisation of the modification, high quality MSMS spectra are a pre-requisite. Most modified peptides, however, are present in sub-stochiometric amounts compared to the unmodified counterparts.By exploiting unique chemical properties of specific PTMs, a series of approaches have been developed to specifically enrich modified peptides2,3. Affinity enrichment procedures have been reported phosphorylation4,5, acetylation6,7, ubiquitinylation8, glycosylation9,10 and others. In contrast, the diverse group of protein carbonylation PTMs is rather poorly-characterised. These modifications are implicated in numerous pathological situations, e.g., Alzheimer’s disease, lung or kidney disease11. Despite this, the importance of such PTMs is only beginning to emerge and very little is known about the biological and clinical ramifications of such protein modifications. A subgroup of carbonylation adducts on proteins are derived from the reactive aldehyde species that occur following the oxidative breakdown of polyunsaturated fatty acids from membrane lipids. Malondialdehyde (MDA) is one of the most abundant reactive aldehyde species and is used as an indirect measure of oxidative stress12,13. Via the two aldehyde functions, MDA can react with the amino groups of proteins14, nucleotides15 and membrane lipids16. This leads to the formation of a variety of covalent Schiff base-type adducts. The most commonly-reported modifications are a transient imidopropene group and a stable, more complex dihydropyridine adduct (Fig. 1A)17,18. Under certain conditions, malondialdehyde can also result in the crosslinking of two moieties19. Previous efforts to identify the protein targets of carbonylation PTMs have primarily relied on their chemical derivatisation with carbonyl reactive compounds20. Amongst these, di-nitrophenylhydrazine (DNPH) is the most widely used21,22. DNPH-derivatisation allows carbonyl-specific visualisation with a DNPH-reactive antibody after two-dimensional gel separation of a proteome19. Target proteins are usually identified after excision of DNPH-positive spots followed by mass spectrometric identification.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 22

Although successfully applied23,24, the method is very tedious. Site localisation is challenging and often results in a high false positive rate.Alternatively, hydrazine derivatisation can be used to introduce a biotin tag that enables gel free enrichment of carbonylated proteins25-28. Alternative tagging chemistry relies on O-(biotinylcarbazoylmethyl) hydroxylamine29 or aminooxy-coupled biotin tags30. The drawbacks of this approach, however, are a potential increase in sample complexity introduced by the derivatisation reaction; and altered peptide fragmentation behaviour particularly when using a biotin probe25. This problem can be partly overcome with a tag that is more amenable to MSMS fragmentation31. Most of these approaches broadly target all protein carbonylation products. Thus, database searches become markedly more complex. A modification-specific antibody for MDA adducts has led to the identification of several putative targets by 2D-gel-MSMS32-36. With respect to site localisation, however, these approaches suffer from the same limitations detailed above. Herein, we present an integrated analytical strategy to identify MDA-modified proteins and peptides. The strategy aims at the selective enrichment of intact proteins with MDA adducts from a complex protein mixture via an antibody directed against MDA-derived epitopes37. A second stage enrichment of the MDA-modified peptides using hydrazine-functionalised agarose beads leads to the identification of the exact site of modification38. Hydrazine groups react with aldehyde and keto groups to form a covalent bond that can be cleaved by acid hydrolysis. As aldehyde groups only occur in peptides with an oxidation-specific modification; the approach is highly specific for this peptide subgroup. The methodology is complemented by an LC-MSMS strategy that was specifically tailored towards peptides with MDA modifications. This procedure allows the selective and comprehensive identification of MDAmodified proteins and the subsequent localisation of the MDA target site(s) within the proteins.


Page 5 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Experimental Section Enrichment of MDA-modified proteins and peptides. Unless otherwise stated, MDA-modified proteins were enriched according to the following procedure. 1F83 was added to the protein mixture at a ratio of 1:50 and incubated for 4 h at 4 °C with gentle rotation. Protein G-coated sepharose beads (GE Healthcare, Little Chalfont, UK) were washed with immunoprecipitation buffer and added to the protein mixture at a ratio of 1:5. Samples were incubated with gentle rotation for a minimum of 16 h at 4 °C. The supernatant was removed and the beads washed with 3×500 µL 250 mM HEPES pH 8, 150 mM NaCl, 5 mM EDTA. To elute the bound proteins, the beads were incubated for 20 min at RT with wash buffer supplemented with 2 % SDS under gentle rotation39. The original protein mixtures, the antibodyenriched samples and the eluted proteins were digested with trypsin according to the filter-aided sample preparation (FASP) protocol as published40,41. The peptides were collected from the filters by washing 1× with 100 µL 0.5 M NaCl and 2× with 100 µL hydrazine coupling buffer (100 mM NaOAc, 150 mM NaCl)38, pH 5.5 to yield a total volume of 350 µL. Peptides were coupled to 100 µL washed hydrazine-functionalized beads (Affy-Gel Hz, Bio-Rad, Berkeley, CA, USA) at 37 °C for 16 h with gentle rotation. Hydrazine-functionalized beads were washed with 5×300 µL 100 mM ammonium bicarbonate. Covalently coupled peptides were released with 2 % trifluoroacetic acid for 2 h at 37 °C with gentle rotation, and purified and concentrated via stop-and-go extraction (STAGE) tips42. Organic solvent was evaporated by vacuum centrifugation and the peptides were dissolved in 5 % formic acid prior to LC-MSMS analysis. Liquid chromatography tandem mass spectrometry. Mass spectrometric analyses were performed on a Q Exactive mass spectrometer (ThermoFisher, Bremen, Germany) coupled to an Agilent 1200 series dual pump HPLC system (Agilent, Santa Clara, CA, USA). Samples were transferred to a trap column (Zorbax 300SB-C18 5 µm, 5 × 0.3 mm, Agilent Biotechnologies, Santa Clara, CA, USA) at a flow rate of 45 µL/min. Separation occurred on a 20 cm 75 µm i. d. analytical column packed with Reprosil C18 (Dr. Maisch, Ammerbuch-Entringen, Germany) over a 60 minute gradient ranging from 12 % to 50 % organic phase at a constant flow rate of 250 nL/min. The mobile phases used were 0.4 %



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 22

formic acid and 90 % acetonitrile plus 0.4 % formic acid. The mass spectrometer was operated in a data-dependent mode with each full scan followed by a maximum of 10 MSMS scans and a 10 s dynamic exclusion, unless specified otherwise. For MS scans, 100 ms were allowed as the maximum ion injection time. For MSMS scans, the maximum ion injection time was set to 300 ms or 500 ms for samples of high and low complexity, respectively. The analyser resolution was 70,000 for MS and 17,500 for MSMS. Overfilling of the C-trap was prevented by setting the automatic gain control to 3×106 and 1×105 for MS and MSMS, respectively. The underfill ratio for MSMS was set to 12 %, corresponding to an intensity threshold of 2.4×104 for peptide fragmentation. Fragmentation was performed by higher collision energy induced dissociation (HCD) at a normalized collision energy (NCE) of 28. The ubiquitous contaminating siloxane ion Si(CH3)2O)6 was used as a single lock mass at m/z 445.120024 for internal mass calibration43. All samples were analysed as technical replicates.


Page 7 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Results and Discussion ESI-MSMS characteristics of MDA-modified peptides. An in vitro-modified model peptide was used to determine the predominant modification variant following the reaction with malondialdehyde and acetaldehyde. After only 5 minutes, the relative amount of the unmodified peptide decreased by about 40 % and approximately one third of the peptide was present with the linear MDA adduct (Fig. 1A, upper structure); however, this adduct was unstable and subsequently disappeared from the later reaction time points. After 60 minutes, approximately 50 % of the peptide was modified with the irreversible MAA modification (Fig. 1A, lower structure); and after three hours, MAA was the dominant modification with close to 100 % of the peptide containing this particular adduct (Fig. 1B). No additional larger adduct masses were observed, indicating that the dihydropyridine adduct represents the end point of the modification reaction. This result is consistent with reports that showed biologically-relevant malondialdehyde modifications tend to be of the more complex dihydropyridine type44-46. The proposed formation reaction of MAA adducts is outlined in Fig. S1. For the purpose of this study, we therefore focused our attention on this adduct type. The fragmentation behaviour of the MAA-modified model peptide following collision-induced dissociation (CID) was then assessed to: (i) confirm the location of the MAA adduct on the side chain of the lysine residue; and (ii) to determine if the MAA adduct results in a neutral loss. The normalised collision energy (NCE) was manually tuned and optimized at 25 % for the model peptide. The resultant MSMS spectrum revealed that the MAA modification was stable under standard peptide fragmentation conditions and no neutral loss was observed. Furthermore, the MAA-modified peptide produced a near complete b- and y-ion series (Fig. 1C) thus enabling unequivocal localization of the modification to the lysine residue. Optimisation of LC-MSMS conditions for MAA-modified peptides. Analysis of the chromatographic properties of an in vitro-modified yeast mitochondrial extract revealed that peptides with an MAA modification eluted at a considerably later retention time (tR) than the non-modified counterparts. The median tR for peptides without an MAA modification was between 40 and 50 minutes compared to approxi-



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 22

mately 60 minutes for modified peptides, with a tendency to elute only during the equilibration phase of the gradient (Fig. 2A and Fig. S2B). Consequently, a large proportion of the modified peptides were not selected for fragmentation by the mass spectrometer. The extended tR of the MAA-modified peptides is a consequence of the increased length of the peptides, since the modification blocks a tryptic site. Additionally, the MAA modification contains a large hydrophobic ring that has the potential to further increase the tR of the peptides. To ensure the complete elution of the MAA-modified peptides, a modified gradient containing a higher percentage of organic solvent (initially 12 % c.f. 8 % in our standard analyses) was implemented. With the modified gradient, all peptides were eluted from the column before the equilibration phase (Fig. S2A). The median tR of the MAA-modified peptides was decreased to 42 minutes (Fig. S2C). In addition, all modified peptides were eluted before the end of the gradient without compromising the elution profile of the non-modified peptides (Fig. 2B). Even following a successful enrichment, MDA-modified peptides are still quite infrequent and of low abundance. Thus, the tandem mass spectrometry settings were optimised to compensate for these very low quantities of input material. In general, samples with a reduced amount of analyte suffered from poor spectral quality that impairs confident peptide-to-spectrum matching. Five different tandem MS methods were assessed on an in vitro-modified yeast mitochondrial extract. Two different quantities containing 2 ng and 0.2 ng of peptide mixture per injection were evaluated. The parameters that contributed the most to increasing peptide-to-spectrum matching were: (i) a reduction in the dynamic exclusion time; and (ii) an increase in maximum ion injection time. An increase in normalised collision energy, however, did not appear to have any beneficial effects (Fig. 3A). For the 2 ng sample, decreasing the dynamic exclusion from 30 to 10 seconds and increasing the maximum injection time from 120 to 300 ms led to the identification of an additional 200 modified and 400 non-modified peptides, respectively (Fig. 3A). This effect was even more pronounced for the 0.2 ng sample. Here, extending the ion accumulation time to a maximum of 500 ms resulted in a 5-fold increase in the number of identified peptides (Fig. 3B).


Page 9 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The beneficial effect of longer ion accumulation times was immediately apparent as an increased number of ions retained in the analyser produced higher quality spectra. The influence of a shorter dynamic exclusion of the ions, however, initially seemed contradictory. Yet it appeared that by allowing an ion that had been previously selected and fragmented to be re-selected after a shorter exclusion period enabled any ion to be sampled more frequently and therefore increased the probability of a high-quality spectrum triggered at the apex of the eluted peak. In a proteomic experiment that is aimed towards achieving a deep coverage of the proteome, such amendments to the MS method as described above would result in far fewer identifications than when a shorter maximum injection time and a longer dynamic exclusion are chosen. In this particular case, such changes are beneficial for a highly-reduced sample containing very low-abundance peptides, since more analysis time is allocated to each single peptide. Immuno-recognition of MAA-modified proteins. Unbiased binding of the antibody used in this study irrespective of the epitope carrier was assessed by probing the binding of the antibody to in vitromodified model proteins with differing characteristics regarding size and composition. Sham-modified proteins were used as a control. The antibody specifically reacted with the MAA-modified version and no staining was visible for the control, sham-modified protein (Fig. S3A). This result was confirmed by ELISA. Only a strong binding to MAA-modified BSA and low-density lipoprotein (LDL) was observed. No binding was apparent to the respective sham-modified controls, nor to LDL modified with the linear MDA adduct only (Fig. S3B). These results confirmed that the anti-MAA 1F83 antibody was suitable for the unbiased immunoprecipitation of MAA-modified proteins. Selective identification of MAA-modified peptides and proteins. To demonstrate the specificity and sensitivity of the two-step enrichment strategy, an in vitro MAA-modified yeast mitochondrial lysate was prepared together with a corresponding sham-modified lysate. An immunoblot analysis confirms the modification and the reactivity towards the MAA-modified but not the sham-modified protein extract (Fig. S3C). The modified yeast mitochondrial extract was spiked into a HEK293 whole cell lysate at a final concentration of 2 % (w/w). The human/yeast protein mixture was then subjected to the two-step



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 22

enrichment procedure (Fig. S4). In the spiked lysate, up to 10,000 different peptides were identified. The proportion of MAA-modified peptides, however, was only 0.4 % (or 40 peptides on average) between the three biological replicates. This result is not surprising as the low-abundance MAA-modified peptides are not observed amidst the overwhelming number of non-modified background peptides. This clearly illustrates the necessity of a dedicated enrichment strategy (Fig. 4A). After the sequential enrichment procedure between 300 and 400 MAA-modified peptides were apparent with an enrichment factor of over 80 %. These numbers are greatly increased compared to the original mixture, thus confirming that the strategy successfully enriches MAA-modified peptides (Fig. 4A). As expected, very few MAA-modified peptides were identified in the samples spiked with sham-modified yeast extract. As the data was filtered to an expected 1 % false discovery rate, these peptides may represent incorrect identifications. Alternatively, the MAA-modified peptides in the sham control may actually embody naturallyoccurring modification events within the yeast or the HEK293 proteomes. When decreasing amounts of the MAA-modified yeast extract, i.e., 1% and 0.5% w/w, were spiked into the background HEK293 proteome; the method still led to the identification of more than 100 MAAmodified peptides (Fig. 4B). This result convincingly demonstrates the high sensitivity of the approach even when minimal amounts of MAA-modified peptides were present in the sample. Quantitation of the precursor area of the MAA-modified peptides that were identified in at least 2 replicates of all three spiked samples (2%, 1% and 0.5% w/w) showed a significantly decreased intensity in the samples that were spiked with lower amounts of modified protein (Fig. 4C). This indicates that the amount measured after enrichment is a reflection of the initial amounts of spiked protein, and thus enabling quantitation between different samples. The data sets from the sequential enrichment were also searched for three additional abundant carbonylation modifications, 4-hydroxynonenal (HNE), 4-oxononenal (4-ONE), and acrolein. None of the other modifications was significantly increased after sequential enrichment, indicating the specificity for MAA-modifications (Fig. 4B). Nonetheless, the specificity can be easily adapted for a different modification by using an antibody with another specificity in the first step of the procedure. Thus the approach is applicable to a range of different carbonylation modifications.


Page 11 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Conclusion Here, we present an integrated biological and chemical LC-ESI-MSMS approach to enrich and identify malondialdehyde-modified proteins and peptides. By fine-tuning the LC-MSMS methodology to a specific subset of peptides with the modification of interest, the number of identified MAA-peptides was greatly increased. Whilst providing valuable information on target proteins and possible interactors, we also showed that antibody enrichment of MAA-modified proteins, is not sufficiently specific to localise the exact site of modification. A sequential enrichment at both the protein and peptide level, however, enables the successful identification and localization of MAA target sites. Most of the published methods to identify carbonylated proteins and assign the carbonylated residues are focused on a spectrum of carbonylation PTMs23,24,26-28. Other approaches that focus on a specific adduct type have used modification-specific antibodies for enrichment32-36. These studies, however, primarily performed the enrichment at the protein level, which often leads to the loss of site information as the modified peptide cannot be identified. Our comprehensive approach represents a significant improvement compared to previously-existing methods to identify MDA-modified proteins. Unequivocal site assignment of the modification on a proteome-wide scale can now be robustly achieved. Our strategy focuses on adducts derived from MDA. In principle, however, the approach is broadly applicable to other carbonylation PTMs. A pre-requisite is the availability of a modification-specific antibody. We anticipate that future studies on MDA and other carbonylation PTMs exploit of the sequential enrichment strategy to both identify target proteins and assign carbonylation sites on a proteome-wide level.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 22

ASSOCIATED CONTENT Supporting Information Description of materials and methods, and additional figures as noted in the main text (PDF). This material is available free of charge via the Internet at http://pubs.acs.org. Proteomics data are deposited to ProteomeXchange via the PRIDE47 repository with the dataset identifier PXD004978.

AUTHOR INFORMATION Corresponding Author * Email: [email protected] Present Addresses † present address: Max-Delbrück-Center for Molecular Medicine, 13092, Berlin, Germany § present address: University of the West Indies, Department of Biological and Chemical Sciences, PO Box 64, Bridgetown, Barbados Notes The authors declare no competing financial interests.

ACKNOWLEDGEMENT The authors would like to thank all members of the Bennett and Binder laboratories for support and advice. Special thanks to Maria Oszvar-Kozma from the Department of Laboratory Medicine, Medical University of Vienna, and Dr. Monika Oberer from the Institute of Molecular Biosciences, KarlFranzens-University in Graz for providing the modified LDL and recombinant bMGL, respectively. Research in our laboratory is supported by the Austrian Academy of Sciences. J.W. and S.R.A. are recipi-


Page 13 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ents of Ph.D. DOC fellowships from the Austrian Academy of Sciences; and C.J.B. is the recipient of a Ph.D. fellowship from the Boehringer Ingelheim Fonds.

References (1) Mann, M.; Jensen, O. N. Nat. Biotechnol. 2003, 21, 255-261. (2) Doll, S.; Burlingame, A. L. ACS Chem. Biol. 2015, 10, 63-71. (3) Lennicke, C.; Rahn, J.; Heimer, N.; Lichtenfels, R.; Wessjohann, L. A.; Seliger, B. Proteomics 2016, 16, 197-213. (4) Larsen, M. R.; Thingholm, T. E.; Jensen, O. N.; Roepstorff, P.; Jørgensen, T. J. D. Mol. Cell. Proteomics 2005, 4, 873-886. (5) Grønborg, M.; Kristiansen, T. Z.; Stensballe, A.; Andersen, J. S.; Ohara, O.; Mann, M.; Jensen, O. N.; Pandey, A. Mol. Cell. Proteomics 2002, 1, 517-527. (6) Guan, K.-L.; Yu, W.; Lin, Y.; Xiong, Y.; Zhao, S. Nat. Protoc. 2010, 5, 1583-1595. (7) Shaw, P. G.; Chaerkady, R.; Zhang, Z.; Davidson, N. E.; Pandey, A. Anal. Chem. 2011, 83, 36233626. (8) Yu, K.; Phu, L.; Varfolomeev, E.; Bustos, D.; Vucic, D.; Kirkpatrick, D. S. J. Mol. Biol. 2015, 427, 2121-2134. (9) Zhang, H.; Li, X.-j.; Martin, D. B.; Aebersold, R. Nat. Biotechnol. 2003, 21, 660-666. (10) Kaji, H.; Saito, H.; Yamauchi, Y.; Shinkawa, T.; Taoka, M.; Hirabayashi, J.; Kasai, K.-i.; Takahashi, N.; Isobe, T. Nat. Biotechnol. 2003, 21, 667-672. (11) Dalle-Donne, I.; Giustarini, D.; Colombo, R.; Rossi, R.; Milzani, A. Trends Mol. Med., 9, 169-176. (12) Esterbauer, H.; Zollern, H. Free Radical Biol. Med. 1989, 7, 197-203. (13) Esterbauer, H. Am. J. Clin. Nutr. 1993, 57, 779S-785S.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 22

(14) Slatter, D. A.; Murray, M.; Bailey, A. J. FEBS Lett., 421, 180-184. (15) Marnett, L. J. Mutat. Res., Fundam. Mol. Mech. Mutagen. 1999, 424, 83-95. (16) Jain, S. K. Biochim. Biophys. Acta, Biomembr. 1988, 937, 205-210. (17) Ishii, T.; Kumazawa, S.; Sakurai, T.; Nakayama, T.; Uchida, K. Chem. Res. Toxicol. 2006, 19, 122129. (18) Tuma, D. J.; Kearley, M. L.; Thiele, G. M.; Worrall, S.; Haver, A.; Klassen, L. W.; Sorrell, M. F. Chem. Res. Toxicol. 2001, 14, 822-832. (19) Zagol-Ikapitte, I.; Sosa, I. R.; Oram, D.; Judd, A.; Amarnath, K.; Amarnath, V.; Stec, D.; Oates, J. A.; Boutaud, O. J. Lipid Res. 2015. (20) Colombo, G.; Clerici, M.; Garavaglia, M. E.; Giustarini, D.; Rossi, R.; Milzani, A.; Dalle-Donne, I. J. Chromatogr. B 2016, 1019, 178-190. (21) Talent, J. M.; Kong, Y.; Gracy, R. W. Anal. Biochem. 1998, 263, 31-38. (22) Ferreira, R.; Domingues, P.; Amado, F.; Vitorino, R. In Proteostasis: Methods and Protocols, Matthiesen, R., Ed.; Springer New York: New York, NY, 2016, pp 349-367. (23) Carpentieri, A.; Gamberi, T.; Modesti, A.; Amoresano, A.; Colombini, B.; Nocella, M.; Bagni, M. A.; Fiaschi, T.; Barolo, L.; Gulisano, M.; Magherini, F. J. Proteome Res. 2016, 15, 3666-3678. (24) Shen, L.; Chen, Y.; Yang, A.; Chen, C.; Liao, L.; Li, S.; Ying, M.; Tian, J.; Liu, Q.; Ni, J. Int. J. Mol. Sci. 2016, 17, 469. (25) Madian, A. G.; Regnier, F. E. J. Proteome Res. 2010, 9, 1330-1343. (26) Madian, A. G.; Myracle, A. D.; Diaz-Maldonado, N.; Rochelle, N. S.; Janle, E. M.; Regnier, F. E. J. Proteome Res. 2011, 10, 3959-3972.


Page 15 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(27) Zhang, H.; He, D.; Yu, J.; Li, M.; Damaris, R. N.; Gupta, R.; Kim, S. T.; Yang, P. Proteomics 2016, 16, 989-1000. (28) Shearn, C. T.; Fritz, K. S.; Shearn, A. H.; Saba, L. M.; Mercer, K. E.; Engi, B.; Galligan, J. J.; Zimniak, P.; Orlicky, D. J.; Ronis, M. J.; Petersen, D. R. Redox Biol. 2016, 7, 68-77. (29) Bollineni, R. C.; Fedorova, M.; Blüher, M.; Hoffmann, R. J. Proteome Res. 2014, 13, 5081-5093. (30) Coffey, C. M.; Gronert, S. Anal. Bioanal. Chem. 2016, 408, 865-874. (31) Afiuni-Zadeh, S.; Rogers, J. C.; Snovida, S. I.; Bomgarden, R. D.; Griffin, T. J. BioTechniques 2016, 60, 186-188, 190, 192-186. (32) Schutt, F.; Bergmann, M.; Holz, F. G.; Kopitz, J. Invest. Ophthalmol. Visual Sci. 2003, 44, 36633668. (33) Dalfó, E.; Portero-Otín, M.; Ayala, V.; Martínez, A.; Pamplona, R.; Ferrer, I. J. Neuropathol. Exp. Neurol. 2005, 64, 816-830. (34) Pamplona, R.; Dalfó, E.; Ayala, V.; Bellmunt, M. J.; Prat, J.; Ferrer, I.; Portero-Otín, M. J. Biol. Chem. 2005, 280, 21522-21530. (35) Yarian, C. S.; Rebrin, I.; Sohal, R. S. Biochem. Biophys. Res. Commun. 2005, 330, 151-156. (36) Choksi, K. B.; Nuss, J. E.; Boylston, W. H.; Rabek, J. P.; Papaconstantinou, J. Free Radical Biol. Med. 2007, 43, 1423-1438. (37) Yamada, S.; Kumazawa, S.; Ishii, T.; Nakayama, T.; Itakura, K.; Shibata, N.; Kobayashi, M.; Sakai, K.; Osawa, T.; Uchida, K. J. Lipid Res. 2001, 42, 1187-1196. (38) Han, B.; Hare, M.; Wickramasekara, S.; Fang, Y.; Maier, C. S. J. Proteomics 2012, 75, 5724-5733. (39) Huber, M. L.; Sacco, R.; Parapatics, K.; Skucha, A.; Khamina, K.; Müller, A. C.; Rudashevskaya, E. L.; Bennett, K. L. J. Proteome Res. 2014, 13, 1147-1155.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 22

(40) Wisniewski, J. R.; Zougman, A.; Nagaraj, N.; Mann, M. Nat. Methods 2009, 6, 359-362. (41) Manza, L. L.; Stamer, S. L.; Ham, A.-J. L.; Codreanu, S. G.; Liebler, D. C. Proteomics 2005, 5, 1742-1745. (42) Rappsilber, J.; Ishihama, Y.; Mann, M. Anal. Chem. 2003, 75, 663-670. (43) Olsen, J. V.; de Godoy, L. M. F.; Li, G.; Macek, B.; Mortensen, P.; Pesch, R.; Makarov, A.; Lange, O.; Horning, S.; Mann, M. Mol. Cell. Proteomics 2005, 4, 2010-2021. (44) Duryee, M. J.; Klassen, L. W.; Schaffert, C. S.; Tuma, D. J.; Hunter, C. D.; Garvin, R. P.; Anderson, D. R.; Thiele, G. M. Free Radical Biol. Med. 2010, 49, 1480-1486. (45) Anderson, D. R.; Duryee, M. J.; Shurmur, S. W.; Um, J. Y.; Bussey, W. D.; Hunter, C. D.; Garvin, R. P.; Sayles, H. R.; Mikuls, T. R.; Klassen, L. W.; Thiele, G. M. PLoS One 2014, 9, e107440. (46) Thiele, G. M.; Duryee, M. J.; Anderson, D. R.; Klassen, L. W.; Mohring, S. M.; Young, K. A.; Benissan-Messan, D.; Sayles, H.; Dusad, A.; Hunter, C. D.; Sokolove, J.; Robinson, W. H.; O'Dell, J. R.; Nicholas, A. P.; Tuma, D. J.; Mikuls, T. R. Arthritis Rheumatol. 2015, 67, 645-655. (47) Vizcaíno, J. A.; Csordas, A.; del-Toro, N.; Dianes, J. A.; Griss, J.; Lavidas, I.; Mayer, G.; PerezRiverol, Y.; Reisinger, F.; Ternent, T.; Xu, Q.-W.; Wang, R.; Hermjakob, H. Nucleic Acids Res. 2016, 44, D447-D456.


Page 17 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure Captions Figure 1. A, structure linear MDA and MAA adduct B, the circular MAA adduct predominates following extended reaction times. C, representative tandem mass spectrum of an MAA-modified model peptide. The MAA modification is stable under standard MSMS fragmentation conditions.

Figure 2. A: MAA-modified peptides have increased retention times with the standard gradient. B, retention time distribution improves with adjusted higher organic solvent gradient. The shaded area represents the equilibration phase.

Figure 3. Improving detection of peptides in low abundant samples. Peptides identified with high input (A) and low input (B) with specified settings. DE: dynamic exclusion, CE: collision energy, IT: maximum injection time

Figure 4: A, Number of peptides identified in the original spiked HEK293 lysate, the antibody-enriched fraction and following sequential enrichment with the antibody and the hydrazine beads. B, number of carbonylated peptides that were identified following enrichment with varying quantities of spiked MAAmodified yeast mitochondria lysate. ONE: 4-oxononenal, HNE: 4-hydroxynonenal C, quantitation of peptides based on precursor area.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1


Page 18 of 22

Page 19 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3


Page 20 of 22

Page 21 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

TOC graphic


Page 22 of 22

A Comprehensive Analytical Strategy To Identify Malondialdehyde

Recommend Documents