TABLE AND FIGURE LEGENDS · Web viewProteins were fixed in 50% ethanol, 10% acetic acid and 40% double distilled reverse osmosis water for 30 min followed by two repeats of 10 min

Proteomic investigation of protein profile changes and amino acid

residue-level modification in cooked lamb longissimus thoracis et

lumborum: the effect of roasting

Supplementary data 1

Tzer-Yang Yu,1,2,* James D. Morton,2 Stefan Clerens,1 and Jolon M. Dyer1-4

1 Food & Bio-Based Products, AgResearch Lincoln Research Centre, Private Bag 4749,

Christchurch, 8140, New Zealand

2 Wine, Food & Molecular Biosciences, Lincoln University, Faculty of Agriculture and Life

Sciences, PO Box 85084, Canterbury, 7647, New Zealand

3 Riddet Institute, Massey University, Palmerston North, 4442, New Zealand

4 Biomolecular Interaction Centre, University of Canterbury, Private Bag 4800, Christchurch,

8140, New Zealand

Corresponding Author

*Tel.: +64-3-321-8814. E-mail address: [email protected] .

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mailto:[email protected]

S1. SUPPLEMENTARY EXPERIMENTAL SECTION

S1.1. Protein Fixing and Staining Procedures for SDS-PAGE gels

Proteins were fixed in 50% ethanol, 10% acetic acid and 40% double distilled reverse osmosis

water for 30 min followed by two repeats of 10 min rinsing in Milli-Q water. Gels were then

stained with the organic solvent-free Coomassie Blue G-250 solution for 80 min (Lawrence &

Besir, 2009) at room temperature. Gel destaining was done in Milli-Q water for 40 min with

Kimwipes (Kimberly-Clark) with one change of water. After excision of the preparative gel

lanes, analytical lanes were stained with colloidal Coomassie Blue G-250 (Candiano et al.,

2004) overnight and then destained with Kimwipes and Milli-Q water. Gel images were taken

using a Nikon D100 digital camera over a light box. Images of the destained gels prior to

excision of the preparative gel lanes are shown in Figures S1-2 to S1-4.

S1.2. Data analysis

S1.2.1. Mascot Search Parameters, the Peptide and Protein Score Threshold for the Creation

of a Small Sequence Database

Peak lists were searched against the combined Ovis aries sequence database (Yu, Morton,

Clerens & Dyer, 2015) using a local Mascot server (Matrix Science) version 2.4. The search

parameters were: Enzyme specificity was set to semi-trypsin with up to two missed cleavages

for trypsin digested samples; for gel samples, Fixed modification was propionamide (C) and

Variable modifications were deamidation (NQ), hydroxylation (KP) and oxidation (M); for

in-solution trypsin digested samples, Fixed modification was carbamidomethylation (C) and

Variable modifications were the same as those of the gel samples except

carbamidomethylation (N-term) was also included; Monoisotopic; MS error tolerance, 0.3 Da;

MS/MS error tolerance, 0.6 Da. Each protein identification was required to contain at least

one peptide with a Mascot score greater than the Mascot identity score at a Significance

Threshold of p <0.05. Peptide rank cut-off was set to 1. Peptides with scores ≤30 or lengths

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below six residues were all rejected. Protein score threshold was set to 45 for gel samples and

46 for in-solution trypsin digested samples, respectively.

S1.2.2. The Mascot Search Approaches for Protein Identification

For the Percolator post-processed searches, each set of peak lists from the triplicate samples

of control or a specific cooking time point that underwent the same sample preparation

workflow (e.g., GeLC) was merged into single files using the ProteinScape software v3.1

(Bruker Daltonics). Merged peak lists were searched against the shortlist database using the

Mascot server with the same parameters described above except: the Mascot Percolator option

was activated; each protein identification was required to contain at least one peptide with a

posterior error probability (PEP) <0.01; peptides with a PEP <0.05 or lengths below eight

residues were all rejected. False discovery rate (FDR) estimation was based on searches

against a decoy database of reversed protein sequence. The search results showed that the

false discovery rate (FDR) of the Percolator post-processed searches were all below 1.0% at

PEP <0.05 except one, which had no hit out of the decoy searches (Table S1-6). At PEP

<0.01, two datasets had an FDR below 0.1% while the rest had no hit out of the decoy

searches (Table S1-6). Protein identification results were compiled into a minimal list using

the “Queries” function of ProteinScape.

For the standard Mascot search, original, unmerged peak lists were searched against the

shortlist database without activating the Percolator. The search parameters were: no enzyme

specificity; for gel samples, Fixed modification was propioamide (C) and Variable

modifications were deamidation (NQ), glutamine to pyro-glutamate (N-term), hydroxylation

(P) and oxidation (M); for in-solution trypsin digested samples, Fixed modification was

carbamidomethylation (C) and Variable modifications were the same as those of the gel

samples except carbamidomethylation (N-term) was also included; Monoisotopic; MS error

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tolerance, 0.3 Da; MS/MS error tolerance, 0.4 Da. Peptides lengths below six residues were

all rejected. Peptide rank cut-off was set to 1. Mascot ion score and Protein score threshold

were set to 40 or 45, respectively. Each protein identification was required to contain at least

one peptide with a Mascot score greater than the Mascot identity score at a Significance

Threshold of p <0.05. Peptide identifications derived from non-collagenous proteins carrying

hydroxylation (P) were excluded for further analysis.

S1.2.3. The Manual Evaluation Procedure for Peptide IDs Carrying a Modification of

Interest

Search results of individual LC-MS/MS runs encompassing the 16 groups of modifications

were imported into Excel and organised by an in-house developed Excel add-in. Mascot

and/or ProteinScape annotated b and y ion peaks as well as those with an ammonia loss (-17

Da) or a water loss (-18 Da) were used to assess the quality of a peptide-spectrum match.

Peptide identification entries (peptide IDs) exhibiting one of the features listed below were

rejected from further analysis:

1) Those with a name containing “keratin”

2) Those with an ion score <40

3) Those with a modified N or C-terminal residue (except for carbarmidomethylated ones)

whose modification exhibits a monoisotopic mass close (±1 Da) to that of the flanking

residue, e.g., HNE (+156.12 Da) and arginine residue (+156.10 Da)

4) Those with ≥ four variable modifications. It should be noted that in this analysis

procedure, the following Variable modifications were not regarded as a “variable

modification”: N-terminal carbarmidomethylation; N-termimal carbarmidomethylation of

C plus N-termimal b-aminoalanine of C, as the combination results in a +40 Da mass

shift that is identical to a known artefact (Geoghegan, Hoth, Tan, Borzilleri, Withka &

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Boyd, 2002); hydroxylation of K or P in collagens; one deamidation of N or Q per

sequence, because they can be either known artefacts or biologically endogenous.

5) Those derived from non-collagenous proteins but carrying a hydroxylation of K and/or P

6) Those derived from a protein that did not meet the protein identification criteria described

previously.

The remaining peptide IDs containing only singly oxidised methionine residue (methionine

sulphoxide) plus the above-mentioned “variable modifications” that were not regarded as a

variable modification (if any) were accepted. Unmodified peptides (defined below)

corresponding to the modified peptides of interest in terms of sequence were also sought as

supporting evidence.

The rest of the candidate peptide IDs with a modification of interest satisfying the following

criteria were accepted:

1) When redundant IDs arising from the same MS/MS spectrum, those with a higher

number of variable modifications within a three ion score margin were rejected.

Otherwise, the criteria shown below were applied to retain only one entry at most,

2) Assignment of major peaks by Mascot or ProteinScape

3) Occurrence of at least three consecutive amino acids

4) Containing peaks that indicate mass shift corresponding to the mass of a modification of

interest, unless the modified residue reported was located at N- or C-terminus and was

not K or R,

5) To support a candidate ID with a variable modification at N- or C-terminus, at least three

unique b- (or those with an ammonia or a water loss) or y- (or those with an ammonia or

a water loss) peaks were required, respectively,

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6) When a modification of interest was reported to locate at N- or C-terminus of a peptide

ID, the ID shall contain no other variable modifications except up to two deamidated

(NQ) and/or one methionine sulphoxide. In the case of collagen derived-peptides,

hydroxylation (KP) was also allowed,

7) Supported by the presence of an unmodified peptide corresponding to the sequence of a

candidate ID. In this context, an “unmodified” peptide also includes:

A. That with a partial sequence longer than six amino acid residues when the reported

modified peptide ID of interest was miss-cleaved,

B. A peptide ID carrying only methionine sulphoxide, deamidation (NQ) and/or the

above-mentioned “variable modifications” that were not regarded as a variable

modification.

S1.2.4. Mascot Percolator post-processed searches for protein modifications

In order to provide larger population of MS/MS spectra for Mascot Percolator post-processed

searching, each set of peak lists from the triplicate in-soluble samples of control or a specific

cooking time point were merged into single files using ProteinScape. Peak lists corresponding

to the section one to eight (counting from the top of a gel) of a single gel lane were also

merged into one file. The ninth sections were omitted because they seemed to contain none to

a very small number of recognisable MS/MS spectra related to meat protein, as suggested by

the standard Mascot search results.

The peak lists were searched against the shortlist database (the database refers to Section 2.8.1

of the main text; The search parameters were: Enzyme specificity was set to trypsin with up to

two missed cleavages; Monoisotopic; for gel samples, Fixed modification was propionamide

(C) and Variable modifications were deamidation (NQ), hydroxylation (P), oxidation (M) and

one additional modification from one of the following: single oxidation of C, F, H, W or Y;

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double oxidation of C, F, H, M, P, W or Y; trioxidation of C, W or Y; γ-glutamic

semialdehyde (R), pyrraline (K), quinone of W or Y), PhIP (C), MG-H1 (R),

malondialdehyde (K), kynurenine (W), hydroxykynurenine (W), HNE (K), GH-1 (R),

dehydroalanine of C or Y, dehydration of T or S, CML, CEL, CMA, argpyrimidine (R),

acetyl (K), aminoadipic acid (K); for in-solution trypsin digested samples, Fixed modification

was carbamidomethylation (C) and Variable modifications were the same as those of the gel

samples except carbamidomethylation (N-term) was also included; MS error tolerance, 1.3

Da; MS/MS error tolerance, 0.3 Da; peptide rank cut-off, one. Peptide IDs longer than eight

residues with a PEP <0.05 were accepted for further analysis. Protein score (after Mascot

Percolator adjustment) threshold was 20.

The purpose of this additional database search was to cover more modified peptides that

might not have been picked up by the standard Mascot search strategy (Section S1.2.3). The

mass error tolerance was expanded to cover potential peptides that were assigned with a

monoisotopic peak within the isotopic distribution. This was the reason why the modifications

of 1 Da were not compared between samples in this study. The enzyme specificity was

changed to “trypsin” from “semi-trypsin” because in some cases the Percolator assessment

appeared to become more stringent when “semi-trypsin” was used as opposed to “trypsin”, as

indicated by the numbers of peptides identified above a given PEP threshold. The high

stringency was not required in this case since the manual evaluation would be applied.

The search results were compiled and manually assessed as indicated in Section S1.2.3.

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TABLE AND FIGURE LEGENDS

Table S1-1 – Record of the meat roasting experiments.

Table S1-2 – Amount of trypsin used for a gel slice based on the stain intensity.

Table S1-3 – Dilution scheme used for a gel slice based on the stain intensity.

Table S1-4 – Dilution schemes for GeLC.

Table S1-5 – List of additional Variable modifications that were divided into 16 groups, each

group for one Mascot search. These modifications were known potential heating

and/or reactive oxygen species-induced modifications.

Table S1-6 – False discovery rate (FDR) of the Mascot Percolator post-processed searches for

protein identification (Section S1.1.2).

Table S1-7 – Percentage of semi-tryptic and non-tryptic peptides: GeLC.

Table S1-8 – Percentage of semi-tryptic and non-tryptic peptides: meat homogenate residue

proteomic samples.

Table S1-9 – A summary of the five regions across the SDS-PAGE gel lanes that showed

distinct changes in banding patterns in response to roasting, along with the

GeLC results that are potentially related to these changes (refer to Section 3.1.1

of the main text).

Fig. S1-1 – Meat patty samples prepared for roasting.

Fig. S1-2 – SDS-PAGE gel image prior to slicing for GeLC: cooking replicate 1.



Fig. S1-5 – The data analysis strategy for protein identification and identifying peptides with

modification of interest.

Fig. S1-6 – Images of the analytical SDS-PAGE gel lanes for cooking replicates 2 and 3.

Fig. S1-7 – Supernatants and insoluble residues from the control and roasted meat

homogenates.

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Table S1-1 – Record of the meat roasting experiments.

Date TreatmentCooking replicate #

Time of meat thawing in the food grade polypropylene container (min)

Meat wet weight (g)

Approx. handling time at room temperature between the end of thawing and the start of cooking (X min)

Approx. handling time at room temperature between the end of thawing and immersion into the liquid nitrogen (X min)

Approx. time to reach back to an oven temperature of 190°C after placing the meat (sec)

Approx. handling time at room temperature between the end of cooling and immersion into the liquid nitrogen (min)

Meat cooked weight (g)

12/03/2014 10 min 1 30 0.60 15 min < X < 20 min N/A 28 1.083 0.15

28/03/2014 10 min 2 30 0.60 15 min < X < 20 min N/A 19 0.67 0.15

29/03/2014 5 min 1 30 0.61 15 min < X < 20 min N/A 23 0.83 0.18

31/03/2014 10 min 3 30 0.61 15 min < X < 20 min N/A 40 2.85 0.15

31/03/2014 0 mina 1 30 0.59 N/A 15 min < X < 20 min N/A N/A N/A

1/04/2014 5 min 2 30 0.59 15 min < X < 20 min N/A 26 3 0.17


2/04/2014 5 min 3 30 0.60 15 min < X < 20 min N/A 32 4.9 0.19


a ”0 min” means a control.

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Table S1-2 – Amount of trypsin used for a gel slice based on the stain intensity.

Trypsin (µg) used for digestion

Stain intensity (%)

0.2 <50.3 5-9.90.4 10-17.90.5 18-21.9

Table S1-3 – Dilution scheme used for a gel slice based on the stain intensity.

Stain intensity (%) Dilution

<13 undiluted (undil.)13 undil.14 1 in 215 1 in 316 1 in 417 1 in 518 1 in 619 1 in 8

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Table S1-4 – Dilution schemes for GeLC.

Cooking replicate 1

Control (0 min) 5 min 10 min

Slice #Dilution scheme

Stain intensity (%) Slice #

Dilution scheme


Dilution scheme

Stain intensity (%)

1 undil. 10.116 1 undil. 12.348 1 undil. 12.1952 undil. 13.232 2 1 in 4 16.376 2 undil. 7.3953 1 in 3 14.76 3 undil. 12.335 3 undil. 5.5784 undil. 12.66 4 undil. 10.85 4 undil. 7.5425 1 in 3 15.361 5 1 in 3 15.262 5 undil. 12.0286 1 in 3 14.868 6 1 in 2 14.244 6 undil. 14.4137 undil. 10.096 7 undil. 10.757 7 undil. 15.098 undil. 6.223 8 undil. 4.731 8 undil. 16.8929 undil. 2.685 9 undil. 3.097 9 undil. 8.866

Total 100.0 100.0 100.0Cooking replicate 2




Dilution scheme


Dilution scheme

Stain intensity (%)

1 undil. 11.955 1 undil. 13.015 1 undil. 13.8942 1 in 2 13.685 2 1 in 4 15.957 2 undil. 9.183 1 in 2 14.133 3 undil. 11.998 3 undil. 7.8664 undil. 12.482 4 undil. 11.661 4 undil. 8.4435 1 in 2 14.337 5 1 in 3 14.525 5 undil. 11.0196 1 in 2 13.582 6 undil. 13.293 6 undil. 13.328

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7 undil. 9.856 7 undil. 10.2 7 undil. 14.5658 undil. 6.08 8 undil. 5.778 8 undil. 15.4069 undil. 3.892 9 undil. 3.571 9 undil. 6.299

Total 100.0 100.0 100.0

Cooking replicate 3




Dilution scheme


Dilution scheme

Stain intensity (%)

1 undil. 7.645 1 undil. 10.581 1 undil. 6.8942 1 in 4 15.975 2 1 in 6 17.765 2 undil. 5.4533 1 in 2 13.756 3 undil. 11.335 3 undil. 5.2784 undil. 12.789 4 undil. 11.431 4 undil. 7.5185 1 in 6 17.54 5 1 in 5 17.458 5 undil. 11.7086 undil. 11.364 6 undil. 10.901 6 undil. 14.5547 undil. 10.107 7 undil. 9.868 7 undil. 18.7228 undil. 6.327 8 undil. 5.964 8 undil. 20.4069 undil. 4.496 9 undil. 4.697 9 undil. 9.466

Total 100.0 100.0 100.0

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Table S1-5 – List of additional Variable modifications that were divided into 16 groups, each group for one Mascot search. These modifications were

known potential heating and/or reactive oxygen species-induced modifications.

Modification Residue Position Formula Monoisotopic mass (Da)

Any amino acid residue to which the mass shift is identical

References Group

Acetyl any N-term H(2) C(2) O 42.010565 no Bienvenut et al. (2012) 1

b-aminoalanine C any H N S(-1) -16.961172 no Van Lancker et al. (2011) 1

Carboxymethyl K any C(2) H(2) O(2) 58.005479 noMeltretter et al. (2013); Sun et al. (2015)

1

Dehydroalanine Y any C(-6) H(-6) O(-1) -94.041865 no Unimod accession #400 1

Imidazolone B R any C(6) H(6) O(4) 142.026609 no Bhonsle et al. (2012) 1

Carboxyethyl K any C(3) H(4) O(2) 72.021129 no Ahmed et al. (1997) 2

Dehydration S any H(-2) O(-1) -18.010565 noWada & Lönnerdal (2014)

2

Dehydration T any H(-2) O(-1) -18.010565 no Unimod accession #23 2

Glu→pyro-Glu E N-term H(-2) O(-1) -18.010565 noHaitinger (1882); Liu et al. (2011)

2

Methylglyoxal-derived hydroimidazolone (MG-H1) R any C(3) H(2) O 54.010565 no

Unimod accession #859; Meltretter et al. (2013); Frolov et al. (2014)

2

Dioxidation FWY any O(2) 31.989829 no Dyer et al. (2009); 3

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Grosvenor et al. (2011); Deb-Choudhury et al. (2014)

Malondialdehyde K any C(3) H(2) O 54.010565 noUnimod accession #319; Estévez (2011)

3

Malondialdehyde R any C(3) H(2) O 54.010565 noUnimod accession #319; Estévez (2011)

3

Carboxymethyl R any C(2) H(2) O(2) 58.005479 no Arena et al. (2014) 4

Didehydro T any H(-2) -2.01565 noStadtman & Levine (2003)

4

Glyoxal-derived hydroimidazolone (G-H1)

R any C(2) O 39.994915 no Arena et al. (2014) 4

Hexose (Amadori compound) any N-term Hexose 162.052823 noArena et al. (2014); Guedes et al. (2009)

4

Formyl K any C O 27.994915 no Arena et al. (2014) 4

Argpyrimidine R any C(5) H(4) O 80.026215 no Bhonsle et al. (2012) 5

Aminoadipic Acid (AAA) K any H(-3) N(-1) O(2) 14.96328 noEstévez (2011); Fan et al. (2009)

5

Nitration (Nitro) FHW any H(-1) N O(2) 44.985078 no Unimod accession #354 5

3-deoxyglucosone (a.k.a. imidazolone A)

RK any C(6) H(8) O(4) 144.042259 no Unimod accession #949; Niwa et al. (1997); Bhonsle et al. (2012);

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Meltretter et al. (2013)

Glycolaldehyde-pyridine K any C(6) H(5) O(2) 109.028954 no Arena et al. (2014) 6

Quinone W any H(-2) O(2) 29.974179 no Grosvenor et al. (2010) 6

Quinone Y any H(-2) O(2) 29.974179 noDeb-Choudhury et al. (2014)

6

Acetyl K any H(2) C(2) O 42.010565 no Arena et al. (2014) 7

Pyrraline K any C(6) H(4) O(2) 108.021129 noUnimod accession #886; Hebling et al. (2013)

7

Oxidation HW O 15.994915 no Grosvenor et al. (2011) 7

4-hydroxy-2-nonenal (HNE) C any C(9) H(16) O(2) 156.11503 arginine Unimod accession #53 8

4-hydroxy-2-nonenal (HNE) H any C(9) H(16) O(2) 156.11503 arginine Suman et al. (2007) 8

4-hydroxy-2-nonenal (HNE) KR any C(9) H(16) O(2) 156.11503 arginineUnimod accession #53; Bernevic et al. (2011)

8

Oxidation Y any O 15.994915 no Grosvenor et al. (2011) 8

1-alkyl-2-formyl-3,4-glycosyl-pyrrole (AFGP) KR any C(12) H(14) O(7) 270.073953 no

Bhonsle et al. (2012); Arena et al. (2014)

9

Dehydroalanine C any H(-2) S(-1) -33.987721 noUnimod accession #368; Rombouts et al. (2010)

9

Kynurenine W any C(-1) O 3.994915 no Grosvenor et al. (2011) 9

Nitration (Nitro) Y any H(-1) N O(2) 44.985078 no Unimod accession #354 9

16

γ-glutamic semialdehyde (GGS) R any C(-1) H(-5) N(-3) O -43.053433 no

Estévez (2011); Roldan et al. (2014)

10

Dioxidation P any O(2) 31.989829 no Unimod accession #425 10

Hex (Amadori compound) K any Hexose 162.052823 noArena et al. (2014); Frolov et al. (2006)

10

N-fructosyl-lysine – 2H2O K any C(6) H(6) O(3) 126.031694 noBhonsle et al. (2012); Arena et al. (2014)

10

Trioxidation C any O(3) 47.984744 no

Jeong et al. (2011); Meltretter et al. (2013); Meltretter et al. (2014)

10

Carboxymethyl C any C(2) H(2) O(2) 58.005479 no Unimod accession #6 11

Hexose R any Hexose 162.052823 no Unimod accession #41 11

N-glycerinyl-lysine K any C(3) H(4) O(3) 88.016044 no Arena et al. (2014) 11

Oxidation F any O 15.994915 noDyer et al. (2009); Deb-Choudhury et al. (2014)

11

Pro→pyro-Glu P any H(-2) O 13.979265 noUnimod accession #359; Guedes et al. (2010)

11

Aldehyde (Met) M any C(-1) H(-4) O S(-1) -32.008456 no Xu & Chance (2007) 12

Carbonyl KR any H(-2) O 13.979265 no Xu & Chance (2007) 12

Dihydroxyimidazolidine R any C(3) H(4) O(2) 72.021129 no Unimod accession #830; Meltretter et al. (2013);

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17

Frolov et al. (2014); Meltretter et al. (2014)

Gln→pyro-Glu Q N-term H(-3) N(-1) -17.026549 no

Deb-Choudhury et al. (2014); Van Lancker et al. (2011)

12

Ornithine R any C(-1) H(-2) N(-2) -42.021798 noArena et al. (2014); Frolov et al. (2014)

13

2-amino-3-methylimidazo[4,5-f]quinolone (IQ; a heterocyclic amine adduct)

C any C(11) H(8) N(4) O 212.069811 no Turesky et al. (1987) 13

N-(2-hydroxy-dioxopentyl)-lysine K any C(5) H(6) O(3) 114.031694 asparagine Arena et al. (2014) 13

N-acetoxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP, a heterocyclic amine adduct)

any C-term C(13) H(10) N(4) O(-1) 206.095632 no Kataoka et al. (2010) 13

PhIP C any C(13) H(10) N(4) 222.090546 no Chepanoske et al. (2004) 13

Dioxidation H any O(2) 31.989829 no N/A 14

Hydroxykynurenine W any C(-1) O(2) 19.989829 no Grosvenor et al. (2011) 14

N-acetoxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP, a heterocyclic amine adduct)

DE any C(13) H(10) N(4) O(-1) 206.095632 no Kataoka et al. (2010) 14

Ring open of His (+5 Da) H any C(-1) H(-1) N(-1) O(2) 4.97893 no Xu & Chance (2007) 14

Lys→Allysine (AAS) K any H(-3) N(-1) O -1.031634 noEstévez (2011); Roldan et al. (2014)

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Oxidation C any O 15.994915 noUnimod accession #35; Jeong et al. (2011)

15

Oxidation R any O 15.994915 no Unimod accession #35 15

Ring open of His (-10 Da) H any C(-1) H(-2) N(-2) O(2) -10.031969 no Xu & Chance (2007) 15

Trioxidation W any O(3) 47.984744 no Grosvenor et al. (2011) 15

α-ketoamide L N-term H(-3) N(-1) O -1.031634 noMeltretter et al. (2013); Meltretter et al. (2014)

16

Dioxidation CM any O(2) 31.989829 noMeltretter et al. (2013); Meltretter et al. (2014)

16

19

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Table S1-6 – FDR of the Mascot Percolator post-processed searches for protein identification (Section S1.1.2).

At PEP <0.05

At PEP <0.01

Dataset Peptide matches # MS/MS spectra

in normal Database

in Decoy FDR

in normal Database

in Decoy FDR

Control GeLC cooking replicates 1-3

Above identity threshold 136045 9492 15 0.16% 6057 0 N/A

Above identity or homology threshold

9492 15 0.16% 6057 0 N/A

5 min boiling GeLC cooking replicates 1-3

Above identity threshold 137597 9919 28 0.28% 6756 3 0.04%


9919 28 0.28% 6756 3 0.04%

10 min boiling GeLC cooking replicates 1-3

Above identity threshold 126775 2216 0 N/A 1039 0 N/A


2216 0 N/A 1039 0 N/A

Control meat homogenate residue (in-solution digested) cooking


20

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replicates 1-3


849 1 0.12% 452 0 N/A

5 min roasting meat homogenate residue (in-solution digested) cooking replicates 1-3



1542 10 0.65% 1190 0 N/A

10 min meat homogenate residue (in-solution digested) cooking replicates 1-3

Above identity threshold 14014 1563 9 0.58% 1300 1 0.08%


1563 9 0.58% 1300 1 0.08%

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250

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Table S1-7 – Percentage of semi-tryptic and non-tryptic peptides: GeLC.

Samples

# qualified semitryptic peptide IDsa

# qualified non-tryptic peptide IDs

# qualified peptide IDs (all types) for the semistryptic peptide stat

% # qualified semi-tryptic peptide IDs/# qualified peptide IDs

% # qualified non-tryptic peptide IDs/# qualified peptide IDs

Control Replicate 1 29 6 943 3.1% 0.64%

(0 min) Replicate 2 30 3 948 3.2% 0.32%

Replicate 3 27 1 939 2.9% 0.11%

Mean 29 3 943 3.0% 0.35%

Stad. Dev. 2 3 5 0.1% 0.27%

5 min Replicate 1 30 8 864 3.5% 0.93%

Replicate 2 31 6 877 3.5% 0.68%

Replicate 3 27 2 967 2.8% 0.21%

Mean 29 5 903 3.3% 0.61%

Stad. Dev. 2 3 56 0.4% 0.37%

10 min Replicate 1 8 3 187 4.3% 1.60%

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Replicate 2 6 0 167 3.6% 0.00%

Replicate 3 5 1 169 3.0% 0.59%

Mean 6 1 174 3.6% 0.73%

Stad. Dev. 2 2 11 0.7% 0.81%a Peptides with the following properties are not true semi-tryptic and thus not included: 1) Carbamidomethyl (N-term) and a G flanking its N-termimus

plus a C-terminal K/R; 2) Protein N-term plus a C-terminal K/R.

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Table S1-8 – Percentage of semi-tryptic and non-tryptic peptides: meat homogenate residue proteomic samples.

Samples# qualified semitryptic peptide IDsa

# qualified non-tryptic peptide IDs

# qualified peptide IDs (all types) for the semistryptic peptide stat

% # qualified semi-tryptic peptide IDs/# qualified peptide IDs

% # qualified non-tryptic peptide IDs/# qualified peptide IDs

Control Replicate 1 4 0 189 2.1% 0.00%

(0 min) Replicate 2 5 0 193 2.6% 0.00%

Replicate 3 1 0 163 0.6% 0.00%

Mean 3 0 182 1.8% 0.00%

Stad. Dev. 2 0 16 1.0% 0.00%

5 min Replicate 1 4 0 220 1.8% 0.00%

Replicate 2 2 1 228 0.9% 0.44%

Replicate 3 4 0 222 1.8% 0.00%

Mean 3 0 223 1.5% 0.15%

Stad. Dev. 1 1 4 0.5% 0.25%

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10 min Replicate 1 5 1 218 2.3% 0.46%

Replicate 2 7 0 176 4.0% 0.00%

Replicate 3 5 0 170 2.9% 0.00%

Mean 6 0 188 3.1% 0.15%

Stad. Dev. 1 1 26 0.8% 0.26%

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Table S1-9 – A summary of the five regions across the SDS-PAGE gel lanes that showed distinct changes in banding patterns in response to roasting,

along with the GeLC results that are potentially related to these changes (refer to Section 3.1.1 of the main text). Note the GeLC results were obtained

from gel sections whose approximate positions were indicated by the red marks on the left hand side of the images.

Image of the representative gel (refer to Fig. 1 in the main text for a larger, untrimmed version; the images of the other replicates were given in Fig. S1-6)

Approx. position in a gel lane

Changes observed (refer to Table S2-1 of Supplementary data 2 for the GeLC results)

Interpretation of the changes observed

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286

1: top gel section/circled by the red box

A. The 5 min samples showed higher stain intensity on the very top of the gel lanes as well as tinge of high Mr smearing. These samples had obviously more peptide IDs derived from myosin heavy chains but around half less from sarcoplasmic/endoplasmic reticulum calcium ATPase 1 isoform 1 compared to the controls.

B. At 10 min, no sarcoplasmic/endoplasmic reticulum calcium ATPase 1 isoform 1 and only a few myosin heavy chain-derived peptides were identified in this gel section.

C. The 5 min and 10 min samples contained more actin-derived peptides in this region than the control samples.

A. The myosin heavy chain probably had undergone protein aggregation/polymerisation due to heating whereas the reduction of the ATPase-derived peptide IDs might be caused by unaccounted protein modification or by an increased competition for data-dependent acquisition when the level of the myosin peptides rose.

B. This could probably be explained by the decreased extractability due to increased cross-link formation.

C. This indicated the protein was also involved in the heat-induced aggregation/polymerisation.

2: Gel section 2/ circled by the orange box

A. The only thick band above the 250 kDa marker in the controls became fainter at 5 min and disappeared at 10 min of roasting. Notable reduction in titin-derived peptides was seen in the gel section at 5 min roasting. At 10 min, most of the titin peptide IDs have gone from the entire gel lanes.

A. This finding is likely to be related to titin, the third most abundant muscle after actin and myosin (Clark, McElhinny, Beckerle & Gregorio, 2002), based on the GeLC results. It is uncertain whether the “lost” titin had turned into aggregate and/or fragments at 5 min roasting, according to the number of peptide IDs in each gel slice.

The lower protein load of the 10 min samples could contribute to the lower number of peptide IDs but this can also be viewed as a direct consequence of the loss of protein extractability in the urea-thiourea solution, as already discussed.

Titin has been shown using SDS-PAGE and Western blots to be more susceptible to aggregation and truncation when heating beef at 73 °C compared to the other major myofibrillar proteins,

27

e.g., the myosin heavy chain (Fritz, Dietrich & Greaser, 1992). In that study, the fragments as well as the high Mr aggregate of titin were both observed at as early as 15 min of the heating.

3: Gel section 3/ circled by the yellow box

A. A thick intense band at an estimated Mr of 135 kDa in control turned into a few thin bands at 5 min. For the control and the 5 min samples, the most abundant protein found in this gel section was myosin heavy chains.

A. The GeLC results from the cooking replicates 2 and 3 indicated the protein may be involved in this band quality change as at least 30 more peptides were found in the control. It is possible that some of the myosin heavy chain in this region at 5 min had aggregated/polymerised and gone to the top of the gel lane, where more myosin heavy chain peptides were found compared to that of the control, as already mentioned. This difference was not obvious in the cooking replicate 1 probably because the gel slice seemed to have incorporated some part of the conspicuous myosin heavy chain band between the 150 kDa and 250 kDa markers.

4: Gel section 4/ circled by the green box

A. The many distinct fine bands near the 75 kDa marker in control were turned to a few bands at 5 min roasting.

A. It appeared myosin heavy chains were again involved in the change as at least 11 more peptides in average were found in the controls than in the 5 min samples.

5: Gel section 8/ circled by the blue box

A. Several not so well defined, faint bands in the controls below the 15 kDa marker diminished at 5 min cooking.

A. Candidates that may account for this finding included myosin heavy chains, pyruvate kinase isozymes M1/M2 isoform 4 (theoretical Mr = 58 kDa), beta-enolase (theoretical Mr = 47 kDa), fructose-bisphosphate aldolase A isoform 1 (theoretical Mr = 39.4 kDa), L-lactate dehydrogenase A chain isoform 1 (theoretical Mr = 36.6 kDa) and histone H4-like (theoretical Mr = 11.4 kDa). The occurrence of these proteins, except the histone, in the low Mr gel section of the control samples was probably the result of post-mortem proteolysis.

At 5 min roasting, these protein fragments probably were involved in aggregation/polymerisation and/or lost as exudate during heating and not identified in the meat. At 10 min, these proteins, except the histone, re-appeared. This time their presence was probably a result of heat-induced protein truncation because the major proportion of the proteins were no longer extractable by this stage.

28

Fig. S1-1 – Meat patty samples prepared for roasting.

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288289

290291292293294295

Control5 min10 min

Preparative lanes

Control 5 min 10 min

Analytical lanes

MW

Fig. S1-2 – SDS-PAGE gel image prior to slicing for GeLC: cooking replicate 1. MW, Mr marker.

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296

297

298

Control5 min10 min

Preparative lanes


Analytical lanes

MW


31

299

300

301

302

Control5 min10 min

Preparative lanes


Analytical lanes

MW


32

303

304

305

306

Fig. S1-5 – The data analysis strategy for protein identification and identifying peptides with modification of interest. Refer to Sections 2.8 (in the

main text) and S1.2 (in this document) for details.

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34

Fig. S1-6 – Images of the

analytical SDS-PAGE gel lanes for

cooking replicates 2 and 3. The left

image corresponds to cooking

replicate 2 and the right image,

cooking replicate 3. Red marks on

the left hand side of the image

indicate the approximate position

of the gel sections from the

preparative lanes sliced for

proteomic analysis. MW, Mr

marker.

311

312

Fig. S1-7. Supernatants and insoluble residues from the control and roasted meat

homogenates (refer to Section 2.4 of the main text). Top row: control (0 min) replicates.

Middle row: 5 min roasting replicates. Bottom row: 10 min roasting replicates. Left column:

supernatants. Right column: wet insoluble residues obtained from 350 µL of

homogenate/sample upon centrifugation (17810 xg, 15 °C, 20 min).

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Documents

TABLE AND FIGURE LEGENDS · Web viewProteins were fixed in 50% ethanol, 10% acetic acid and 40% double distilled reverse osmosis water for 30 min followed by two repeats of 10 min