MS Identification of Species-Specific Muscle Peptides in

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LC-MS/MS identification of species-specific muscle peptides in processed animal proteins Daniela Marchis, Alessandra Altomare, Marilena Gili, Federica Ostorero, Amina Khadjavi, Cristiano Corona, Giuseppe Ru, Benedetta Cappelletti, Silvia Gianelli, Francesca Amadeo, Cristiano Rumio, Marina Carini, Giancarlo Aldini, and Cristina Casalone J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04639 • Publication Date (Web): 10 Nov 2017 Downloaded from http://pubs.acs.org on November 10, 2017

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Journal of Agricultural and Food Chemistry

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LC-MS/MS Identification of Species-specific Muscle Peptides in Processed Animal Proteins

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Daniela Marchis *†┴, Alessandra Altomare ‡┴, Marilena Gili †, Federica Ostorero †, Amina Khadjavi

3

†

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Cristiano Rumio #, Marina Carini ‡, Giancarlo Aldini ‡, Cristina Casalone †

, Cristiano Corona †, Giuseppe Ru †, Benedetta Cappelletti §, Silvia Gianelli ‡, Francesca Amadeo ‡,

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†

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10154, Torino, Italy

8

‡

9

20133, Milano, Italy

Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle D’Aosta, via Bologna 148,

Department of Pharmaceutical Sciences, Università degli Studi di Milano, Via Mangiagalli 25,

10

§

11

#

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Trentacoste 2, 20134, Milano, Italy

Italian Ministry of Health, Viale Giorgio Ribotta 5, 00144, Roma, Italy Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Via

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*Corresponding

author:

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[email protected]

tel

+39

011

2686252;

fax

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ACS Paragon Plus Environment

+39

011

2686237;

e-mail:


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ABSTRACT

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An innovative analytical strategy has been applied to identify signature peptides able to distinguish

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among processed animal proteins (PAPs) derived from bovine, pig, fish and milk products.

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Proteomics was first used to elucidate the proteome of each source. Starting from the identified

31

proteins and using a funnel based approach, a set of abundant and well characterized peptides with

32

suitable physical-chemical properties (signature peptides) and specific for each source were

33

selected. An on-target LC-ESI-MS/MS method (MRM mode) was set-up using standard peptides

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and was then applied to selectively identify the PAP source and also to distinguish proteins from

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bovine carcass and milk proteins. We believe that the method described meets the request of the

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European Commission which has developed a strategy for gradually lifting the “total ban” towards

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“species to species ban”, therefore requiring official methods for species-specific discrimination in

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feed.

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KEYWORDS: Processed animal proteins (PAPs), Bovine spongiform encephalopathy (BSE),

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signature peptides, LC-QqLIT MS, bovine, pig, fish.

42 43 44 45 46 47 48 49 50 51 52


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INTRODUCTION

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Animal by-products are generally accepted as carcasses, hides, bones, meat trimmings, blood, fatty

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tissues, horns, feet, hoofs or internal organs. In the past, meat by-products, the main source for meat

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and bone meal, have been associated with exposure to the agent of bovine spongiform

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encephalopathy (BSE),

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specific legislation has been created regarding the handling and treatment of said by-products to at

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least partially inactivate the prions. 3 In particular, under European legislation animal by-products

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have been classified into three categories based on their potential risk to animals and the

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environment. Categories 1-3 can be disposed of by incineration or rendering followed by landfill

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disposal and only category 3 by-products may be used in the production of animal feedstuffs.

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Processed animal proteins (PAPs) are animal proteins derived entirely from category 3 material

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after specific regulated treatments. 3, 4

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In the EU, PAPs are subject to strict controls to avoid any possible exposure of ruminants to prions.

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An EU wide ban on the use of mammalian meat and bone meal for ruminants was first implemented

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in 1994, then, to further restrict the possibility of cross-contamination in feed production, in January

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2001 the feed ban was extended (“total feed ban”) to all farmed animals. 5-7 The latter measure has

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played a major role in counteracting the circulation of the BSE agent leading to the EU-wide

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decline of the BSE epidemic.

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Commission to develop an exit strategy through two subsequent TSE Road Maps: 10, 11 within TSE

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Road Map II a lift of the feed ban was expected to develop into a “species to species” ban, and since

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June 2013 the total feed ban of PAPs has been partially lifted, allowing the use of non ruminant

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PAPs in aquaculture. 12 The review of the total feed ban was envisaged as control tools were made

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available both for official and self-monitoring laboratories. EU Regulation 51/2013 13 lays down the

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official methods for determination of constituents of animal origin for the official control of feed.

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According to this regulation, banned PAPs can be detected by light microscopy and polymerase

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chain reaction (PCR). 13 Light microscopy identifies structures on the basis of their morphology and

1

the most known animal prion disease.

8, 9

2

In order to mitigate that risk,

3

The success in combatting the disease led the European



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enables identification of particles (such as bones, cartilages, muscle fibres, etc.),14-18 while PCR is

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able to detect and identify the presence of specific animal DNA in feed.19-21 Nevertheless, even

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combined, these methods sometimes do not succeed in determining the origin of the PAPs.

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Moreover, although PCR analysis can detect ruminant DNA, it is unable to discriminate between

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ruminant material from different origins (muscle and bones vs milk products, which are allowed to

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be used). Thus, there is the risk of obtaining positive ruminant PCR signals from these authorized

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materials.

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addition of milk products in feed could mask a possible presence of ruminant PAP, and leave the

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door open to potential fraud. As highlighted above, the presence of ruminant PAP in the feed chain

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could be of some concern being a known risk factor for BSE.

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Therefore, the recognition of species-specific markers for detection of PAPs, and tissue-specific

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markers for an intra-specific discrimination of milk and meat products is highly desirable.

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Moreover, both microscopy and PCR are qualitative methods. A tolerance level for PAP in feed for

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farmed animals would be of great interest due to its implications in an international commercial

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context. Different proteomics methodologies have been applied to study food products for species

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identification,

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contamination of pork and horse meat in beef matrix, using a shotgun proteomics approach to select

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specific biomarker peptides.25 Recently, some researchers have focused their interest specifically on

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PAPs. These are very heterogeneous and difficult matrices, due to the rendering treatments,

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according to Annex VIII Regulation (EC) 142/2011.4 Peptide biomarkers of bovine plasma, blood

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products and processed bovine proteins were identified using high-pressure liquid chromatography

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Fish feed can contain both non ruminant PAP and milk products. At present, the

23, 24

and mass spectrometry techniques have been used to recognize trace

24, 26

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coupled to electrospray ionization and tandem mass spectrometry in PAPs.

However, the

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bovine selected peptides were from haemoglobin α and heat shock protein β-1 from blood products.

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24

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become leaky, allowing for the paracellular transfer of blood components into milk and vice

Blood proteins can be present in milk, as during an immune response, the blood-milk barrier can


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versa.27,

28

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bovine heat stress response,29, 30 and somatic cells are widely known to be present in milk.

Heat shock proteins can be produced by mammary somatic cells, as involved in the

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In this study, proteomics was applied to identify signature peptides able to distinguish among

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PAPs derived from bovine, pig, fish and milk products. A liquid chromatography–tandem

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quadrupole-linear ion trap (LC–QqLIT MS) method was then developed in order to identify such

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signature peptides in PAPs and to confirm their selectivity. The proposed method was found

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suitable to distinguish among PAPs from different species and also to distinguish proteins for

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different tissues originating from the same species, as in the case of PAP from bovine carcass and

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milk proteins.

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MATERIALS AND METHODS

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Samples

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Two samples of commercial milk for zootechnical use, two samples of commercial whey powder,

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one commercial fish meal, one pure commercial pork meal and one pure bovine meal provided by

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the European Reference Laboratory on animal proteins in feed (EURL AP) were used. The purity of

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each PAP was verified performing both pork and ruminant PCR analyses, according to the EURL

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AP protocols.

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Reagents

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Formic acid, trifluoroacetic acid (TFA) and acetonitrile were LC-MS grade; sodium dodecyl sulfate

123

(SDS), and all other chemicals were analytical grade and purchased from Sigma-Aldrich (Milan,

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Italy). Ultrapure water was prepared by a Milli-Q purification system (Millipore, Bedford, MA).

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Any KD ™ Mini Protean TGX precast gel, Standard Precision Plus prestained protein standards,

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Laemmli sample buffer (2X / 4X), Running buffer and Bio-Safe Coomassie, together with the

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threo-1,4-dimercapto-2,3-butanediol (DTT) and iodoacetamide (IAA) were supplied by Bio-Rad

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Laboratories, Inc. (Segrate, Italy). Trypsin was purchased from Roche Diagnostics SpA (Monza,

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Italy). ACS Paragon Plus Environment


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Standard peptides were synthesized by Primm Srl (Milano, Italy); peptides identification was

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carried out by determining their molecular weights by MALDI-TOF analysis; purity (>95%) was

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assessed by HPLC-UV analysis (λ 214 nm).

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Digestion buffer was 50 mM ammonium bicarbonate; destaining solution was prepared mixing

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acetonitrile with digestion buffer (1:1 v/v ratio); reducing solution was 10 mM DTT in digestion

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buffer; alkylating solution was 55 mM iodoacetamide in digestion buffer; extraction solution (3%

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TFA/30% acetonitrile in MilliQ H2O).

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Sample Preparation

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Protein extraction - Each sample was prepared in two replicates as follows: 800 µL of Laemmli

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sample buffer 2X were added to 50 mg of PAP and incubated overnight at 85 °C in shaking

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conditions (800 rpm) in a thermomixer (Eppendorf, Stevenage, UK). Samples were then centrifuged

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at 22,000 x g for 30 min. Supernatant was transferred to another Eppendorf and diluted 1:6 (v/v) in

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cold (-20°C) acetone and kept 1 h at -20 °C; after centrifugation (15 min at 20,000 x g, 4 °C),

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supernatant was discarded and the pellet was washed with the same volume of cold (-20 °C)

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acetone. After centrifugation, the first replicate was dissolved in 100 µL of SDS 1% solution to

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determine the protein concentration by Bradford Assay.

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The second replicate was used for SDS-PAGE analysis: 100 µL of Laemmli sample buffer 1X was

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added to the pellet and incubated at 37 °C in the thermomixer under shaking conditions at 1000 rpm

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overnight, until complete dissolution. In the case that the pellet did not completely dissolve, an

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aliquot of 100 µL ultrapure water was added.

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SDS-PAGE (reducing conditions) – 20 µL of raw extract in Laemmli sample buffer was mixed with

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10 µL of Laemmli sample buffer 4X and 10 µL of 200 mM DTT and heated at 95 °C for 5 min.

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Gel electrophoresis was performed using precast gel placed in a Mini-PROTEAN Tetra

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electrophoresis cell (Bio-Rad Laboratories, Inc.) filled with running buffer and using a protein

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mixture (Precision Plus Protein Standards) as standard. The electrophoretic run was conducted at

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200 V (constant) for a variable time of about 30-40 min. Following the run, the gel was immersed in ACS Paragon Plus Environment

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ultrapure water to remove any trace of running buffer and then stained for 1 h with Bio-Safe

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Coomassie. After removing the excess stain in ultrapure water (2 h, under shaking) the images of

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gels were acquired using a GS800 densitometer (Bio-Rad Laboratories, Inc).

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In-gel tryptic digestion - Each band (ca. 1 cm width x 0.5 cm height) from the entire lane (10 bands

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for proteomic studies and 5 for signature peptides identification by tandem MS) was cut using a

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scalpel, finely chopped, transferred to an Eppendorf and washed with 200 µL of ultrapure water. An

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aliquot of 200 µL of destaining solution was added to each gel portion and heated at 37 °C for 10

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min in the thermomixer (1400 rpm); the destaining solution was then discarded and this step was

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repeated until destaining was completed. Each gel portion was completely covered with acetonitrile

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and heated at 37 °C for 5 min in the thermomixer (1400 rpm). Acetonitrile was removed and 200

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µL of reducing solution was added and heated for 1 h at 56 °C in the thermomixer (300 rpm). After

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removal of the reducing solution, the pieces of gel were washed with 100 µL of digestion buffer.

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Gel portions were dried again with acetonitrile. Thiol residues were alkylated adding 200 µL of

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alkylating solution for 45 min at room temperature in the dark. The alkylating solution was

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discarded and the residue was washed in digestion buffer as previously described. 200 µL of

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proteolytic enzyme solution, containing trypsin at a final concentration of 5 ng/µL in digestion

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buffer was added to the gel portions and incubated overnight at 37 °C in the thermomixer (600

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rpm).

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The tryptic mixtures were acidified with formic acid up to a final concentration of 1%. To guarantee

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better protein detection, an additional peptide extraction step was performed: 100 µL of extraction

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solution was added to each gel portion and mixed in the thermomixer for 10 min at 37 °C at high

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speed shaking (1400 rpm). The step was repeated twice followed by two steps using acetonitrile.

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The four extracted fractions were then collected, mixed, dried in the Speed Vac (Martin Christ.,

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Osterode am Harz, Germany) at 37 °C and finally stored at -20 °C until the time of analysis.

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Signature peptides identification



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Analytical Methods - Peptides from the in-gel digestion were separated by reversed-phase (RP)

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nanoscale capillary liquid chromatography (nanoLC) and analyzed by electrospray tandem mass

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spectrometry (ESI-MS/MS). The digests were resuspended in 20 µL of A (0.1% HCOOH),

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vortexed for 15 min, centrifuged and loaded into 96-well multiwell plates to be analyzed. For each

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analysis 5 µL of sample was injected onto a column by means of an autosampler; the column used

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was a 75mm x 10cm, 2.7 mm particles, pores 100 Å, C18HALO PicoFrit column (New Objective,

187

USA). Samples were loaded onto the fused silica column at 400 nL/min of mobile phase consisting

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of 99% A and 1% B (0.1% HCOOH in CH3CN) for 15 min. Peptide separation was performed with

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a 55 min linear gradient of 1-35% B. The separative gradient was followed by 5 min at 80% B to

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rinse the column, and 15 min of 99% A and 1% B served to re-equilibrate the column to the initial

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conditions. The nano-chromatographic system, an UltiMate 3000 RSLCnano System (Dionex,

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Thermo Scientific Inc., Milan, Italy), was connected to an LTQ-Orbitrap XL mass spectrometer

193

(Thermo Scientific) equipped with a nanospray ion source (dynamic nanospray probe, Thermo

194

Scientific) set as follows: positive ion mode, spray voltage 1.8 Kv; capillary temperature 220 °C,

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capillary voltage 35 V; tube lens offset 120 V. The LTQ-Orbitrap XL mass spectrometer was

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operated in data-dependent acquisition mode (DDA) to acquire both the full MS spectra and the

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MS/MS spectra. Full MS spectra were acquired in "profile" mode, by the Orbitrap (FT) analyzer, in

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a scanning range from m/z 300-1500, using a capillary temperature of 220 °C, AGC target = 5x105

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and resolving power 60,000 (FWHM at m/z 400). Tandem mass spectra MS/MS were acquired by

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the Linear Ion Trap (LTQ) in CID mode, automatically set to fragment the nine most intense ions in

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each full MS spectrum (exceeding 1x104 counts) under the following conditions: centroid mode,

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normal mode, isolation width of the precursor ion of m/z 2.5, AGC target 1x104 and normalized

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collision energy of 35 eV. Dynamic exclusion was enabled (exclusion dynamics for 45 s for those

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ions observed 3 times in 30 s). Charge state screening and monoisotopic precursor selection were

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enabled, singly and unassigned charged ions were not fragmented. Xcalibur software (version 2.0.7,

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Thermo Scientific) was used to control the mass spectrometer. ACS Paragon Plus Environment

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Data processing - Proteins were identified using Proteome Discoverer 1.3 software (Thermo

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Scientific), implemented with the algorithm SEQUEST. The databases used for data analysis were

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downloaded

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UniProt_SusScrofa.fasta (pig proteins), and a merged FASTA file obtained by combining all the

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available fish species proteome databases (Lepisosteus oculatus, Danio rerio, Astyanax mexicanus,

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Gasterosteus aculeatus, Takifugu rubripes, Xiphophorus maculatus, Oreochromis niloticus,

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Tetraodon nigroviridis, Oryzias latipes, Poecilia formosa) for fish proteins; the search parameters

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set in the workflow were all the experimental parameters used for mass spectra acquisition: mass

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range between 350.0 Da - 5000.0 Da; any activation type mode; total intensity threshold 1; S/N

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threshold 5; 5 ppm as precursor mass tolerance; and 0.5 Da as fragment mass tolerance; trypsin was

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set as the proteolytic enzyme and 2 was the number of missed cleavages. Carbamidomethyation of

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cysteine (+57.021 Da) was set as fixed modification while the oxidation of methionine (+15.995

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Da) as variable modification.

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To ensure the lowest number of false positives, the mass values experimentally recorded were

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further processed through a combined search with the Database Decoy, where the protein sequences

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are inverted and randomized. This operation allows the calculation of the false discovery rate (FDR)

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for each match, so that all the proteins out of range of FDR between to 0.01 (strict) and 0.05

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(relaxed) were rejected.

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Once the task of matching with the UniProt database was completed, the resulting listing of proteins

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was filtered under stringent conditions to ensure the lowest error probability (0.5%): only peptides

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with medium/high confidence were accepted and all proteins recognized with less than two peptides

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were excluded.

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Identification of proteins to selectively distinguish between bovine PAP and milk products.

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The Proteome Discoverer merging tool was applied to the lists of proteins found in bovine PAP and

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milk products to identify their unique gene products. The candidate proteins were then filtered by

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excluding all those proteins with a coverage of less than 20%, all enzymatic proteins (because it is

from

the

UniProt

web

site:

UniProt_BosTaurus.fasta


(bovine

proteins),


233

assumed that they have more than one conserved domain), and all the proteins previously detected,

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by our research group, in bovine colostrum.31 An in depth bibliographic search allowed us to further

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refine this list, excluding proteins previously identified in milk (bovine/human - breast/mature -

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milk).

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Identification of signature peptides - Identification of the signature peptides started from the multi-

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consensus tabular reports obtained by merging all the protein-matches resulting from the MS data

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elaboration (Proteome Discoverer 1.3). Uncharacterized proteins were not considered. Proteins

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were then screened on the basis of their protein coverage (>20%), the score value (>200), the

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number of unique peptides identified and the PSM (Peptide Spectral Match) number; the evaluation

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of the last parameter is a good indicator of the abundance and good ionization properties of the

243

peptide. For each selected protein, the complete panel of peptides was refined considering several

244

factors: 1) the amino acids (aa) contained in the peptides (the presence of aa undergoing known

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post translational modifications (PTMs) should be avoided) and the sequence of the peptide

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(selected peptides should not contain in their sequence the proteolytic cleavage sites recognized by

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trypsin or reactive or labile amino acid residues); 2) the protein-uniqueness of a peptide sequence

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(prototypic-peptide); 3) the species-specificity; 4) the physicochemical properties of a peptide, that

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can affect mass spectrometric sensitivity for a given mass spectrometer 32 and 5) the peptide length

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(only peptides with a sequence composed of less than 20 aminoacidic residues were considered).

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The final candidate peptides were aligned in the BLAST browser to exclude the conserved

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sequences with the species examined.

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LC-MS/MS meal extracts analysis

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Analytical Method Development by LC-ESI-TSQ Quantum Triple Quadrupole

255

Chromatographic condition. The analyses were performed on a reversed-phase 75 x 2.1 mm, i.d.

256

2.6 µm, 100 Å, Kinetex Core-Shell Technology - C18 column (Phenomenex, Milan, Italy) with a

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Kinetex security-guard column (Phenomenex, Milan, Italy), by using a Surveyor system (Thermo

258

Scientific) equipped with an autosampler kept at 8 °C working at a constant flow rate (300 µL/min). ACS Paragon Plus Environment

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10 µL of standard solution was injected and peptides were eluted with a 38 min multistep gradient

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of A (0.1% HCOOH) and B (0.1% HCOOH in CH3CN): 0-3 min, 3% B isocratic; 3-28 min, 3-70%

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B; 28-30 min, 70-95% B; 30-33 min, 95% B isocratic; and then 5 min 3% B isocratic.

262

Characterization of MS/MS product ions for Multiple Reaction Monitoring analysis. A multiple

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reaction monitoring (MRM) method was set up for the quantitative analyses. Signature peptides (20

264

µM) dissolved in 3% B/97% A were infused into the mass spectrometer at a flow rate of 10 µL/min

265

to characterize the product ions of each compound. The fragmentation was carried out using CID

266

mode. The MS/MS analyses were performed with a TSQ Quantum Triple Quadrupole (Thermo

267

Scientific) mass spectrometer fitted with an electrospray (ESI) interface operating in positive ion

268

mode and with the following source parameters: capillary temperature, 270 °C; spray voltage 4.5

269

kV; capillary voltage, 35 V; and tube lens voltage 211 V. The parameters influencing the transitions

270

were optimized as follows: argon gas pressure in the collision Q2, 1.0 mTorr; peak full width at

271

half-maximum (fwhm), m/z 0.70 at Q1 and Q3; scan width for all MRM channels, m/z 0.5; scan

272

time 0.675 s, skimmer offset 10V.

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Linearity evaluation of standard peptides. Samples for linearity evaluation were obtained starting

274

from a stock solution containing all the peptides which was then diluted with a solvent composed of

275

3% B (0.1% HCOOH in CH3CN) and 97% A (0.1% HCOOH) to the following final concentrations:

276

10.000, 5.000, 2.500, 1.250, 1.000, 0.500, 0.025, 0.010 µM. Those standard solutions (10 µL each)

277

were injected into the LC-ESI-MS system and the analytes were separated by using the

278

chromatographic method above described. Three different MRM analytical methods were set-up:

279

parent ions were separately analyzed using three different methods, corresponding to the bovine

280

meal, pig meal, and fish meal signature peptide transitions respectively; 3 transitions for each parent

281

ion were included. Using a linear regression, seven linear curves were obtained from the areas

282

under the peak of the seven peptide signatures. Data processing was performed by Xcalibur 2.0

283

software.

284

Application of the method using a LC-ESI-5500 QTRAP ACS Paragon Plus Environment


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Chromatographic and mass spectrometer conditions. The chromatographic separation was

286

performed performed on a reversed-phase 50 x 2.1 mm, i.d. 1.7 µm, 100 Å, Kinetex Core-Shell

287

Technology - C18 column (Phenomenex, Milan, Italy) with a Kinetex security-guard column

288

(Phenomenex, Milan, Italy), by using a Shimadzu Prominence HPLC system (Shimadzu S.R.L.

289

Italy, Milan, Italy) equipped with an autosampler kept at 8°C working at a constant flow rate (350

290

µL/min). 10 µL of standard solution was injected and peptides were eluted with a 15 min multistep

291

gradient of A (0.1% HCOOH) and B (0.1% HCOOH in CH3CN): 0-0.5 min, 3% B isocratic; 0.5-10

292

min, 3-45% B; 10-10.2 min, 45-95% B; 10.2-12 min, 95% B isocratic; and then 3 min 3% B

293

isocratic.

294

A multiple reaction monitoring (MRM) method was used. MS/MS analysis was performed with a

295

5500 QTRAP (Sciex Italy, Milan, Italy) mass spectrometer fitted with an turbo ion spray (TIS)

296

interface operating in positive ion mode and with the following source parameters: spray voltage

297

4.5 kV; source temperature (TEM) 450 °C; gas 1 and 2 at 50 and 55 psi, respectively; and curtain

298

gas at 30 psi. The parameters influencing the transitions were optimized as follows: nitrogen gas

299

pressure in the collision Q2, 10 mTorr; total scan time working in scheduled MRM was 0.4 s.

300

Beside the MRM acquisition mode, QTRAP instrument allows the full mass spectra registration to

301

confirm the amino acid sequence of each target peptide.

302

In order to test the enhanced instrument sensitivity in respect to the previous analysis (TSQ

303

Quantum Triple Quadrupole), standard peptide mixtures at three different concentrations (10.0nM,

304

1.0nM 0.1nM) were finally injected.

305 306

RESULTS AND DISCUSSION

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Analytical strategy overview

308

To unequivocally identify the sources of bovine, pig and fish processed animal proteins (PAP) and

309

bovine milk proteins, the first step was a proteomic approach aimed at elucidating the proteome of

310

each source. Starting from the identified proteins and using a funnel based approach, a set of ACS Paragon Plus Environment

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abundant and well characterized peptides with suitable physical-chemical properties (signature

312

peptides) and specific for each source were selected. An on-target LC-ESI-MS/MS method (MRM

313

mode) was then set-up using standard peptides and was then applied to selectively identify the PAP

314

source; this method was then successfully applied to verify the selectivity of the signature peptides

315

in different PAP sources and blank matrices (feedstuffs without PAP), to unequivocally identify

316

bovine PAP. The results for each step are reported in detail.

317

Proteomic approach

318

The proteomic approach consists of three steps: protein extraction, protein separation by SDS-

319

PAGE electrophoresis and digestion, protein sequencing by LC-ESI-MS.

320

Protein extraction - Several attempts were made to extract the maximum amount of protein from

321

PAP and the yield of protein extraction was evaluated by a densitometric analysis of the SDS-gel

322

electrophoresis patterns (data not shown). Several parameters were considered, such as the relative

323

percentage of SDS, temperature and time of extraction, and the protein precipitation agent. The

324

most suitable conditions in terms of protein extraction efficiency were obtained by treating PAP

325

samples with Laemmli sample buffer 2X at 85 °C (1200 rpm shaking) overnight to dissolve proteins

326

which were then isolated after acetone precipitation. It should be noted that such harsh conditions

327

for dissolving proteins are needed, due to the heat treatment used for PAP preparation, which

328

greatly denatures and covalently modifies the protein substrates, thus affecting their water

329

solubility. Acetone was selected as the protein precipitation agent, in part due to its ability to

330

dissolve lipids which are known to interfere with electrophoretic separation.

331

SDS-PAGE gel electrophoresis - Proteins were then separated by 1D SDS-PAGE electrophoresis

332

with the aim of splitting the proteins extracted from each source into 10 samples which were

333

individually injected into the LC-MS; this approach permits the reduction of the number of peptides

334

to be analyzed in each data dependent LC-MS run, thus increasing the rate of peptide identification;

335

gel electrophoresis was also used in order to efficiently remove SDS which would greatly affect ESI

336

ionization. The SDS-PAGE profiles in reducing conditions of proteins extracted from PAP derived ACS Paragon Plus Environment


337

from pig /fish/bovine are shown in Figure 1: all the lanes relative to PAP samples are characterized

338

by a quite smeared profile as was expected due to the rendering process for PAP preparation which

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greatly affects protein integrity; however this does not represent a limiting aspect since here the

340

SDS-PAGE is not applied for an analytical purpose but to separate the entire proteome in 10

341

fractions; by contrast, the protein profile of the two batches of milk proteins shows more defined

342

bands. Figure 2 also shows the ten segments along each electrophoretic track which were cut to

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enable in-gel protein digestion.

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Protein identification by MS - Peptide sequence identification was carried out by LC-ESI-MS

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analysis in data dependent scan mode and proteins were identified by using Proteome Discoverer

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and setting high peptide confidence as filter (Peptide RANK 1). Only proteins with at least two

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peptides assigned were considered. For each PAP source, a list of proteins was generated by

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merging all the proteins identified in the 10 segments and deleting the redundancies by using the

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merging tool. Starting from the complete list of proteins a step by step approach (here named funnel

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based approach since based on a progressive exclusion of hits) was used in order to identify specific

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and suitable proteins for each source. As an example, Figure 2A shows the funnel approach we used

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for bovine PAP proteins. The initial list contains 179 unique gene products. We firstly excluded

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proteins which are also contained in milk products and for this a list of proteins contained in milk

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products was compiled by retrieving milk proteins from literature and from our database of

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colostrum proteins.31,33 Proteins with a sequence coverage 20) and characterized by a low PSM value (

MS Identification of Species-Specific Muscle Peptides in

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