A biotin enrichment strategy identifies novel carbonylated amino acids in proteins from human plasma

    A biotin enrichment strategy identifies novel carbonylated amino acids in proteins from human plasma Jesper F. Havelund, Katarzyna Wojdyla, Michael J. Davies, Ole N. Jensen, Ian Max Møller, Adelina Rogowska-Wrzesinska PII: DOI: Reference:

S1874-3919(16)30547-4 doi:10.1016/j.jprot.2016.12.019 JPROT 2748

To appear in:

Journal of Proteomics

Received date: Revised date: Accepted date:

10 May 2016 14 November 2016 30 December 2016

Please cite this article as: Havelund Jesper F., Wojdyla Katarzyna, Davies Michael J., Jensen Ole N., Møller Ian Max, Rogowska-Wrzesinska Adelina, A biotin enrichment strategy identifies novel carbonylated amino acids in proteins from human plasma, Journal of Proteomics (2017), doi:10.1016/j.jprot.2016.12.019

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A bioitin enrichment strategy identifies novel carbonylated amino

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acids in proteins from human plasma

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Authors

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Jesper F. Havelund1,2, Katarzyna Wojdyla1,3, Michael J. Davies4, Ole N. Jensen1, Ian Max Møller2, Adelina Rogowska-Wrzesinska1*

Department of Biochemistry and Molecular Biology and VILLUM Center for Bioanalytical

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Sciences, University of Southern Denmark, Campusvej 55, DK-5230 Odense, Denmark Department of Molecular Biology and Genetics, Science and Technology, Aarhus

Department of Biomedical Sciences, Panum Institute, University of Copenhagen,

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Present address: The Babraham Institute, CB22 3AT Cambridge, United Kingdom

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University, Forsøgsvej 1, DK-4200 Slagelse, Denmark

Blegdamsvej 3, Copenhagen 2200, Denmark

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Corresponding Author

* Adelina Rogowska-Wrzesinska: [email protected]; Campusvej 55, 5230 Odense M, Denmark; phone no. +45 6550 2351

Running title Mapping novel carbonyls in proteins

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ACCEPTED MANUSCRIPT List of Abbreviations ROS - reactive oxygen species

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biotin-lc-hydrazide - biotin-long chain-hydrazide

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MCO – metal ion-catalysed oxidation

MCO-BSA - BSA that had been subjected to metal ion-catalysed oxidation

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HCD - higher-energy collisional dissociation

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NHS - N-hydroxysuccinimide

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nLC-MS/MS – nano liquid chromatography combined with tandem mass spectrometry

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ACCEPTED MANUSCRIPT Abstract Protein carbonylation is an irreversible protein oxidation correlated with oxidative stress,

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various diseases and ageing. Here we describe a peptide-centric approach for identification

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and characterisation of up to 14 different types of carbonylated amino acids in proteins. The

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modified residues are derivatised with biotin-hydrazide, enriched and characterised by tandem mass spectrometry. The strength of the method lies in an improved elution of biotinylated peptides from monomeric avidin resin using hot water (95oC) and increased sensitivity

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achieved by reduction of analyte losses during sample preparation and chromatography. For

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the first time MS/MS data analysis utilising diagnostic biotin fragment ions is used to pinpoint sites of biotin labelling and improve the confidence of carbonyl peptide assignments. We

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identified a total of 125 carbonylated residues in bovine serum albumin after extensive in

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vitro metal ion-catalysed oxidation. Furthermore, we assigned 133 carbonylated sites in 36 proteins in native human plasma protein samples. The optimised workflow enabled detection

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of 10 hitherto undetected types of carbonylated amino acids in proteins: aldehyde and ketone modifications of leucine, valine, alanine, isoleucine, glutamine, lysine and glutamic acid (+14

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Da), an oxidised form of methionine - aspartate semialdehyde (-32 Da) - and decarboxylated glutamic acid and aspartic acid (-30 Da).

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ACCEPTED MANUSCRIPT Biological significance Proteomic tools provide a promising way to decode disease mechanisms at the protein level

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and help to understand how carbonylation affects protein structure and function. The

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challenge for future research is to identify the type and nature of oxidised residues to gain a

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deeper understanding of the mechanism(s) governing carbonylation in cells and organisms and assess their role in disease.

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Highlights

A peptide centric, MS-based approach for identification of carbonylated residues in

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proteins

Hot water (95ºC) treatment efficiently elutes biotinylated peptides from monomeric avidin

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“Novel” carbonylated amino acids are detected in MCO-BSA and human plasma

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Biotin fragment ions in MS/MS increase the reliability of carbonyl peptide identifications

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Keywords

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Protein oxidation; biotin-hydrazide; metal ion-catalysed oxidation; oxidative stress;

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ACCEPTED MANUSCRIPT Introduction All cells continuously produce reactive oxygen species (ROS) as a by-product of aerobic

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metabolism [1, 2]. Some of these ROS are highly reactive and can damage cellular

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components, including proteins by oxidation. Protein carbonylation is a commonly studied

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irreversible protein oxidation [2] and it is an important marker of protein damage, related to oxidative stress, disease and ageing [3-5].

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One of the most common mechanisms of protein carbonylation appears to be metal ioncatalysed oxidation (MCO). MCO of the side chains of the amino acids proline, arginine,

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lysine, and threonine induces formation of glutamic semialdehyde, aminoadipic semialdehyde and 2-amino-3-ketobutyric acid, and these are considered to be the main direct protein-

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derived carbonyl products in biological samples [6]. Carbonylation of arginine, lysine,

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cysteine, and histidine can also occur via reaction with carbohydrates and lipids containing

pathway [8].

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reactive carbonyl groups [7]. Carbonyl derivatives can also arise through an α-amidation

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Identification of specific carbonylated and functionally impaired protein(s), and accurate characterisation of the modification type(s) and affected amino acid residues is required in order to assess the role of protein carbonylation in health and disease. Mass spectrometry is a superior tool for such studies because of its sensitivity and specificity, which allows characterisation and sequencing of peptides and their carbonylated derivatives. However, protein/peptide carbonyls are typically formed at very low stoichiometry [9, 10] and the accompanying MS signal intensity is frequently reduced due to low ionisation efficiency of modified peptides and ion signal suppression from unmodified peptides. Despite significant advances, mass spectrometry-based methods for analysis of carbonylated proteins are still mostly applied at the protein level, which hampers the identification of endogenously carbonylated peptides and significantly reduces sensitivity of the methods due to signal 5

ACCEPTED MANUSCRIPT suppression by the presence of the more abundant non-carbonylated peptides [11-14]. Peptide-centric approaches for large-scale analysis of protein carbonylation do exist [15, 16].

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However, in these studies carbonyl-specific labelling is performed at the peptide level, after

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overnight proteolysis at elevated temperatures, typically 37°C. This increases the risk of

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artefactual labelling, and false positive results. Even for true positives it can be difficult to distinguish between primary carbonylation and secondary products generated via damage propagation. These methodologies are laborious and time consuming, requiring both expert

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technical knowledge and comprehensive bioinformatics analysis which makes their

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implementation challenging.

We here present an optimised biotin-based protocol for identification of carbonylated residues

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in proteins, where carbonyl labelling is performed at the protein level, and subsequent

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enrichment of carbonylated peptides is achieved at the peptide level. The method described provides improved elution efficiency of biotinylated peptides from monomeric avidin resin,

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minimized sample losses during sample preparation and chromatographic steps and facilitation of semi-automatic in silico evaluation of identification of modified peptides by the

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use of diagnostic biotin fragment ions. This optimised method is applied to both single proteins and complex biological mixtures. The enhanced sensitivity of this protocol has allowed the identification of multiple carbonylated residues in untreated (non-oxidised) human plasma and additional carbonylated species, that have previously been observed only with individual amino acids and short peptides.

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ACCEPTED MANUSCRIPT Experimental Procedures Materials

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Spin filters, 10 kDa cut-off (Merck Millipore: UFC5003xx), bovine serum albumin (Sigma-

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Aldrich: A9647), monomeric Avidin UltraLink Resin (Pierce – Thermo Scientific: 53146),

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monomeric avidin magnetic beads (Bioclone Inc.: MMI-102), N-hydroxysuccinimide-biotin (NHS-biotin) (Pierce – Thermo Scientific: 21339), biotin-long chain (lc)-hydrazide (Pierce –

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Thermo Scientific: 21340), biotin-hydrazide (Pierce – Thermo Scientific: 21339), glass wool (Sigma-Aldrich: 2-0411), Ac-YVKD-CHO and Ac-WEHD-CHO peptides with aldehyde

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groups on aspartic acid (Bachem: N-1355 and N-1630), C8 sorbent (3M EmporeTM: 2214), Poros R2 and R3 column material (Applied Biosystems: 1-1128 and 1-1339), ReproSil-Pur

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C18-AQ-3 μm column material (Dr. Maisch: r13.aq). All other reagents were purchased from

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Sigma-Aldrich in reagent grade quality. Pooled human plasma samples from 100 healthy

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donors (unknown age and gender) was provided by Odense University Hospital. Samples were collected in 10 ml BD Vacutainer™ Plastic Blood Collection Tubes with dipotassium EDTA and left for maximum 1 h at 21˚C and maximum 24 h at 4˚C. After incubation the

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samples were centrifuged at 3000 g for 10 min at 4˚C. The supernatant was collected and stored at -20˚C. Subsequently the samples were thawed at 4˚C, pooled, aliquoted and stored at -80˚C. Samples with visible lipemia were excluded. Optimisation and testing of elution conditions using model peptides β-Lactoglobulin digested with trypsin and labelled with NHS-biotin and two model aldehyde peptides Ac-YVKD-CHO and Ac-WEHD-CHO labelled with biotin-hydrazide and biotinlong chain (lc)-hydrazide were used. Enrichment of biotinylated model peptides was performed using monomeric avidin immobilised on magnetic beads. Evaluation of enrichment efficiency was performed using Ac-YVKD-CHO tagged with biotin-lc-hydrazide and dimethyl labelling [17]. All experiments were monitored using MALDI MS performed on a 7

ACCEPTED MANUSCRIPT Bruker Ultraflex Extreme MALDI TOF/TOF (Bruker Daltonics). Before MALDI MS analysis all samples were desalted using in-house made micro-columns packed with POROS

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20R2 RP (Applied Biosystems) as described previously [18]. Additional experimental details

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are provided in Supplementary Methods.

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Metal ion-catalysed oxidation of BSA

Metal ion-catalysed oxidation (MCO) was performed as described previously [19], with the

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exception of the BSA concentration, which was kept at 3.5 mg/ml to prevent overloading of the spin filters. Briefly, oxidation was carried out in 50 mM HEPES-KOH, pH 7.4, containing

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100 mM KCl and 10 mM MgCl2 using ascorbic acid/FeCl3 at a final concentration of 25 mM/100 µM and incubation overnight (15 h) at 37ºC. Formation of carbonyls was monitored

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using an OxyBlot kit (Merck Millipore) [20]. Four samples were prepared and analysed

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independently.

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Biotinylation of carbonylated residues in MCO-BSA Following BSA oxidation the buffer was changed to 3% ACN, 0.1% TFA, pH 1.8 using 10 kDa spin filters following the manufacturer’s instructions. Briefly, the sample was centrifuged

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to remove the MCO buffer and 10 times volume of a new buffer was added to the sample on the spin filter and centrifuged again. This procedure was repeated at least three times with the MCO-BSA recovered by centrifugation. SDS and biotin-lc-hydrazide (dissolved in DMSO) were added to the MCO-BSA to a final concentration of 1% and 5 mM, respectively, in a reaction volume of 250 µl. Labelling was carried out for 2 h at 37ºC using gentle shaking. Finally, the hydrazone bonds were reduced using 60 mM sodium cyanoborohydride in 250 µl ice-cold PBS (pH 7.4) and incubated at 4ºC for 30 min. Trypsin digestion The biotin-lc-hydrazide labelling buffer was changed to a denaturing buffer (8 M urea, 50 mM ammonium bicarbonate, pH 8.0) using spin filters, with excess free biotin-lc-hydrazide 8

ACCEPTED MANUSCRIPT removed using at least 4 buffer washes. Dithiothreitol and iodoacetamide (10 mM, 40 min and 20 mM, 40 min in the dark) were then added to reduce and alkylate cysteines, with the BSA

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retained on the spin filter membrane. The samples on the filter were then centrifuged to

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remove excess reagents, and the residual buffer diluted with 50 mM ammonium bicarbonate

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buffer to give a final concentration of 1 M urea. The protein recovered from the spin filter was digested with trypsin (1:50 w/w enzyme to protein, 15 h, 23ºC, gentle shaking). The samples

POROS 20R2/R3 [18, 21].

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Enrichment of biotinylated MCO-BSA

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were then desalted using in-house made micro-columns packed with reverse phase material

Biotinylated MCO-BSA samples were resuspended in PBS, pH 7.4 and enriched using

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Monomeric Avidin UltraLink resin using home-made micro-columns. Briefly, a P200 tip was

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blocked with glass wool at the tip. Monomeric avidin resin (~60 µl slurry) was added, and the column packed to a depth of 1.5 cm by applying gentle pressure. The column was washed

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with 150 µl of ddH2O and equilibrated with 2 x 150 µl PBS (pH 7.4). Biotinylated peptides in 150 µl of PBS was loaded on to the column over 5 min by applying gentle pressure, and

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washed with PBS (1 ml, to remove unbound peptides), and ddH2O (150 µl, to remove salts). Biotinylated peptides were then rapidly eluted (10 s) using two portions of ddH2O (100 µl) at 95ºC. The two fractions were then pooled, vacuum dried and analysed by nLC-MS/MS. nLC-MS/MS The biotinylated peptides were resuspended (5 µl 0.1% formic acid) and loaded on an EASY nLC system (Thermo Fisher Scientific) equipped with a pre-column (100 μm i.d. x 2 cm, 5 µm particles) and analytical column (75 μm i.d. x 15 cm, 3 µm particles) packed in-house with C18 ReproSil reverse-phase material in fused silica. Peptides were eluted, at a flow rate of 250 nl/min, using a 60 min gradient (eluent A: 0.1% formic acid; eluent B: 0.1% formic acid, 90% acetonitrile) consisting of 14-38% of eluent B, before washing with 100% of eluent 9

ACCEPTED MANUSCRIPT B for 12 min. The flow from the analytical column was coupled to a LTQ Orbitrap XL mass spectrometer operated in positive ion mode with data-dependent acquisition. A full ion scan

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(m/z 350–1500) was acquired at resolution of 30,000 before the five most intense precursor

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ions (with z > +1, intensity > 15,000 counts) were selected for collision-induced dissociation

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(CID) fragmentation using a normalised collision energy at 35. These ions were then dynamically excluded for 30 s.

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Data processing and analysis

The raw files were converted into mgf files using MassMatrix Mass Spectrometric Data File

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Conversion Tools, version 3.9 [22], and processed using an in-house developed Python (Python Software Foundation, version 2.7.3) script (provided in supplementary materials) to

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remove MS/MS spectra that did not contain the expected biotin-lc diagnostic fragment ions of

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m/z 227 and 340. Inclusion criteria for analysis were as follows: a) for MS/MS spectra with precursor ions smaller than m/z 715, the presence of both reporter ions was required with a

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precision of 0.16 and 0.22 centred on m/z 227.1350 and m/z 340.1801, respectively; only reporter ions of intensity >30 counts were considered; b) for MS/MS spectra with precursor

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ions m/z >715, but <1210, only the m/z 340 ion was required; only reporter ions of intensity >30 counts were considered; c) all MS/MS spectra with precursor ions of m/z >1210 were included. The diagnostic ions were then removed from the spectra. In addition, all signals below an intensity of 6 counts were removed from the spectra. The specific criteria set for the different precursor ions are defined by the ion trap mass analyser used for fragmentation and detection. This places an upper limit on the ratio between the precursor's mass-to-charge ratio (m/z) and the lowest trapped fragment ion (the "one third rule" [23]). Therefore for peptides with m/z ratio higher than 715 only the m/z 340 fragment ion will be observed, and for peptides with an m/z ratio higher than 1210, no biotin fragment ions will be observed.

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ACCEPTED MANUSCRIPT The extracted mgf files were analysed using Proteome Discoverer v1.4 (Thermo Scientific) and searched against an in-house database containing 36 standard-protein sequences

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(downloaded from UniProt database May 2014), including bovine serum albumin with

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accession number P02769, using an in-house MASCOT server (v2.3.02, Matrix Science Ltd.).

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Database searches were performed with the following parameters: tryptic or semi-tryptic digestion with up to three missed trypsin cleavages, precursor mass tolerance of 6 ppm, fragment mass tolerance of 0.8 Da, carbamidomethylation of cysteines (+57.02146 Da) as a

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fixed modification, and oxidation (+15.99492 Da) of methionine as a variable modification.

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The biotin-tagged samples were searched including the following variable modifications labelled with biotin-lc-hydrazide tag: aminoadipic semialdehyde (K) (+354.17256 Da),

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glutamic semialdehyde (R) (+312.15076 Da), glutamic semialdehyde (P) (+371.19911 Da), 2-

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amino-3-ketobutyric acid (T) (+353.18855 Da), decarboxylation of E and D (E-30, D-30) (+325.19363 Da), aspartate semialdehyde (M-32)(+323.19574 Da) and +14 Da carbonylation

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of LVKIEAQ (+369.18346 Da). Data for all the modifications are provided in Table 1. Mascot Percolator was used to estimate Peptide-Spectrum Match False Discovery Rate and

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only peptides with a percolator q-score ≤ 0.01 were included in the analysis. All rank 1 peptides (Mascot score >10), present in at least two replicates, were accepted and manually validated. Annotated spectra are provided in Supplementary File 1. Detection of carbonylated residues in human plasma Analysis of human plasma carbonylated peptides (five experimental replicates) was performed as described above, with minor modifications. Briefly, 50 µl of randomised plasma pooled from 100 healthy persons (~3.5 mg of protein) was mixed with 450 µl of 3% ACN, 0.1% TFA, pH 1.8 buffer, transferred to 10K spin filter and centrifuged at 14,000 g for 10 min to leave ~50 µl on the filter. This was repeated twice. Finally 50 µl of 3% ACN, 0.1% TFA, 1% SDS, pH 1.8 buffer was added on top of the filter membrane. Labelling of carbonylated 11

ACCEPTED MANUSCRIPT residues used biotin-lc-hydrazide dissolved in DMSO (50 mM stock) at a final concentration of 5 mM, for 2 h at 37ºC with gentle shaking. The hydrazones were then reduced using 60

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mM sodium cyanoborohydride in 250 µl ice-cold PBS (pH 7.4) at 4ºC for 30 min. Digestion,

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reduction, alkylation, enrichment of biotinylated peptides and nLC-MS/MS analysis were

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carried out as described above, except that the flow from the analytical column was coupled to a qExactive HF spectrometer (Thermo Fisher) operated in positive ion mode with datadependent acquisition. A full ion scan (m/z 400–1600) was acquired at resolution of 120,000

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and automatic gain control of 3 x 106 before the 12 most intense precursor ions (z > +1,

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intensity > 2e4 counts) were selected for higher-energy collisional dissociation (HCD) fragmentation (normalised collision energy 30, AGC 1e5). MS/MS spectra were recorded with a dynamic scan range of m/z 200-2000, with the target ions selected for fragmentation

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were dynamically excluded for 20 s. Data processing was carried out as described above with the Pyton script modified to the high resolution instrument and HCD fragmentation mode.

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Inclusion criteria were the presence of a diagnostic ion (m/z 227.0841 or m/z 340.1701) within a range of m/z 0.01. Database searches were performed as above with the exception of:

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SwissProt database, taxonomy: Homo sapiens (April 2015, number of sequences after taxonomy: 20340), including common contaminants from the GPM database [24], precursor mass tolerance 6 ppm, fragment mass tolerance 0.01 Da, target FDR 0.01. All rank 1 peptides present in at least two replicates were accepted and manually validated. Annotated spectra are provided in Supplementary File 2. Data availability The mass spectrometry proteomics data have been deposited to the Proteome Xchange Consortium via the PRIDE partner repository with the dataset identifier PXD002966.

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ACCEPTED MANUSCRIPT Results Protein oxidative generates multiple low intensity modifications on amino acid side chains [2,

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9, 10]. Detection of these modified amino acid residues with high sensitivity using mass

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spectrometry requires isolation or enrichment of the modified peptides prior to analysis.

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To achieve this an already published protocol in which biotin hydrazide and monomeric avidin were used for labelling of carbonylated amino acids has been adapted and developed

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[12, 25], with increased sensitivity being achieved by: 1) increasing the efficiency of elution of the biotinylated peptides from the monomeric avidin resin; 2) minimizing sample losses

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during preparation; 3) use of diagnostic biotin fragment ions in MS/MS to increase the confidence of correct assignment of modified peptides and amino acid residues.

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Peptide centric protocol for analysis of carbonylated residues in proteins In order to achieve optimal enrichment of the biotinylated peptides using monomeric avidin

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resin, three literature protocols [26, 27] were tested using NHS-biotin labelled peptides from trypsin-digested β-lactoglobulin and MALDI analysis (Figure S1). The highest recovery was

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achieved using 95oC water (Figure S1a-c). Using this approach, a model aldehyde-containing peptide (Ac-WEHD-CHO) labelled with biotin-hydrazide and biotin-long chain (lc)-hydrazide could be efficiently enriched from a peptide mixture to a 100 times molar excess (Figure S1de). The recovery was high and reproducible (76 + 2%, n = 2) (Supplementary methods, Figure S2). Those pilot experiments allowed us to develop strategy that was later on tested using LCMS-MS approach (see next section). Using 95oC water for elution, a workflow (Figure 1a and S3) was developed for the identification of carbonylated sites in BSA generated by metal ion-catalysed oxidation (MCOBSA) following a published oxidation protocol [28]. Immediately after oxidation, carbonylated residues were labelled with biotin-lc-hydrazide, and the resulting derivatized protein was digested into peptides using trypsin endopeptidase. The carbonylated peptides in 13

ACCEPTED MANUSCRIPT the peptide mixture were then separated from unmodified species by monomeric avidin affinity chromatography using home-made micro (60 µl) columns packed with of Monomeric

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Avidin UltraLink Resin Biotinylated peptides were eluted using 95oC water, dried and

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analysed by nLC-MS/MS (for further details see Materials and Methods).

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The hot-water method outperforms the classical biotin elution protocols Application of this hot-water protocol to MCO-BSA was tested against two well established

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alternative methods (0.1 M glycine, pH 2.8 buffer; and 2 mM D-biotin in PBS buffer, pH 7.4; [26]), and a recently published elution buffer (0.4% formic acid in 30% aqueous acetonitrile;

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[15]). This last protocol showed a similar performance to the 2 mM D-biotin in PBS buffer, pH 7.4 system, and was omitted from further experiments. The hot water elution allowed the

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identification of ~60% more arginine/lysine/proline/threonine carbonylation sites (57 vs 35)

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compared to the other two methods combined (Figure 1b). The overlap between the three methods was very high, with only 7 out of a total of 57 sites not observed using the new

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method. Addition of SDS, which enhances protein unfolding and denaturation increased the number of recovered modified peptides by 16% (Figure 1c), suggesting that some carbonyl

unfolding.

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sites are buried within the protein structure and are only tagged efficiently on protein

Biotin fragment ions increase the reliability of carbonyl peptide identification Collision-induced dissociation (CID) MS/MS of biotin-lc-hydrazide produces two major fragment ions at m/z 227.1350 and m/z 340.1801 (Figure 2a and b) which were also observed in MS/MS spectra of biotinylated peptides (Figure 2c). These fragment ions complicate the elucidation of the sequence of modified peptides and have been reported to decrease the efficiency of the search engines [26, 29]. To circumvent this a Python script was developed, which extracts MS/MS spectra containing at least one of these ions and removes them from the spectra. Application of this script to the MCO BSA samples and randomised human 14

ACCEPTED MANUSCRIPT plasma samples (see below) indicates that the number of MS/MS spectra that do not contain these diagnostic ions varies between sample types (72% for MCO BSA, 13% for plasma).

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Comparison of the data obtained using processed (biotin fragment ions removed) and

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unprocessed data did not show a significant increase in the number of PTMs assigned to

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arginine/lysine/proline/threonine modified peptides. Using our inclusion criteria for analysis (see Materials and Methods) we found only 10 and 27 modified peptides assigned to MCO

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BSA and randomised plasma samples. At the same time 5 and 11 modified peptides observed in unprocessed data were not identified in processed spectra, but these identifications could

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not be confirmed manually and were omitted from further analysis. The removal of the biotin fragment ions does not therefore appear to influence significantly the number of modified

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peptides identified.

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The removal of the biotin fragment ions from the MS/MS spectra increased the peptide ion

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scores obtained by database searching for 80% and 95% of peptides in MCO BSA and human plasma samples respectively (Figure S4). For the peptides identified from human plasma, the Mascot scores increased by 12% on average. For the MCO-BSA samples the improvement

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was smaller (4%) and mainly observed for spectra with scores below 20. Similar experiments using biotin-hydrazide produced only a single major fragment ion at m/z 227.1350, due to the absence of the spacer, making this less diagnostic In all subsequent experiments biotin-lc-hydrazide was therefore used. Characterisation of carbonylated peptides in MCO-BSA The optimised protocol was applied to MCO-BSA to map carbonylated residues (see Table 1). In addition to the Python script inclusion criteria, only peptides detected in at least 2 of 4 independent experiments were considered as positive identifications (Table S1A). All MS/MS spectra were manually evaluated to confirm the location of the modified residue. Examples

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ACCEPTED MANUSCRIPT are presented in Figure 3. Using these strict criteria 65 unique carbonylated residues were detected, with the most frequently modified being lysine (Figure 4a).

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Enrichment of carbonylated peptides from human plasma

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The developed protocol was applied to pooled native human plasma, from 100 healthy

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individuals [24], to examine whether this method could identify endogenous carbonylation in untreated biological materials. 50 µl of the pooled plasma analysed as described in the

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Materials and Methods. Five experimental replicas were examined and only modified peptides observed in at least 2 independent experiments were considered (Supplementary

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Table S1B). 167 carbonylated peptides derived from 36 different proteins were identified (Table 2). All tandem mass spectra from modified residues were manually validated

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(Supplementary File 2). The most frequently observed carbonylations were on lysine (78) and

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threonine (25) (Figure 4b). Serum albumin and apolipoprotein A-I both exhibited a high degree of modification (33 and 17 modified sites, respectively) (Figure S5a-b). Comparison of

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the carbonylation sites observed for MCO-BSA and HSA showed that 51% of sites (17 of 31) identified in HSA were also observed in MCO-BSA (Figure S6).

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Most of the carbonylated proteins identified in the pooled human plasma are present at concentrations ranging from 0.01-1.0 mg/ml, with HSA being the most abundant (35-50 mg/ml). These proteins include those involved in the immune response (11), inhibition of plasma proteases (6), lipid transport (5) and iron transport / recycling (3) (Supplementary Table S1C). “Novel” carbonylated amino acids detected in MCO-BSA and human plasma The biotin-lc-hydrazide labelling strategy outlined above was subsequently utilised to search for modifications to other amino acid residues previously reported to be converted into hydrazide-reactive aldehydes or ketones/lactams [9], but which have not been previously identified on large peptides or proteins. Analysis of the MCO-BSA data allowed aldehyde and 16

ACCEPTED MANUSCRIPT ketone modifications on leucine, valine, alanine, isoleucine, glutamine, lysine and glutamic acid (+14 Da), an oxidised form of methionine - aspartate semialdehyde (-32 Da) - and

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decarboxylated glutamic acid and aspartic acid (-30 Da) to be detected (Figure 4c and d). The

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localisation of these modified residues in the BSA primary structure is presented in Figure

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S5c. The number of additional modifications detected on the proteins from human plasma was lower (15 sites on 5 different proteins), with decarboxylation of glutamic acid (6 sites) the most frequently observed additional modification (Figure 4e), as seen for MCO-BSA.

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glutamic acid are presented in Figure 5.

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Representative tandem mass spectra identifying carbonylation of leucine, methionine and

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ACCEPTED MANUSCRIPT Discussion Protein carbonylation is an important marker of protein damage, related to oxidative stress,

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disease and ageing [3-5]. Therefore there is a need for sensitive tools to detect and map

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protein carbonylation sites. Despite significant advances, development of such methods has

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been challenging, with the success rate of identification of carbonylated sites being relatively low. Thus only 25, 50 and 0% of the enriched and identified proteins contained carbonylated

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residues in the studies reported in [13], [30] and [31] respectively.

Enrichment at peptide level is crucial for efficient site identification

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Most studies designed to locate sites of protein carbonylation have applied some form of enrichment in the experimental workflow [11, 30, 32], as the ions from low-abundance

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carbonylated peptides in non-enriched samples are often suppressed by unmodified peptides

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and thus overlooked (Figure S1d-e). This phenomenon is also observed protein

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phosphorylation studies [33].

Most of the enrichment methods reported to date, have been applied at the protein level,

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resulting in the majority of the analysed peptides being not carbonylated. This is likely to contribute to a low detection rate. Enrichment at the peptide level should eliminate unmodified peptides, and increase the detection rate of modified species and site identification [33]. The unique mass of the carbonylated group increases the likelihood of unambiguous identification, a factor of crucial significance with regard to answering questions with regard to cause / effect of protein carbonylation in cell function and disease. Effective and efficient elution of the modified peptides from the enrichment matrix is critical to peptide enrichment methods. We hypothesised that the strong binding between avidin and biotin was a potential reason for the low recovery of biotinylated peptides and the poor results obtained in subsequent mass spectrometry analysis. It has been shown that water at 95oC is highly effective at eluting biotinylated peptides from monomeric avidin resin (Figure S1), as 18

ACCEPTED MANUSCRIPT expected on the basis of previous data using this approach to elute biotinylated DNA and regenerate streptavidin beads [27]. The simple approach gives a higher recovery (+60%) of

T

biotinylated peptides compared to previous method for MCO-BSA (Figure 1). The use of

IP

water also eliminates further clean-up and desalting steps before MS analysis (Figure 1,

low sample volumes (e.g. 50 µl) to be employed.

SC R

Supplementary Figure S3). The use of centrifugal spin filters and micro-columns also allows

NU

These advances and the simplicity of this procedure has been shown to result in a high identification rate of carbonylation sites, when compared to protein fractionation protocols

MA

and targeted mass spectrometry [15, 29, 30, 34, 35]. The number of direct arginine/lysine/threonine/proline carbonylation sites identified in the current study from

D

native (untreated) human plasma is higher than those reported previously (Supplementary

TE

Table S1D). However higher levels have been reported previously when species arising from lipid and carbohydrate oxidation products are included [15]. The latter species, which are

CE P

often termed advanced lipid peroxidation endproducts (ALEs) and advanced glycation endproducts (AGEs) arise predominantly from secondary reactions of lipid- and sugar-derived

AC

carbonyls with protein nucleophiles (mainly cysteine, lysine, histidine, arginine and Nterminal amino groups), which have not been examined in the current study. The highest cumulative number of carbonylation sites identified on plasma proteins has come from a study on lean and obese patients with and without Type 2 diabetes mellitus [15]. However the plasma proteins from the control group in this previous study yielded only 94 carbonylation sites and the direct protein modifications that are the focus of the current study, constituted only 39% (37 sites) of total. There is therefore considerable scope to apply the method reported here to these additional (secondary) protein-bound carbonyls in such samples, and this is likely to significantly boost the total number of species detected, albeit at the expense of much greater analytical effort.

19

ACCEPTED MANUSCRIPT Potential advantages of carbonyl labelling at the protein level Peptide-centric approaches for the analysis of protein carbonylation have been reported

T

previously [15, 16], but performing carbonyl-labelling after proteolysis (often carried out

IP

overnight), increases the risk of artifactual oxidation and labelling. Labelling at the protein

SC R

level allows sample derivatisation very rapidly after the material has been obtained. Any subsequent (artifactual) modifications will not be identified in this new approach, potentially

NU

limiting false positives.

Biotin fragment ions provide a means for validation of biotin labelling

MA

The presence of biotin fragment ions is known to decrease MS/MS identification ion scores [26, 29], and this has been confirmed here (Supplementary Figure S4). This effect appears to

D

be relatively small and occurs mainly with peptides with a low ion score (poor MS/MS

TE

spectra). However in silico extraction of spectra containing these (diagnostic) fragment ions, and their subsequent removal, can be turned into an advantage. The first step acts as semi-

CE P

automated validation of the presence of a carbonyl function, on top of the enrichment procedure, as this only occurs with species tagged by biotin. The second step simplifies the

AC

spectra, making database searching (e.g. with Mascot) more efficient. This increases the individual peptide ion score and the robustness of the identification, as it is unlikely that a peptide containing a biotin fragment ion, and a characteristic mass increase corresponding to a carbonylation modification labelled with biotin-lc-hydrazine, would be a false positive. Mapping of carbonylation sites in MCO-BSA In this study, 65 unique carbonylated residues have been identified on arginine/lysine /threonine and proline residues for MCO-BSA (Figures 4, 5), with lysine the most frequently modified and threonine the least, consistent with previous literature [36, 37]. The majority of these sites are present in protein regions rich in these residues, and previously reported to be sensitive to oxidation (Supplementary Figure S5c; cf. 26 sites reported previously for the 20

ACCEPTED MANUSCRIPT same oxidation system [28]). Five MCO-induced carbonylation sites have been previously identified in human serum albumin [38].

T

Carbonylated proteins and human plasma

IP

Protein carbonylation has been reported to be associated with a number of pathologies and

SC R

ageing [39]. The mapping of carbonylation sites on proteins obtained from biological samples, e.g. body fluids, is therefore of interest, and potential clinical importance. The

NU

approach reported here appears to allow analysis of (at least some) complex biological matrices such as human plasma (Figure 4, Table 2). As the material examined was pooled

MA

material from 100 healthy donors, it is likely that the 114 carbonylated sites identified on arginine/lysine/threonine/proline residues on the 34 proteins is representative of the in vivo

D

physiological plasma carbonylation pool. These have been mapped to specific proteins

TE

including HSA and apolipoprotein A-I. This data may indicate that these proteins are particularly susceptible to oxidation because they contain a large number of readily oxidised

CE P

residues, are particularly targeted by oxidants (e.g. as a result of high affinity binding of metal ions or other oxidant systems), or merely reflect their high abundance in plasma. The last of

proteins.

AC

these factors is likely to be, at least partly, responsible as they are both high abundance

Identification of “novel” carbonylated amino acids Aminoadipic semialdehyde (and its further oxidation product, 2-aminoadipic acid; from lysine), glutamic semialdehyde (from arginine, proline) and 2-amino-3-ketobutyric acid (threonine) have been previously considered to be the major carbonyl oxidation products generated on proteins as a result of metal-ion catalysed oxidation (Table 1) [39]. However, studies on free amino acids and small peptides have shown that most amino acids can be oxidised to carbonyl-containing species, though not all of these have been detected on proteins (reviewed in [2] and [9]). 21

ACCEPTED MANUSCRIPT In this study the biotin labelling strategy has been used to identify 10 additional carbonylation products on MCO-BSA and human plasma proteins (Table 1, Figures 5 and 6). These

T

additional species include conversion of methionine to aspartate semialdehyde and subsequent

IP

derivatisation (Supplementary Figure S7). For the MCO-treated BSA, 65 different sites have

SC R

been identified with such additional carbonyl modifications (Figure 4). Five of these sites have been shown to be modified in multiple ways, e.g. Glu-30 and Glu-125 give both a +14 a.m.u. product and a decarboxylated species; and Lys-36, Lys-256 and Lys-412 give both

NU

aminoadipic semialdehyde and a +14 a.m.u. carbonyl (Supplementary Table S1A). These data

MA

reinforce the conclusion that these residues are important sites of oxidation, and also indicate the problems in quantifying damage using data for single oxidation products (e.g. relying only on +14 a.m.u. products). Additional carbonyl products formed from glutamic acid, leucine

TE

D

and methionine were also detected on human plasma proteins (Figure 4) consistent with previous reports on the heterogeneity of protein oxidation products generated in vivo [40].

CE P

The low frequency of these modifications on the human plasma proteins compared to MCOBSA, may indicate that these are of low abundance, but it should be noted that such in vivo

AC

data are pool sizes, and reflect differences between the rates of formation and removal, which potentially confounds analysis of their importance. Thus, a modification may be formed rapidly and / or to high levels, but also removed rapidly, resulting in a low steady state level. It remains an open question whether and which carbonylation products have biological significance. For MCO-BSA, we have identified more carbonylations on glutamic acid and leucine residues than on arginine, proline and lysine (Figure 4), despite the latter being reported to be the major carbonyl products generated by protein MCO [37]. This may indicate that these additional carbonyl products are of biological importance. Proposed mechanisms of formation of carbonyl amino acids Multiple oxidants can give rise to protein carbonyls (reviewed in [39, 40]). For the MCO system studied here, the initiating species is likely to be the “free” hydroxyl radicals (HO.), 22

ACCEPTED MANUSCRIPT HO. generated within a solvent cage, or a metal-ion oxo complex. Irrespective of the identity of this species, the initial reaction with the target amino acids that give rise to the carbonyl

T

compounds detected here (see Table 1) is likely to be hydrogen abstraction to give a carbon-

IP

centred radical (R.). Carbon-centred radicals undergo rapid (diffusion-controlled, k ~ 109 M-1

SC R

s-1; reviewed [40]) reaction with molecular O2 to yield peroxyl radicals (ROO.), with this being the major fate of most R. when the radical flux is low, as is the case in most biological systems (see Figure 6; reviewed in [40]). The subsequent reactions of these ROO. are

NU

dependent on the local structure.

MA

When ROO. are generated at primary or secondary carbon sites, these species can undergo termination reactions with another ROO. (or HOO.) via the Russell mechanism [41] with this

D

yielding one mole of carbonyl and one mole of alcohol (see Figure 6). The resulting alcohols

TE

have been detected in model systems [42-44], and also in normal and pathological tissue samples [45-47]. These reactions are the likely source of the carbonyls detected on leucine

CE P

(+14), isoleucine (+14), valine (+14), alanine (+14), glutamic acid (+14), lysine (+14) and glutamine (+14) (Figure 6; reviewed in [40]).

AC

Neighbouring heteroatoms with labile X-H bonds (e.g. O-H or N-H) can result in alternative reactions of ROO., with this resulting in the elimination of HOO. and direct formation of a carbonyl group (e.g. with threonine, to give 2-amino-3-ketobutyric acid), or an imine (-C=N-) when the neighbouring group is –NH2 (Figure 6) (e.g. [48, 49]). Imines are unstable in aqueous solution and undergo ready hydrolysis to give a carbonyl species (in the case of lysine this yields aminoadipic semialdehyde and ammonium ions; with arginine, glutamic semialdehyde and a guanidine group; and for proline the ring-opened product glutamic semialdehyde) (Figure 6). Related chemistry may underlie the loss of the thiomethyl group from methionine (to give the -32 a.m.u. species) via formation, and subsequent reaction, of a

23

ACCEPTED MANUSCRIPT carbon-centred radical formed on a neighbouring C-H to the sulphur centre (i.e. at the carbon).

T

As a result of the high concentration of C-H (or S-H) bonds in proteins (up to molar

IP

concentrations), protein ROO. also undergo rapid hydrogen atom abstraction reactions to give

SC R

hydroperoxides (ROOH) (Figure 6). These are major intermediates in radical-mediated protein oxidation (reviewed in: [40, 50]), and can undergo metal-ion catalysed (pseudo-

NU

Fenton) reactions to give alkoxyl radicals. The latter can then undergo hydrogen atom abstraction reactions (to give alcohols, which have been detected in a range of biological

MA

samples [42-47]), or fragmentation, some of which give rise to additional carbonyl groups. These alkoxyl fragmentation reactions provide a mechanism for the observed loss of the

D

carboxyl groups from aspartic and glutamic acids (Figure 6; D-30 and E-30 species). This

TE

type of fragmentation has been reported for irradiated amino acids and small peptides, as well

CE P

as some proteins, with CO2-. released on hydroperoxide decomposition to alkoxyl radicals by metal ions [51, 52].

The additional carbonyl compounds reported here on proteins are

therefore consistent with the chemistry of peroxyl and alkoxyl radicals generated on proteins.

AC

The consequences of the formation of these products have yet to be elucidated, but it is interesting to note that the formation of some of these carbonyls results in changes to protein charge (e.g. decreased positive charge from loss of the amine and guanidine groups from lysine and arginine, and decreased negative charge from aspartic and glutamic acid decarboxylation), the formation of carbonyls that can give rise to Schiff base cross-links with other biological materials, and also changes in residues that define protein structure (e.g. ring opening of P residues). These events may therefore have consequences for protein function (e.g. interaction with binding partners or receptors) or protein conformation and activity, which remain to be elucidated.

24

ACCEPTED MANUSCRIPT Funding Sources The authors were financially supported by Danish Council for Independent Research - Natural

T

Sciences (FNU) (to IMM), Graduate School of Science and Technology, Aarhus University

IP

(to JFH), Sino-Danish Center For Education and Research (to KW), Department of

SC R

Biochemistry and Molecular Biology, University of Southern Denmark (to ARW), Novo Nordisk Foundation (NNF13OC0004294 to MJD) and a generous grant from the VILLUM

NU

Foundation to the VILLUM Center for Bioanalytical Sciences (ONJ)

MA

Acknowledgements

We are grateful to Hans Christian Beck and Odense University Hospital for providing the

AC

CE P

TE

D

human plasma sample.

25

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Fu, S.L. and R.T. Dean, Structural characterization of the products of hydroxylradical damage to leucine and their detection on proteins. Biochem J, 1997. 324 ( Pt 1): p. 41-8. Pietzsch, J. and R. Bergmann, Analysis of 6-hydroxy-2-aminocaproic acid (HACA) as a specific marker of protein oxidation: the use of N(O,S)-ethoxycarbonyl trifluoroethyl ester derivatives and gas chromatography/mass spectrometry. Amino Acids, 2004. 26(1): p. 45-51. Fu, S., et al., The hydroxyl radical in lens nuclear cataractogenesis. J Biol Chem, 1998. 273(44): p. 28603-9. Fu, S., et al., Evidence for roles of radicals in protein oxidation in advanced human atherosclerotic plaque. Biochem J, 1998. 333 ( Pt 3): p. 519-25. Pietzsch, J., Measurement of 5-hydroxy-2-aminovaleric acid as a specific marker of iron-mediated oxidation of proline and arginine side-chain residues of low-density lipoprotein apolipoprotein B-100. Biochem Biophys Res Commun, 2000. 270(3): p. 852-7. Behrens, G. and G. Koltzenburg, Elimination of Ammonium Ion from the AlphaHydroxyalkyl Radicals of Serine and Threonine in Aqueous-Solution and the Difference in the Reaction-Mechanism. Zeitschrift Fur Naturforschung C-a Journal of Biosciences, 1985. 40(11-12): p. 785-797. Bothe, E., et al., Ho2 Elimination from Alpha-Hydroxyalkylperoxyl Radicals in Aqueous-Solution. Photochemistry and Photobiology, 1978. 28(4-5): p. 639-644. Gebicki, J.M., Protein hydroperoxides as new reactive oxygen species. Redox Report, 1997. 3(2): p. 99-110. Davies, M.J., S. Fu, and R.T. Dean, Protein hydroperoxides can give rise to reactive free radicals. Biochem J, 1995. 305 ( Pt 2): p. 643-9. Davies, M.J., Protein and peptide alkoxyl radicals can give rise to C-terminal decarboxylation and backbone cleavage. Arch Biochem Biophys, 1996. 336(1): p. 163-72.

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43.

28

ACCEPTED MANUSCRIPT Tables

K

K

Modified structure (name)

Unmodified structure

IP

Modific. symbol

SC R

Residue

T

Table 1 Structures and masses of carbonylated residues identified in this study.

Composition change

Mass change (Da)

-3H -1N +1O

-1.03

+O

+15.99

-5H -1C -3N +1O

-43.05

-2H

2.02

-4H -1C -1S +1O

-32.01

-2H -1C -1O

-30.10

P

MA

P*

NU

Aminoadipic semialdehyde

R

M

E

T

AC

T

CE P

TE

R

D

Glutamic semialdehyde

Glutamic semialdehyde

2-Amino-3-ketobutyric acid

M-32

Aspartate 4-semialdehyde

E-30 4-aminobutyraldehyde

D

D-30

-2H -1C -1O

-30.10

L

L+14

-2H +1O

+13.98

29

K+14

V

V+14

A

A+14

I

I+14

Q

Q+14

E

E+14

-2H +1O

+13.98

-2H +1O

+13.98

-2H +1O

+13.98

-2H +1O

+13.98

-2H +1O

+13.98

-2H +1O

+13.98

AC

CE P

TE

D

MA

NU

SC R

IP

K

T

ACCEPTED MANUSCRIPT

30

ACCEPTED MANUSCRIPT Table 2. Carbonylated proteins identified in untreated human plasma.

Protein

No of carbonylated Protein name

Modification type sites

Serum albumin

34

P02647

Apolipoprotein A-I

17

P01009

Alpha-1-antitrypsin

P02671

Fibrinogen alpha chain

P02787

Serotransferrin

P00738

Haptoglobin

P01834

Ig kappa chain C region

P01023

Alpha-2-macroglobulin

P02652

Apolipoprotein A-II

P01011

Alpha-1-antichymotrypsin

P01024

Complement C3

P0CG05

SC R

P02768

IP

T

Accession

K, P, R, T, E-30, M-32, L+14 K, P, R, M-32 K,P,T

9

K,T

6

K

NU

11

K,T

5

K,T

4

K,T

4

K

3

K, T

3

K, P, T

Ig lambda-2 chain C regions

3

K,P

P02763

Alpha-1-acid glycoprotein 1

2

K, T

P01019

Angiotensinogen

2

K, L+14

P00450

Ceruloplasmin

2

K

P01857

Ig gamma-1 chain C region

2

K,P

Kininogen-1

2

K, T

Antithrombin-III

1

K

P06727

Apolipoprotein A-IV

1

K

P04114

Apolipoprotein B-100

1

K

P02656

Apolipoprotein C-III

1

K

P00751

Complement factor B

1

K

P01606

Ig kappa chain V-I region OU

1

T

P01871

Ig mu chain C region

1

K

1

K

1

K

P01008

D

TE

CE P

AC

P01042

MA

5

Immunoglobulin lambda-like B9A064 polypeptide 5 Inter-alpha-trypsin inhibitor heavy Q14624 chain H4

31

ACCEPTED MANUSCRIPT Serum amyloid P-component

1

K

P02766

Transthyretin

1

T

P01876

Ig alpha-1 chain C region

1

K

P02675

Fibrinogen beta chain

1

T

P02679

Fibrinogen gamma chain

1

P04433

Ig kappa chain V-III region VG

1

P25311

Zinc-alpha-2-glycoprotein

1

P69905

Hemoglobin subunit alpha

Q02487

Desmocollin-2

Q86V48

Leucine zipper protein 1

SC R

IP

T

P02743

T K

1

L+14

1

L+14

1

K

NU MA

D TE CE P AC

32

T

ACCEPTED MANUSCRIPT Figure legend Figure 1 Optimised experimental conditions for identification of carbonylated residues. a)

T

overview over experimental workflow, for details see the Methods section and Figure S3; b)

IP

overlap between all R/K/T/P-carbonylated tryptic peptides between different elution

SC R

conditions shows that the hot water method outperforms the classical biotin elution protocols (0.1 M glycine, pH 2.8 or 2 mM D-biotin in PBS, pH 7.4); c) addition of SDS during biotinhydrazide labelling step increases the number of identified R/K/T/P tryptic peptides by 16%.

NU

Only peptides found in 2 out of 4 experiments are considered.

MA

Figure 2. CID induces fragmentation of biotin-lc-hydrazide at the amide group and generates fragment ions, which can be used to support the identification and assignment of peptide

D

carbonylation site. a) Structure of biotin-lc-hydrazide, dashed lines indicate the fragmentation

TE

sites; b) ESI MS/MS spectrum of singly charged biotin-lc-hydrazide ion (m/z 372 Da). Two

CE P

fragment ions at m/z 227 and m/z 340 are observed; c) ESI MS/MS spectrum of peptide carrying carbonylated R (glutamic semialdehyde) and biotin-lc-hydrazide-tagged showing the

AC

presence of diagnostic ions – marked with dashed line boxes. Figure 3 Tandem mass spectra of biotin-lc-hydrazide-tagged carbonylated peptides with different types of modification. a) carbonylation of K to aminoadipic semialdehyde; b) and c) carbonylation of R and P to glutamic semialdehyde; d) carbonylation of T to 2-amino-3ketobutyric acid. The biotin fragment ions have been in silico removed from the spectra. Figure 4 Number of carbonylation sites identified. R/K/T/P carbonylation sites in a) MCOBSA and b) human plasma; c) +14 carbonylation of L, V, K, I, E, A and Q in MCO-BSA; d) decarboxylation of D and E, carbonylation of M in MCO-BSA; e) decarboxylation of E, carbonylation of L and M inhuman plasma.

33

ACCEPTED MANUSCRIPT Figure 5 Representative tandem mass spectra of biotin-lc-hydrazide-tagged carbonylated peptides carrying “novel” types of modifications detected in human plasma. a) +14

T

carbonylation of L; b) M-32 – carbonylation of M and c) E-30 – decarboxylation of E. #1 and

IP

#2 indicate biotin-lc fragment ions m/z 227 and m/z 340, respectively.

SC R

Figure 6 Proposed mechanisms of formation of carbonyl groups on amino acid side-chains in

AC

CE P

TE

D

MA

NU

proteins.

34

AC

CE P

TE

D

MA

NU

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IP

T

ACCEPTED MANUSCRIPT

35

ACCEPTED MANUSCRIPT Figure 2 b)

H N

NH2

NH

S

80 60 40 20

O

44

T

O

227.2

0

100

340 m/z

AC

CE P

TE

D

MA

NU

c)

36

336.2

IP

H N O

H 2N

340.2 100

227 m/z

SC R

Biotin-LC-hydrazide Biotin-lc-hydrazide

Relative Abundance

a)

150

200

250 m/z

354.2 300

350

ACCEPTED MANUSCRIPT Figure 3 Extracted from : C:\Users\adelinar\Desktop\MCO_BSA from Jesper\Jesper workflow based search\Obc08647.RAW #5537 y13 RT:y12y11y10 47.94 y9 ITMS, [email protected], z=+2, Mono m /z=1024.06531 Da, MH+=2047.12334 Da, Match Tol.=0.8 Da

AEFVEVTK LVTDLTK

a)

b5 b6 b7 b8

b₈⁺ 1258.38

y₅⁺ 577.27 y₆⁺ 676.31

2 0 00

1 5 00

y₃⁺ 361.33

y₄⁺ 476.27

b₁₂²⁺ 844.02 y₁₃²⁺ 924.16

b₇⁺ 776.29

500

y₈⁺ b₈⁺-H₂O 1240.59 1271.65

0 400

600

b₉⁺, y₉⁺ 1372.52

SC R

2 5 00

800

b9b10b11b12b13b14

b₁₀⁺, y₁₀⁺ 1471.58

T

y₇⁺ 789.42

3 0 00

Intensity [counts]

b₁₄²⁺ 951.16

IP

3 5 00

1 0 00

y8 y7 y6 y5 y4 y3 -1

1 0 00

1 2 00

1 4 00

b₁₁⁺ 1571.65

b₁₂⁺ 1686.56

b₁₃⁺ 1799.61

1 6 00

1 8 00

2 0 00

b)

Intensity [counts]

5000

4000

3000

y₅²⁺ 488.90

a₄²⁺-H₂O, a₂⁺ 215.09

IETMR EK

0

b₆⁺ 1072.25

300 400 500 600based search\Obc08677.RAW 700 800 Extracted 200 from : C:\Users\adelinar\Desktop\MCO_BSA from Jesper\Jesper workflow #2112 RT: 26.70 900 ITMS, [email protected], z=+2, Mono m /z=838.96326 Da, MH+=1676.91924 Da, Match Tol.=0.8mDa /z 6000

CE P

c) 4000

3000

2000

1000

b₄⁺ 465.24

b₂⁺ 251.16

b₅⁺ 594.29

1000

y10y9 y8 y7

b₉²⁺ 709.59

y₅⁺ 615.34

AC

Intensity [counts]

5000

b5 b6

b₅⁺ 943.31

y₄⁺ 875.37

TE

b₃⁺ 344.12

b2 b3

y₃⁺ 744.29

D

y₂⁺ b₂⁺ 243.07 276.16

y2 -43

y₅⁺ 976.42

2000

1000

y5 y4 y3

[M+2H]²⁺-H₂O, [M+2H]²⁺-NH₃ 600.76

MA

6000

NU

Extracted from : C:\Users\adelinar\Desktop\MCO_BSA from Jesper\Jesper workflow basedmsearch\Obc08672.RAW #1879 RT: 24.13 /z ITMS, [email protected], z=+2, Mono m /z=609.81665 Da, MH+=1218.62602 Da, Match Tol.=0.8 Da

1100

y6 y5 y4

HLVDEP+16QNLIK [M+2H]²⁺-H₂O, [M+2H]²⁺-NH₃ 830.65

b2

b₁₀²⁺ 766.04

y₇⁺ 1212.50

a₉²⁺ 695.31

b4 b5 b6

b7 b8 b9b10

y₉⁺ 1426.55 y₈⁺ 1327.49

b₁₀⁺ 1530.49

0 400

600

800

1000

1200

1400

1600

m search\Obc08677.RAW /z Extracted from : C:\Users\adelinar\Desktop\MCO_BSA from Jesper\Jesper workflow based #2831 RT: 32.26 ITMS, [email protected], z=+2, Mono m /z=758.91479 Da, MH+=1516.82231 Da, Match Tol.=0.8 Da

y8 y7 3000

d)

LVNELT +2 EFAK

[M+2H]²⁺-H₂O, [M+2H]²⁺-NH₃ 750.10

b5 b6

2500

Intensity [counts]

y6 y5 y4 y3

b7b8 b9

2000

y₈²⁺ 653.00

1500

500

y₃⁺ 365.26

y₄⁺ 494.22

b₉⁺ 1370.46

y₅⁺ 948.41

1000

b₅⁺ 569.27

y₆⁺ 1061.50

b₇⁺ 1152.32

b₈⁺ 1299.39

0 400

600

800

1000

m /z

37

1200

10

0

20

c) 20

0 0

20

d)

10

0 40

T

IP

SC R

NU

10

12

e)

10

8

6

4

2

0

MA

No of unique carbonylated sites

a)

D

No of unique carbonylated sites 20

TE

CE P

AC

No of unique carbonylated sites

ACCEPTED MANUSCRIPT

Figure 4 80

b)

60

38

ACCEPTED MANUSCRIPT Figure 5

Extracted from : C:\Users\adelinar\Desktop\random ized plasm a_workflow test\workflow final quant\QHF0009.raw #18962 RT: 39.71 FTMS, [email protected], z=+2, Mono m /z=759.90161 Da, MH+=1518.79595 Da, Match Tol.=0.02 Da

140

y₂⁺ 218.14996 #1

y9 y8 y7 y6 y5 y4 y3 y2

L VNEVTEFAK +14

T

#2

b1 b2 b3 b4 b5 b6 b7

100 80

IP

b₁⁺ 483.27444

y₈⁺ y₉⁺ 937.46844 1036.52759

60

y₅⁺ 595.30542

40 20

SC R

Intensity [counts] (10^3)

120

y₃⁺ 365.21735

y₆⁺ 694.37396

0

[M+2H]²⁺ 759.90070

b₇⁺ 1154.57507

NU

2 0:0C:\Users\adelinar\Desktop\random 300 4 0 0 ized plasm 5 0a_workflow 0 600 700 0 0 JespersWorkflow 900 1000 1100 1 2 0 0#21784 RT: 50.59 Extracted from test\New-workflow_based on new8new 12-08-2015\forgotten files\QHF0010.raw FTMS, [email protected], z=+2, Mono m /z=833.41638 Da, MH+=1665.82549 Da, Match Tol.=0.02 m /z Da 400

#2

300

y₂⁺-H₂O 258.14468 200

y₄⁺ 522.29309

#1

y9 y8 y7 y6 y5 y4 y3 y2

y10

AVM-32DDFAAFVEK

y₉⁺ 1041.48718

MA

Intensity [counts] (10^3)

y₃⁺ 375.22275

b3 b4 b5 b6 b7 b8 b9

[M+2H]²⁺-NH₃ 824.91052

b₃⁺ 625.34882

b₈⁺JespersWorkflow 12-08-2015\QHF0009.raw #19322 RT: 40.32 Extracted from : C:\Users\adelinar\Desktop\random ized plasm a_workflow test\New-workflow_based on new new 100 y₈⁺ Da y₇⁺Da, Match Tol.=0.02 FTMS, [email protected], z=+2, Mono m /z=737.90619 Da, MH+=1474.80510 1144.53589 b₉⁺-H₂O y₁₀⁺

D

811.43225

200

TE

0 400

600

y₂⁺ 218.14981

#1 60

#2

800

1273.60205

1000

1495.71399

1200

1400

1600

m /z

y8

y₆⁺ 694.38245

CE P

80

y7 y6 y5 y4 y3 y2

LVNE VTEFAK -30

b2

b4 b5 b6 b7 b8

y₈⁺ 1262.65796

y₅⁺ 595.30554

y₃⁺

40

y₄⁺ I will two more modification typesy₇⁺ here. 365.21976put b₄⁺ 494.26071 b₅⁺

20

0 200

AC

Intensity [counts] (10^3)

100

926.47083

400

781.43927

600

800

1148.61450

880.49353

b₉⁺-NH₃ 1311.67249

1000

m /z

39

1200

1400

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

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IP

T

Figure 6

40

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Graphical abstract

41