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Identification of rosmarinic acid-adducted sites in meat proteins in a gel model under oxidative stress by Triple TOF MS/MS Chang-bo Tang, Wan-gang Zhang, Yao-song Wang, Lu-juan Xing, Xing-lian Xu, and Guang-hong Zhou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02438 • Publication Date (Web): 03 Aug 2016 Downloaded from http://pubs.acs.org on August 4, 2016

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Journal of Agricultural and Food Chemistry

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Identification of rosmarinic acid-adducted sites in

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meat proteins in a gel model under oxidative stress

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by Triple TOF MS/MS

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Chang-bo Tanga ,b, Wan-gang Zhanga, Yao-song Wangc, Lu-juan Xinga, Xing-lian Xua and

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Guang-hong Zhoua,*

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a

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Control, Key Laboratory of Meat Processing and Quality Control, Ministry of Education, Key

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Laboratory of Animal Products Processing, Ministry of Agriculture, College of Food Science

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and Technology, Nanjing Agricultural University, Nanjing 210095, China

Jiangsu Collaborative Innovation Center of Meat Production and Processing, Quality and Safety

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b

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Vocational University, Suzhou 215104, China

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c

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210037, China

Department of Food Nutrition and Detection, College of Education and Humanity, Suzhou

College of Light Industry Science and Engineering, Nanjing Forestry University, Nanjing

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ACS Paragon Plus Environment

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ABSTRACT: Triple TOF MS/MS was used to identify adducts between rosmarinic acid

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(RosA)-derived quinones and meat proteins in a gel model under oxidative stress. Seventy-five

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RosA-modified peptides responded to 67 proteins with adduction of RosA. RosA conjugated

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with different amino acids in proteins, and His, Arg and Lys adducts with RosA were identified

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for the first time in meat. A total of 8 peptides containing Cys, 14 peptides containing His, 48

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peptides containing Arg, 64peptides containing Lys and 5 peptides containing N-terminal, which

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participated in adduction reaction with RosA, were identified respectively. Seventy-seven

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adduction sites were subdivided into all adducted proteins including 2 N-terminal adduction sites,

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3 Cys adduction sites, 4 His adduction sites, 29 Arg adduction sites and 39 Lys adduction sites.

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Site occupancy analyses showed that approximately 80.597% of the proteins carried a single

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RosA-modified site, 14.925% retained two sites, 1.492% contained three sites, and the rest 2.985%

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had four or more sites. Large-scale Triple TOF MS/MS mapping of RosA-adducted sites reveals

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the adduction regulations of quinone and different amino acids as well as the adduction ratios,

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which clarify phenol-protein adductions and pave the way for industrial meat processing and

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preservation.

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KEYWORDS: Triple TOF MS/MS, Total meat proteins, Rosmarinic acid, Adducts

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INTRODUCTION

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Polyphenols, which are originally extracted from natural plants, play important roles in food

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products as antioxidants1,2 and preservatives.3,4However, phenols are prone to oxidization into

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quinones under oxidative stress.5 As reactive electrophilic intermediates, quinones continue to

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react with nucleophilic group thiols6 and amines7-9 to form adducts through Michael addition

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reaction.


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Quinones oxidized from phenolic compounds can react with cysteine in proteins including

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myofibrillar protein isolate,10 α-lactalbumin and lysozyme,11 bovine serum albumin,12whey

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protein13and myoglobin14 to form quinone-thiol adducts. The potential reactions between alkali

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amino acids of myoglobin and several phenolic and related compounds have already been

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reported.15,16 Under variable reaction conditions such as oxidative stress and enzymes, adduction

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sites become more complex. Nikolantonaki et al.17identified three adducts between

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catechin/epicatechin and 1-dodecanethiol in the solution of a wine model. Kroll et al.18found a

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bovine serum albumin(BSA)-quercetin quinone was able to further react with free side chains of

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nucleophilic proteins to cross-link protein molecules leading to polymerization.

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In recent years, the influences of adducts on meat quality and human health have gradually

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been recognized. Jongberg et al.19 reported that the adducts formed between 4-MC and thiol

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groups blocked disulfide bond formation in meat proteins which affected the tenderness20,21 and

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water holding capacity (WHC)22 of meat products. Cao et al.23showed that chlorogenic acid

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influenced the possibly of myofibrillar protein gelation due to quinone-protein associations. Such

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covalent interactions also exert negative effects on the digestibility of diverse food proteins.13 On

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the cellular level, Michael adduct formation of quinone has been implicated in various diseases24-

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25

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leading to cytotoxicity. Phenols become less toxic after adduction reactions,26 and quinone-thiol

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adducts formed between quinones and cellular nucleophiles27-29 still function as redox-cycling

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agents.30,31.

as the adduction disrupts correct formation of disulfide bonds and makes protein misfolding

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To determine adducts in meat products, Jongberg et al.10hydrolyzed myofibrillar protein

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isolates(6NHCl, 110°C,22h) after incubation and identified a cysteine-quinone adduct (m/z

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244.2). Using MALDI-TOF/TOF MS, Tang et al.32 digested myofibrillar proteins into peptides


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and identified an adducted peptide (LEDEC*SELK). Besides, they found rosmarinic acid (RosA)

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generated a Cys949 adduction site in myosin. However, the adduction sites of meat proteins

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remain largely unknown, thus requiring an effective phenol-proteomics approach for large-scale

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comprehensive mapping of quinone adduction sites in meat proteins. Therefore, it is of great

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significance to better understand protein-phenol adduction to comprehend the chemistry behind

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their textures and biological effects. With high resolution and mass accuracy, Triple TOF has

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been applied in phytochemical and proteomics fields.33,34

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In this study, we established a meat gel model under oxidative stress by adding RosA, the main

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phenol of natural extracts,35,36 aiming to map all the RosA-adducted sites in meat proteins and to

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identify these proteins. In addition, we analyzed the attribution of these modified sites and RosA

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adduction referring to different amino acids by employing a high LC-MS/MS system, Triple

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TOF 6600 coupled with EksigentLC (Triple TOF-based MS).The adduction regulations of

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quinone and different amino acids were revealed, which is helpful to develop molecular

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processing techniques by applying these phytochemicals in meat products.

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MATERIALS AND METHODS

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Materials

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Longissimus muscle was purchased from Walmart supermarket. RosA (96%, MW: 360.33),

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piperazine-N,N'-bis (2-ethanesulfonic acid) (PIPES), (±)-6-Hydroxy-2,5,7,8-

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tetramethylchromane-2-carboxylic acid (Trolox), iodoacetamide and1,4-dithiothreitol(DTT)were

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purchased from Sigma-Aldrich (Shanghai,China) and were all of pharma grade. Hydrochloric

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acid, sodium hydroxide, acetonitrile, sodium dihydrogen phosphate, sodium chloride,

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magnesium chloride, ethylene-bis(oxyethylenenitrilo)tetraacetic acid and hydrogen peroxide


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(30%) were purchased from Jiancheng Chemical Regent Co., Ltd. (Nanjing, China), and were at

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least of analytical grade.

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Methods

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Extraction of total meat proteins

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Total meat proteins (TMP) were extracted from fresh pork longissimus muscle purchased from

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Walmart supermarket according to the method of Joo et al.37 with slight modifications. TMP

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were extracted from 10 g muscles using 200 ml of ice-cold 1.1 M potassium iodide in 0.1 M

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phosphate buffer (pH 7.2).The samples were minced, homogenized on ice in a blender (GM 200,

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Retsch, Germany) at the speed of 4000 rpm for 40 s, and then left on a shaker (KS 260, Orbital

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shakers, USA) at the speed of 130 rpm in a 4°Ccompartment overnight. Afterwards, they were

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centrifuged at 1500×g for 20 min. After protein concentration of supernatants was determined by

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Biuret method,38 they were frozen at -80°C and lyophilized.

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Oxidative treatments with RosA

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TMP sample (final concentration: 20 mg/mL) was prepared by thorough dispersion with gentle stirring into 15 mM PIPES buffer containing 0.6M NaCl (pH 6.25). Samples with RosA at

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final concentrations of 120µmol/g protein were oxidatively stressed with a hydroxyl radical

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(•OH)-generating system (10 µM FeCl3, 100 µM ascorbic acid, and 1mM H2O2) by incubation at

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4°C for 12 h. Oxidation was terminated by adding Trolox (1 mM). A sample containing RosA

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without oxidative stress was used as the non-oxidized control.

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Trypsin digestion


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Protein (100µl) was resuspended in 100µl of 8M urea without pH adjustment,39 reduced with

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20mM DTT at 60°C for 1 h, and then alkylated with 40mM iodoacetamide at room temperature

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for 30 min while protected from light. Alkylation reactions were quenched by 10mM DTT. The

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samples were diluted to 2M urea with HPLC-grade water, the protein concentrations were

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determined by modified Lowry’s assay (DC Assay Kit, Cat. 500-0111, BioRad) using BSA to

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construct calibration curve. Appropriate amount of trypsin was then added at an

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enzyme/substrate ratio of 1:100. Digestion was performed in 100mM triethylammonium

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bicarbonate (pH 8, T7408, Sigma) at 37°C for 18 h. The digested proteins were desalted by a

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C18 column and dried in spin vacuum.

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Triple TOF MS/MS analysis

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Each dried peptide sample was dissolved in 12 µl of 0.1% formic acid, and analyzed by

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nanoLC-MS/MS using an Eksigentekspert™ nanoLC 425 systems coupled to AB Sciex Triple

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TOF 6600 System.40 After the sample was loaded, peptide was trapped (ChromXPnanoLC Trap

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column 350µm id × 0.5 mm, ChromXPC18 3µm 120Å) and eluted into a reverse-phase C18

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column (ChromXPnanoLC column 75µm id×15 cm, ChromXPC18 3µm 120Å) at a flow rate of

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300nL/min using a linear gradient of acetonitrile (3-36%) in 0.1% formic acid with a total

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runtime of 30 min including mobile phase equilibration. MS and MS/MS spectra were recorded

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in the “high-sensitivity” and positive-ion mode with a resolution of ~35,000 full-width half-

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maximum. Typically, the nanospray needle voltage was 2,300 V in the HPLC-MS mode. After

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acquisition of 5-6 samples, TOF MS and TOF MS/MS spectra were automatically calibrated

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during dynamic LC-MS and MS/MS auto-calibration acquisitions injected with25 µmol alcohol

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dehydrogenase. For collision-induced dissociation MS/MS, mass window for precursor ion

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selection of the quadrupole mass analyzer was set at±2 m/z. The precursor ions were fragmented


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in a collision cell using nitrogen as the collision gas. Advanced information-dependent

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acquisition was used on Triple TOF 6600 to obtain MS/MS spectra for the 20 most abundant and

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multiple-charged (z = 2, 3 or 4) precursor ions following each survey MS1 scan (typically

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allowing 250 ms of acquisition per MS/MS). Dynamic exclusion was set for 30s after 2 repetitive

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occurrences.

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Standard mascot search

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Raw data files were converted to Mascot generic format (MGF) and m/z XML format using

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Open MS. The MGF files were searched against UniProt, NCBI and common MS contaminant

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databases using Mascot 2.5 (Matrix Science) Software.41 Tolerances for MS1 and MS2 errors

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were 50ppm and 0.05Da, respectively. A maximum of 2 trypsin miss-cleavages were allowed.

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Peptides were assumed to have a charge of 2+, 3+or 4+carbamidomethyl (C, +57.02) as a fixed

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modification with oxidation (M, +15.99) and RosA (CHKR, + 358.068868) being the only

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variable modifications. ESI-QUAD was selected as the instrument. The mass input was assumed

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to be monoisotopic. Decoy database was used to control the false discovery rate at below 1%.42

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Only the proteins containing at least two unique peptides were identified.34 All the unique

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sequences of RosA-adducted peptides were submitted online to WebLogo43 to extract the

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adducted site motif of TMPs.

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RESULTS AND DISCUSSION

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Identification of modified proteins and peptides


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The 590RosA-modified peptides and 246 RosA-modified proteins were identified (Table 1 and

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Table S1). To improve the reliability, the peptide data were filtered with score ≥10. Seventy-five

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RosA-modified peptides responded to 67 proteins with adduction of RosA.

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The 67 modified proteins were classified on the basis of functions and locations.44 In the

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myofibrillar protein family, 20 proteins were identified including heavy myosin chain, titin,

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actin, filamin and kinesin. Among 27 adducted peptides, 21 carried a single adduction site and 6

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carried 2 sites. In the sarcoplasmic protein family, 39 proteins were identified and adducted on

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40 peptides. Except for one peptide carrying two sites and one carrying three sites, the other

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peptides all contained a single adduction site. Six members from the membrane protein family

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and 2 other proteins were also identified with each one containing a single adduction site. Four

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MS/MS spectra of RosA-adducted peptides on Cys, His, Lys and Arg are shown in Figure1, and

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all other spectra are exhibited in Figure S1.RosA-adducted peptides on His, Lys and Arg were

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successfully identified for the first time in meat. Model adducts reactions between RosA and

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specific amino acids(cysteine, lysine, arginine and histidine) on myofibrillar proteins are also

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displayed in Figure 2, which provide the chemical structures of corresponding adducts (RosA-

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Cys, RosA-Lys, RosA-Arg and RosA-His).

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As the structural proteins that compose myofibrils,45 myofibrillar proteins play important roles

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in controlling the quality of meat processing including WHC46,47 and binding properties.48Myosin

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is the most abundant myofibrillar component, constituting approximately 43% of myofibrillar

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proteins in mammalian and avian muscle tissues.49We identified six myosin heavy chain protein

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isoforms with RosA modification and four of which including myosin heavy chain slow isoform

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1, myosin heavy chain 2a, heavy chain 2b and myosin heavy chain 2x were the main members in

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skeletal muscle.47They all had more than two adduction sites, and myosin heavy chain


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2xcontainedup to four sites. In postnatal growing pigs, four MyHC isoforms (1, 2a, 2x, 2b) are

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expressed in skeletal muscle,50 the transition of which follows an obligatory pathway in the rank

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order of 1↔2a↔2x↔2b.51,52 Higher proportion of oxidized types muscle fibers(MyHC1 and 2a)

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was associated with smaller muscle color, marbling scores and intramuscular fat content, muscle

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tenderness, muscle fiber diameter and higher water retention properties of muscle,53-56 while the

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glycolytic muscle fiber (MyHC2b) was the opposite and MyHC2x was in the middle. Myosin

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heavy chain 2b (alternative name: myosin-4) has been determined to be modified with RosA at

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Cys949 site (adducted peptide LEDEC*SELK, m/z 1425.73).32Besides the above adduction site,

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we obtained a Lys1111 site (adducted peptide KIK*ELQAR, m/z). A large number of sulfhydryl

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groups in myosin are located in MHC57 thus promoting the modification reaction. Kirshenbaum

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et al. also found myosin fragment 1 Cys-697 and Cys-707 modified ATP and actin binding site

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interactions.58Adduction of Cys949 site may block disulfide group from influencing the gel

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quality during meat processing.

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Cytoskeletal beta-actin which constituted about 22% of the myofibrillar mass59was identified

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at His110 site (adducted peptide VAPEEH*PVLLTEAPLNPK, m/z 1425.73) (Table 2). Two

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subtypes of titin protein, including titin and titin-like as cytoskeletal proteins, were also

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identified. Titin had two adduction sites (LKVEAVK*IK, KSKVTLSALK*) and titin-like had

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three sites (KPEPEKK*VPPPGLK, KLSDTSTLVGDAVELR*, KEAPPAK*).

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Sarcoplasmic proteins which may impact the formation and texture properties of protein gels

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by stimulating the setting effect of MP,60 mainly comprise enzymes related with energy-

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producing metabolism.61 As a crucial enzyme in glycolysis, 6-phosphofructokinase was

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identified with RosA modification. Phosphofructokinase, pyruvate kinase (PK) and hexokinase

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(HK) are key regulatory enzymes of glycolysis.62 Having at least two active thiol groups,


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adenosine monophosphate deaminase 1 catalyzes the deamination of AMP to inosine

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monophosphate as a part of purine catabolism.63We found an adducted peptide

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KVDTH*IH*AAAC*MNQK* in which thiol group was blocked to evidently affect the

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functions. Arylating quinones induce endoplasmic reticulum (ER) stress by activating the

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pancreatic ER kinase signaling pathway including elF2, ATF4, and C-EBP homologous

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proteins.26Since a large number of RosA-adducted proteins are related to metabolic processes,

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sufficient energy is produced through the metabolism of proteins and carbohydrates for queen

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spawning and larval growth which may be realized by regulating enzymatic efficiency.

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Six identified members of membrane protein family were modified with RosA, including

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transmembrane protein 38B; PREDICTED: olfactory receptor 5AP2-like; PREDICTED: LOW

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QUALITY PROTEIN: desmoplakin; PREDICTED: regulating synaptic membrane exocytosis

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protein 1 isoform X6; PREDICTED: transmembrane channel-like 1 isoform X1 and

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PREDICTED: MAGUKp55 subfamily member 6 isoform X2.

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Mapping of adduction sites of peptides

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Overall, RosA-adducted proteins carried 75 modified peptides. According to the peptide

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identification, RosA conjugated with different amino acids such as arginine (R/Arg), cysteine

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(C/Cys), histidine (H/His), lysine (K/Lys) and N-terminal sites. Of 38 identified peptides

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containing Cys (Figure 3), 8 participated in the adduction reaction. A total of 318 peptides

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containing His were identified, of which 14 were involved in adduction reaction. The 1099

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identified peptides containing Arg and 1248 containing Lys, and 48 and 64 participated in the

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adduction reaction respectively. Thirty-one peptides containing N-terminal were identified of

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which 5 were involved in the adduction reaction.


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The ratio of adduction reaction on peptides containing Cys was 21.05% and that of adduction

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reaction sites on Cys was 7.89% (Figure 4). The ratio of adduction reaction on peptides

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containing Lys was 5.12%, and that of adduction reaction sites on Lys was 3.53%. The ratio of

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adduction reaction on peptides containing His was 4.37%, and that of adduction reaction sites on

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His was 1.26%. The ratio of adduction reaction on peptides containing Arg was 4.36%, and that

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of adduction reaction sites on Arg was 3.73%. The ratio of adduction reaction on peptides

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containing N-terminal was 16.13%, and that of adduction reaction sites on N-terminal was

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6.45%.

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Arg and Lys were the most abundant alkin amino acids (Arg 6.78, Lys 6.04 g/100g total amino

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acids) in fetal pigs,64 being consistent with our data (Figure4). Nevertheless, the ratio of Cys

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herein was extremely lower. Disulfanyl65and phenolic adducts conjugated with two peptides

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containing Cys66 were produced under oxidative stress and we identified the peptides containing

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Cys. The percentages of adducted peptides containing Cys and N-terminal exceeded those of

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other alkali amino acids. The peptides containing alkali amino acids, considerably located inside

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the proteins, were barely under adduction reaction owing to steric hindrance.

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Figure 4 shows the ratios of adducted peptides containing and reacting onamino acids. The two

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ratios of Cys, His and N-terminal peptides differed significantly, whereas those of Lys and Arg

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did not, indicating that Lys and Arg may be more reactive than other amino acids (C, H and N-

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terminal) during the adduction reactions.

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Seventy-seven adduction sites were subdivided into all the adducted proteins, 2 N-terminal

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adduction sites, 3 Cys adduction sites, 4 His adduction sites, 29 Arg adduction sites and 39 Lys

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adduction sites, with the ratios of 2.597%, 3.896%, 5.194%, 37.662%and 50.649%, respectively

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(Figure 5). Site occupancy analyses showed that approximately 80.597% of the proteins carried a


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single RosA-modified site, 14.925% retained two sites, 1.492% contained three sites, and the rest

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2.985% had four or more sites (Figure 6).

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Large-scale Triple TOF MS/MS mapping of RosA-adducted sites revealed the adduction

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regulations of quinone and different amino acids as well as the adduction ratios, providing

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evidence for clarifying phenol-protein adductions. Compared with Cys as the focus for meat

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quality in previous literatures, Lys and Arg have higher adduction quantities (Figure 3) and

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reaction efficiencies (Figure 4). Thus, alkaline amino acids may play more important roles in

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adduct reactions and affect meat quality more obviously.

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The regulation of adduction can be used to regulate adducts formed between phenolics and

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meat proteins and then to influence the meat quality in molecular processing. For example, active

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amino acids (e.g.Lys, Arg) can be added in meat products to react with RosA firstly, and to avoid

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the formation of protein-RosA adducts, which significantly affects the meat quality by disturbing

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the order of gel network.23

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To better understand the sequence motif of adduction sites in the meat proteins, the

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surrounding sequences (ten amino acids to both termini) were compared. As shown in Figure 7,

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about 80% were the X-K/R-X motif, of which L-K-E and E-K-L accounted for one third. The

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others were the X-H/C-X motif in downstream (positive values) RosA-adducted sites, the K-

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linked sequence motif was X-K-E/L in K-adducted proteins(X = any amino acid, K/R = lysine or

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arginine, H/C= histidine or cysteine).

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Half of the adduction sites were located on Lys, 37% of them were located on Arg, and the rest

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were on Cys, His and N-terminal. Most proteins (~80%) carried a single adduction site, 15%

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contained two or three sites, and only a few had four or five sites. Identifying the conservative


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motif of amino acid sequences of RosA-adducted peptides may have structural and functional

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importance to future studies on meat proteins.

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Abbreviation list:

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Triple TOF MS/MS: Triple time-of-flight tandem mass spectrometer

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RosA: Rosmarinic acid

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Cys: Cysteine

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His: Histidine

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Arg: Arginine

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Lys: Lysine

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DTT: 1,4-dithiothreitol

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The supporting Information. Spectra for RosA-adducted peptides (Figure S1) and RosA-

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modified peptides (Table S1) are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

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Corresponding Author

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Guang-hong Zhou

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E-mail address: [email protected]

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Notes

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The authors declare no competing financial interest.

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Author Contributions


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The manuscript was written through contributions of all authors. All authors have given approval

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to the final version of the manuscript.

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ACKNOWLEDGMENTS

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The research was funded by the National Natural Science Funds of China (No. 31501509) and

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the 111 Project “Innovation of Meat Quality and Safety Control &Nutrition Exchanging

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Foundation” (No.B14023).

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470 471


21


472 Classification

Page 22 of 35

Table1. RosA-adducted proteins in a myofibrillar protein gel model under oxidative stress Accession

Protein description

Coverage (%)

pI

MW(Da)

Score

gi|12060489

myosin heavy chain slow isoform

54.68

5.59

223298.4

21767.85

gi|311273875

PREDICTED: microtubule-associated protein 1B isoform X1

3.96

4.72

270632.5

54.35

gi|335302447

PREDICTED: kinesin heavy chain isoform 5C isoform X1

3.34

5.88

109335.7

16.89

gi|335308411

PREDICTED: kinesin family member 3B

6.69

7.30

85189.22

39.48

gi|350593665

PREDICTED: LOW QUALITY PROTEIN: titin

14.12

5.52

631336.5

2140.56

gi|45269029

cytoskeletal beta actin, partial

54.95

5.55

44792.16

13864.09

gi|5360746

myosin heavy chain 2a

56.21

5.64

223150.3

49622.44

gi|5360748

myosin heavy chain 2b

70.11

5.59

223236.2

105349.2

Myofibrillar

gi|5360750

myosin heavy chain 2x

68.44

5.60

223173.3

57780.98

proteins

gi|545813772

PREDICTED: CAP-Gly domain-containing linker protein 2

9.70

5.73

81569.95

35.71

gi|545826249

PREDICTED: kinesin heavy chain isoform 5A-like

9.14

5.37

93657.66

45.85

gi|545841359

LOW QUALITY PROTEIN: myosin, heavy chain 6, cardiac muscle, alpha

45.65

5.30

205773.8

18396.76

gi|545860458

PREDICTED: LOW QUALITY PROTEIN: myosin-13-like

31.32

5.50

222434.8

12188.89

gi|545874878

PREDICTED: titin-like, partial

21.29

5.37

701564.7

4826.57

gi|545877574

PREDICTED: LOW QUALITY PROTEIN: actin, beta-like 2

32.36

5.08

34497.31

3312.52

gi|545882663

PREDICTED: filamin-C isoform X5

13.06

5.67

292665.2

1009.11

gi|417515907

plectin, partial

19.82

5.57

402915.8

855.82

gi|311274402

PREDICTED: filensin

12.78

5.38

74963.25

70.58


22

Page 23 of 35


gi|545858751

PREDICTED: collagen alpha-1(I) chain-like

10.27

8.19

100006.1

234.66

gi|85002144

myocyte enhancer factor 2C

4.39

8.69

46986.54

40.80

Sarcoplasmic

gi|194042470

PREDICTED: kinesin family member 20B isoform X1 [Susscrofa]

9.18

5.42

207044.9

82.33

proteins

gi|294494655

SAM pointed domain-containing ets transcription factor

9.55

5.88

37247.2

26.37

gi|311260254

PREDICTED: inositol 1,4,5-trisphosphate receptor type 3

3.97

6.11

303803.9

43.63

gi|335290158

PREDICTED: U4/U6 small nuclear ribonucleoprotein Prp31

17.84

5.41

55321.8

82.28

gi|350587974

serine-rich coiled-coil domain-containing protein 1-like, partial

11.67

8.58

62784.92

27.04

gi|350590109

PREDICTED: lysosomal alpha-glucosidase isoform X1

2.65

5.69

104891.4

26.14

gi|350594341

PREDICTED: DNA excision repair protein ERCC-8

7.58

6.06

43942.17

66.92

gi|417515756

transcription initiation factor TFIID subunit 1 isoform 2

4.06

4.98

212554.2

181.94

gi|311276001

PREDICTED: nance-Horan syndrome protein isoform X1

3.81

6.39

174870.5

115.70

gi|335280808

PREDICTED: WD repeat-containing protein 31 isoform X1

5.72

8.79

40832.84

89.02

gi|346986428

heat shock 90kD protein 1, beta

15.33

4.96

83253.17

135.64

gi|347543741

ankyrin repeat domain-containing protein 10

15.00

5.56

44179.07

14.26

gi|350587373

PREDICTED: condensin complex subunit 3 isoform X1

5.92

5.31

113977.2

77.61

gi|456753975

UPF0556 protein C19orf10 precursor

9.20

8.53

18911.36

13.08

gi|545838985

PREDICTED: prefoldin subunit 6 isoform X1

11.41

9.78

20612.52

41.89

gi|545844733

PREDICTED: FRY-like

2.87

4.95

207058.7

42.31

gi|545893182

PREDICTED: olfactory receptor 1J4-like

5.10

8.63

21250.39

14.21

gi|75038608

calsarcin 3

35.92

6.86

26468.85

254.2

gi|545820546

PREDICTED: eukaryotic translation initiation factor 3 subunit E

4.34

5.66

56599.12

20.56


23


Membrane

Page 24 of 35

gi|545828510

PREDICTED: centrosomal protein of 290 kDa isoform X7

7.37

5.79

295106.5

132.82

gi|545830058

PREDICTED: tudor domain-containing protein 12-like

7.53

6.49

148927.1

31.81

gi|545832091

PREDICTED: splicing factor, arginine/serine-rich 19 isoform X2

5.96

9.42

139684.8

23.12

gi|545832513

PREDICTED: LOW QUALITY PROTEIN: zinc finger protein 615-like

8.81

9.23

95041.62

50.51

gi|545832547

PREDICTED: cationic amino acid transporter 3-like isoform X3

5.61

8.84

75747.87

32.31

gi|545861130

peptide-N(4)-(N-acetyl- glucosaminyl)asparagine amidase isoform X1

3.20

6.78

71878.43

27.43

gi|545861503

PREDICTED: deleted in lung and esophageal cancer protein 1, partial

2.06

5.94

177546.5

52.99

gi|545894709

human immunodeficiency virus type I enhancer binding protein 3

4.78

9.04

252229.9

43.31

gi|106647532

antithrombin protein

18.57

5.84

52386.04

84.76

gi|164707699

adenosine monophosphate deaminase 1

8.17

6.53

86509.95

109.21

gi|311255461

mitogen-activated protein kinase kinase kinase 12 isoform X1

6.50

5.93

95897.1

19.00

gi|359465576

A-kinase anchor protein 9

9.26

4.97

452291

323.4

gi|385648284

topoisomerase (DNA) II beta 180kDa

6.13

8.30

181978.9

58.82

gi|545800482

PREDICTED: RNA polymerase-associated protein LEO1-like

2.72

5.53

38295.48

17.61

gi|545823356

PREDICTED: E3 SUMO-protein ligase PIAS3 isoform X2

5.82

8.21

67226.48

46.5

gi|545834167

PREDICTED: leucine zipper protein 1 isoform X2

10.07

8.76

119770.8

55.14

gi|545841000

PREDICTED: poly [ADP-ribose] polymerase 6 isoform X10

46.77

7.64

65650.54

919.72

gi|545863823

acyl-CoA dehydrogenase family member 9, mitochondrial-like

24.38

6.61

17705.45

18.40

gi|545867651

PREDICTED: 6-phosphofructokinase, liver type

5.40

8.10

82772.98

47.59

gi|545870350

PREDICTED: probable E3 ubiquitin-protein ligase HERC4, partial

5.82

7.91

29853.42

365.07

gi|298104070

transmembrane protein 38B

14.14

9.34

32476.97

17.47


24

Page 25 of 35

proteins

Other Proteins


gi|311247798

PREDICTED: olfactory receptor 5AP2-like

2.52

8.73

35442.35

16.96

gi|311259696

PREDICTED: LOW QUALITY PROTEIN: desmoplakin

8.02

6.51

331204.3

230.55

gi|545798332

regulating synaptic membrane exocytosis protein 1 isoform X6

9.10

9.52

112564.7

42.89

gi|545803416

PREDICTED: transmembrane channel-like 1 isoform X1

7.25

6.21

87469.57

55.10

gi|545883649

PREDICTED: MAGUKp55 subfamily member 6 isoform X2

6.08

6.08

64381.12

153.10

gi|545833251

PREDICTED: uncharacterized protein LOC100623546

6.29

8.35

206632.6

93.69

gi|545890348


10.25

9.37

577032.9

435.51

Adduction

Mass error

473 474 475

Table2. RosA-adducted peptides digested from RosA-adducted proteins

Accession

Protein description

Styled peptide sequence

Score

Charge

site

(ppm)

gi|12060489

myosin heavy chain slow isoform

KLK*ELQAR

31.65

2

1109

-0.23

K*

17.10

4

234

25.76

KEQTPGKGTLEDQIIQANPALEAFGNA

gi|311273875

PREDICTED: microtubule-associated protein 1B isoform X1

AEGAEK*QGADVKPKVAK

18.82

3

623

-4.80

gi|335302447

PREDICTED: kinesin heavy chain isoform 5C isoform X1

LQDAEEMK*

14.13

2

700

-13.15

gi|335308411

PREDICTED: kinesin family member 3B

RPVSAVGYK*R*PLSQHAR

19.48

4

636,637

37.40

gi|350593665

PREDICTED: LOW QUALITY PROTEIN: titin

LKVEAVK*IK

15.42

2

2713

-12.59

KSKVTLSALK*

10.67

3

5488

-39.87

gi|45269029

cytoskeletal beta actin

VAPEEH*PVLLTEAPLNPK

21.16

3

130

49.54

gi|5360746

myosin heavy chain 2a

QAEEAEEQSNTNLSK*FR*

12.28

4

1899,1901

-39.42


25


gi|5360748

gi|5360750

myosin heavy chain 2b

myosin heavy chain 2x

Page 26 of 35

LEDEC*SELK

33.94

2

951

8.90

KIK*ELQAR

31.65

2

1111

-0.23

VRELEGEVESEQK*R

23.38

3

1835

-45.13

NLTEEMAGLDETIAK*LTK*

16.31

4

995,998

-37.87

VRELEGEVESEQKR*

14.03

3

1836

-44.20

gi|545813772

PREDICTED: CAP-Gly domain-containing linker protein 2

QEAERLR*EK*

18.65

3

664,666

-48.39

gi|545826249

PREDICTED: kinesin heavy chain isoform 5A-like

HEQSK*QDLKGLEETVAR

13.78

4

710

29.96

PREDICTED: LOW QUALITY PROTEIN: myosin, heavy chain 6, cardiac gi|545841359

muscle, alpha

IEDMoAMLTFLH*EPAVLFNLK*

10.1

3

97,106

45.97

gi|545860458

PREDICTED: LOW QUALITY PROTEIN: myosin-13-like

QAEEAEEQANTQLSRCR*

30.97

3

1901

29.45

gi|545874878

PREDICTED: titin-like

KPEPEKK*VPPPGLK

20.5

4

5731

-4.55

KLSDTSTLVGDAVELR*

15.77

2

2721

39.27

KEAPPAK*

13.32

2

4221

3.915

gi|545877574

PREDICTED: LOW QUALITY PROTEIN: actin, beta-like 2

VAPDEH*PILLTEAPLNPK

21.16

3

102

49.54

gi|545882663

PREDICTED: filamin-C isoform X5

VCAYGPGLK*GGLVGTPAPFSIDTK*

10.1

3

1095,1110

36.49

gi|417515907

plectin, partial [Susscrofa]

QQLVASMEEARRR*

14.79

4

1578

43.23

gi|311274402

PREDICTED: filensin

EVLC*LLQAQK

13.21

2

232

37.58

gi|545858751

PREDICTED: collagen alpha-1(I) chain-like

AEKGGRSSPARPR*

11.47

3

962

9.60

gi|85002144

myocyte enhancer factor 2C

KINEDLDLMISR*

19.59

3

130

29.41

gi|194042470

PREDICTED: kinesin family member 20B isoform X1

LMoQAKIDELR*

22.22

3

957

24.44

gi|294494655

SAM pointed domain-containing ets transcription factor

MoGSASPGLSSGPPSR*

18.24

2

15

24.82

gi|311260254

PREDICTED: inositol 1,4,5-trisphosphate receptor type 3

KMoLLQNYLQNRK*

16.77

4

1669

22.34

gi|335290158

PREDICTED: U4/U6 small nuclear ribonucleoprotein Prp31

YSKRFPELESLVPNALDYIR*

20.14

4

137

22.87

gi|350587974

PREDICTED: serine-rich coiled-coil domain-containing protein 1-like

EVLLQITELPVMoNGR*

19.29

4

361

45.01


26

Page 27 of 35


gi|350590109

PREDICTED: lysosomal alpha-glucosidase isoform X1 [Susscrofa]

KQPMoALAVALTTSGK*

19.91

4

845

49.12

gi|350594341

PREDICTED: DNA excision repair protein ERCC-8

LGFLSARQAGLEDPLR*LR

19.95

3

17

21.23

gi|417515756

transcription initiation factor TFIID subunit 1 isoform 2 [Susscrofa]

LMoPPPPPPPGPMKK*

19.42

3

168

-4.88

gi|311276001

PREDICTED: nance-Horan syndrome protein isoform X1

TISGIPR*R*

10.25

3

382,383

45.02

gi|335280808

PREDICTED: WD repeat-containing protein 31 isoform X1 [Susscrofa]

YSSPDGFIEER*

25.62

2

48

39.88

gi|346986428

heat shock 90kD protein 1, beta [Susscrofa]

VKEVVK*K

12.62

2

203

9.39

gi|347543741

ankyrin repeat domain-containing protein 10

EFAVLTDVK*SSSSVSSTLTNGGVR

13.41

4

256

-41.63

gi|350587373

PREDICTED: condensin complex subunit 3 isoform X1

IKIQLEK*

15.66

2

898

-5.43

gi|456753975

UPF0556 protein C19orf10 precursor [Susscrofa]

AEVRGAEIEYGMoAYSK*

13.08

2

126

1.56

gi|545838985

PREDICTED: prefoldin subunit 6 isoform X1

ASGAVSSQHSLFTSGFTNTER*

17.28

4

26

-0.42

gi|545844733

PREDICTED: FRY-like

SNSLR*LSLIGDR

12.87

2

773

20.50

gi|545893182

PREDICTED: olfactory receptor 1J4-like

NRDMKGALGK*

14.21

3

186

39.34

M*IPKEQKGPVVTAMoGDLTEPAPLL gi|75038608

calsarcin 3

DLGK

12.52

4

1

10.22

gi|545820546

PREDICTED: eukaryotic translation initiation factor 3 subunit E

LASEILMQNWDAAMEDLTRLK*

20.56

4

232

15.72

gi|545828510

PREDICTED: centrosomal protein of 290 kDa isoform X7

QKAYDKMLR*

16.94

3

2121

18.40

QSLIEELQK*

14.46

4

2162

-0.31

gi|545830058

PREDICTED: tudor domain-containing protein 12-like

AEILSTGMoGIDNPEHVQQLKK*

20.45

4

1100

33.08

gi|545832091

PREDICTED: splicing factor, arginine/serine-rich 19 isoform X2

ICHSKSGEINPVKVSNLVR*

13.4

4

1281

11.50

gi|545832513

PREDICTED: LOW QUALITY PROTEIN: zinc finger protein 615-like

M*VKVFERSEESVCR

21.75

2

1

17.84

gi|545832547

PREDICTED: cationic amino acid transporter 3-like isoform X3

AALLTR*AR

21.99

2

7

9.19

PREDICTED: peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagine gi|545861130

amidase isoform X1

ASVEQLQKIR*

27.35

2

25

-24.28

gi|545861503

PREDICTED: deleted in lung and esophageal cancer protein 1

KPNLRPQMoAR*

24.49

4

511

44.69


27


Page 28 of 35

PREDICTED: LOW QUALITY PROTEIN: human immunodeficiency gi|545894709

virus type I enhancer binding protein 3

QRAGGGRSDHLSPPR*

27.23

2

2253

43.51

gi|106647532

antithrombin protein

AKLPGIVAEGR*DDLYVSDAFHK

16.01

3

392

-46.91

303,305,309 ,

gi|164707699

adenosine monophosphate deaminase 1

KVDTH*IH*AAAC*MNQK*

13.01

3

313

36.41

PREDICTED: mitogen-activated protein kinase kinase kinase 12 isoform gi|311255461

X1

AGSQHLTPAALLYR*

15.09

3

739

44.27

gi|359465576

A-kinase anchor protein 9

DK*EELEDLK

14.19

2

3234

40.10

gi|385648284

topoisomerase (DNA) II beta 180kDa

KNKAGVSVKPFQVK*

13.05

2

370

-21.56

gi|545800482

PREDICTED: RNA polymerase-associated protein LEO1-like

K*VTLPLANR

17.61

2

192

-20.25

gi|545823356

PREDICTED: E3 SUMO-protein ligase PIAS3 isoform X2

QLTAGTLLQKLRAK*

10.49

2

283

10.81

gi|545834167

PREDICTED: leucine zipper protein 1 isoform X2

SKAIIKPVIIDK*

15.05

4

763

-40.30

FLR

13.01

3

12

3.29

HGAVGSK*LMoLQQGTAVDISSAGQT gi|545841000

PREDICTED: poly [ADP-ribose] polymerase 6 isoform X10

PREDICTED: acyl-CoA dehydrogenase family member 9, mitochondrialgi|545863823

like

EGKIPNETLEKLK*

13.37

3

102

37.29

gi|545867651

PREDICTED: 6-phosphofructokinase, liver type

AMoDEKR*FDEAIQLR

10.28

2

366

35.43

gi|545870350

PREDICTED: probable E3 ubiquitin-protein ligase HERC4

VPSCLPK*IMGIDTLVR

15.8

3

133

15.76

gi|298104070

transmembrane protein 38B

GAGGSIITNFELLVK*

15.85

3

166

26.92

gi|311247798

PREDICTED: olfactory receptor 5AP2-like

KVLHK*QIL

16.96

2

314

15.14

gi|311259696

PREDICTED: LOW QUALITY PROTEIN: desmoplakin

SQLQISNNR*

11.33

3

1770

43.14

K*LKSTIQR

17.1

3

780

34.24

12.45

4

720

-22.73

PREDICTED: regulating synaptic membrane exocytosis protein 1 isoform gi|545798332

X6

QLSNPGLVIAVVLVMoALTIYYLNAT gi|545803416

PREDICTED: transmembrane channel-like 1 isoform X1 [Susscrofa]

AK*GQK


28

Page 29 of 35


gi|545883649

PREDICTED: MAGUKp55 subfamily member 6 isoform X2

EAGLKFSKGEILQIVNR*

27.37

4

321

2.51

gi|545833251


MoDLLAEQRK*

13.54

3

116

44.87

gi|545890348


LPLESFRISTVK*

14.33

4

3740

28.34

476 477 478

479 480 481 482

Figure1.Representative spectra of RosA-modified peptides on (A) C: LEDEC*SELK (B)K: KIK*ELQAR (C) R: ASVEQLQKIR*(D) H: VAPEEH*PVLLTEAPLNPK

483

* = RosA Adduction


29


Page 30 of 35

HO

O

O

HO

oxidation

O O

HO

HS

OH

+

OH

O

HO

OH

OH OH

O

NH2

OH

Rosmarinic acid

O

OH

Cysteine

HO

O O

HO

H2N

OH

OH

Rosmarinic acid

oxidation

OH NH2

O O

HO

OH OH

O

NH2

+

OH

O

S

RosA-Cys adduct

O

HO

O

OH

Lysine

OH

O

NH

RosA-Lys adduct

OH NH2

HO

HO

O O

HO

NH2

OH OH

O

+

HN

O

O OH oxidation

NH

O

HO

OH OH

O

NH2

OH

OH

Arginine

Rosmarinic acid

RosA-Arg adduct

NH

HN HO

HO

O O

HO O

OH OH

Rosmarinic acid

N

+ N H

OH

oxidation

NH

O

HO

OH OH

O

NH2

Histidine

OH NH2

O

O OH

O

OH

RosA-His adduct

N

NH2 OH

N

484

O

485

Figure 2.Model adduct reactions between RosA and specific amino acids(cysteine, lysine,

486

arginine and histidine) on myofibrillar proteins.

487


30

Page 31 of 35


1400 Addcution peptides containing special amino acids Total peptides containing special amino acids 1200

Number

1000

800

600

400

200

0 N-terminal

488

C

H

R

K

Special amino acids

489

Figure3. Distribution of total peptides and adducted peptides separately on the sites of N-

490

terminal, Cys, His, Arg and Lys.

491


31


25

Page 32 of 35

Addcution peptides containing special amino acids Addcution peptides on special amino acids

Relative content (%)

20

15

10

5

0 N-terminal

492

C

H

R

K

Special amino acids

493

Figure4. Ratios of adducted peptides containing *special amino acids or reaction on *special

494

amino acids separately on the sites of N-terminal, Cys, His, Arg and Lys.

495

*Special amino acidsrepresent eachamino acid below the column separately.

496


32

Page 33 of 35


Number of adduction site on N-terminal Number of adduction site on C Number of adduction site on H Number of adduction site on R Number of adduction site on K R adduction site 37.66%

29 H adduction site 5.19%

4

C adduction site 3.90%

3 2

39

N-terminal addcution site 2.60%

K adduction site 50.65%

497 498

Figure5.Distribution of adduction sites in total meat proteins. “2” is the amount of N-terminal

499

adduction sites, “3” is the amount of C adduction sites, “4” is the amount of H adduction

500

sites, ”29” is the amount of R adduction sites and “39” is the amount of K adduction sites.


33


Page 34 of 35

Proteins carrying>=4 adduction sites Proteins carrying 3adduction sites Proteins carrying 2 adduction sites Proteins carrying 1adduction site

2 adduction sites proteins 14.93%

10

1 2

54

3 adduction sites proteins 1.49% >=4 adduction sites proteins 2.99%

1 adduction site proteins 80.60%

501 502

Figure 6. Distribution of adducted meat proteins carrying different numbers of adduction sites.

503

“1”, “2” and “3” are the adducted proteins carrying 1,2 and 3 adduction sites, respectively.“>=4”

504

is the adducted proteins carryingfour or more adduction sites.

505 506

Figure 7. RosA-adducted site motif in TMPs.


34

Page 35 of 35


507

Graphic for table of contents

508

Adducts between rosmarinic acid (RosA) and meat proteins were identified.

509

RosA adducted with different amino acids and adduction ratio was analyzed.

510

Mechanism of Adduction sites in adducted proteins was analyzed.

511

512


35

Identification of Rosmarinic Acid-Adducted Sites in ... - ACS Publications

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