Data on proteomic analysis of milk extracellular vesicles from bovine leukemia virus-infected cattle

Milk extracellular vesicles (EVs) are nanoparticles that contain proteins, mRNAs, microRNAs, DNAs, and lipids that involved in several biological functions. Milk EVs provide proteins that could represent relevant novel biomarkers for monitoring of different diseases such as breast cancer and mastitis in humans and animals, respectively. Bovine leukemia virus (BLV) is an oncogenic virus that causes progressive B-cell lymphosarcoma in cattle. Here, we aimed to identify proteins in milk EVs from BLV-infected cattle compared with those from uninfcetd cattle. Proteomic analysis was performed by using a comprehensive nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) approach. Identified proteins were analyzed by using a proteomic software, Scaffold-Data Independent Acquisition (Scaffold-DIA).


a b s t r a c t
Milk extracellular vesicles (EVs) are nanoparticles that contain proteins, mRNAs, microRNAs, DNAs, and lipids that involved in several biological functions. Milk EVs provide proteins that could represent relevant novel biomarkers for monitoring of different diseases such as breast cancer and mastitis in humans and animals, respectively. Bovine leukemia virus (BLV) is an oncogenic virus that causes progressive B-cell lymphosarcoma in cattle. Here, we aimed to identify proteins in milk EVs from BLV-infected cattle compared with those from uninfcetd cattle. Proteomic analysis was performed by using a comprehensive nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) approach. Identified proteins were analyzed by using a proteomic software, Scaffold-Data Independent Acquisition (Scaffold-DIA

Value of the Data
• The proteomic dataset of bovine milk-derived extracellular vesicles (EVs) will expand the knowledge and create a solid ground for future investigations of bovine leukemia viral (BLV) infection in cattle. • Identified proteins will give researchers an initial reference database for the understanding of pathogenesis of BLV infection in cattle. • The bovine milk-derived EV proteomic dataset can be used to classify the possible biological properties of proteins/peptides in bovine milk, or to grant access to useful information for the isolation of desired proteins.

Data Description
Milk extracellular vesicles (EVs) were isolated from three bovine leukemia virus (BLV)infected cattle with high proviral load (HPL) along with high percentage of lactate dehydrogenase isozymes (HLDH) 2 + 3 and uninfected cattle ( Table 1 ). The morphology of milk EVs were observed by transmission electron microscopy (TEM) analysis (Data not shown). The proteomic landscape of milk EVs from BLV-infected and uninfected cattle was analyzed by nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) with an Ultimate 30 0 0 RSLC nano HPLC system connected to a Q Exactive mass spectrometry (Thermo Fisher Scientific, Waltham, MA, USA). Identification and quantification of proteins were performed by using Scaffold-Dat a Independent Acquisition (Scaffold-DIA) software.

Sample collection
Blood was collected from Holstein dairy cattle in vacuum blood collection tubes with or without an anti-coagulant (VP-AS076 K, VP-NA050 K, and VP-H070 K, Terumo, Tokyo, Japan). Plasma was separated by centrifugation with MX-307 (Tomy Seiko, Tokyo, Japan) at 2500 × g for 15 min at 25 °C followed by serum separation from clotted blood by centrifugation at 30 0 0 × g for 15 min at 25 °C.

Anti-BLV antibody detection
Anti-BLV antibodies were detected in serum samples by enzyme-linked immunosorbent assay (ELISA) performed using an anti-BLV antibody ELISA kit (JNC, Tokyo, Japan) according to the manufacturer's instructions.

BLV DNA detection
After hemolysis of red blood cells with 0.83% ammonium chloride, white blood cells (WBCs) were isolated and DNA was extracted using QIAamp DNA Mini Kit (51304, Qiagen, Hilden, Germany) following the manufacturer's instructions. WBCs DNA concentration was measured by using a spectrophotometer, NanoDropLite (Thermo Fisher Scientific, Waltham, MA, USA). Nested polymerase chain reaction (nested PCR) was carried out to amplify the envelope or pX region of BLV [ 1 , 2 ]. Nested PCR was performed in a total reaction volume of 20 μL containing 0.

BLV copy number measurement
BLV copy number was measured from extracted WBCs DNA by quantitative real-time PCR. The reaction mixture contained 10 μL of THUNDERBIRD Probe qPCR Mix (A4250K, Toyobo, Osaka, Japan), 0.3 μL of CoCoMo-BLV Primer/Probe (A803, Riken Genesis, Tokyo, Japan), 5 μL of DNA template, and PCR grade water was added up to 20 μL. For the proviral quantification, BLV BoLA-DRA gene Plasmid DNA was used from the kit (A804, Riken Genesis) and BLV proviral DNA was measured by a Thermal Cycler Dice Real Time System III (TP970, Takara Bio) according to the manufacturer's instructions. BLV copies of > 30,0 0 0/10 5 WBCs DNA was considered as HPL in BLV-infected cattle ( Table 1 ).

Sample collection
Milk samples were collected from three BLV-infected cattle with HPL + HLDH and three uninfected cattle based on the hematological parameters ( Table 1 ). The milk samples were placed in a cool box to maintain the temperature and were transported quickly to the laboratory.

Isolation and detection of milk EVs
Milk EVs were isolated as previously described [ 3 , 4 ]. Milk EV morphological features were detected by TEM analysis [5] .

Proteomic analysis of milk EVs
For the proteomic analysis of milk EVs, a comprehensive nanoLC-MS/MS method was performed by using an UltiMate 30 0 0 RSLC nano System (Thermo Fisher Scientific) coupled with Q Exactive mass spectrometer (Thermo Fisher Scientific) as described previously [6] . All procedures of proteomic analyses were carried out by an assigned company (Hakarel, Osaka, Japan). The identified proteins were further analyzed by using a proteomic software Scaffold-DIA (Portland, OR, USA) considering the false discovery rate > 1%. The obtained MS/MS spectra was evaluated based on the UniPort database for Bos taurus ( https://www.uniprot.org/proteomes/UP0 0 0 0 09136 ) for annotation of the accession number of each identified EV protein. The proteins without a gene name or those having a dual gene name were omitted from our datasets.

Ethics Statement
The experiments were conducted in accordance with all relevant guidelines and regulations of the Gifu University Animal Care and Use Committee (Approval numbers: 17046 and 2019-234).

Funding
This study was supported by a regulatory research project for food safety, animal health, and plant protection ( JPJ008617.17935709 ) granted by the Ministry of Agriculture, Forestry, and Fisheries, Japan, and by the OGAWA Science and Technology Foundation , the Morinaga Foundation for Health and Nutrition, and the Sasakawa Scientific Research Grant from The Japan Science Society .

Declaration of Competing Interest
The authors do not have any competing financial interests or personal relationships to declare that could influence the work reported herein.