The protocol was developed with inspiration from that by Rappsilber et?al . plasma samples were analyzed in triplicates. The plasma proteins were unfolded and reduced. To increase the chance of detecting low-molecular-weight proteins and peptides, we filtered samples using a 50?K MWCO filter to deplete larger, abundant proteins, including albumin, and thereby increase the depth of the analysis. The proteins in the permeate were then tryptically digested, desalted, and analyzed with LC-MS/MS.Data source locationCaptures were performed by The Scandinavian Brown Bear Research Project. The bears were sampled in region Dalarna in central Sweden.Data accessibilityRaw and analyzed mass spectrometric proteomics data and the databases used for protein identification have been deposited to ProteomeXchange:where the retentate following filtration using a 30?K MWCO filter was analyzed . In this study, we aimed to expand the coverage of the bear plasma proteome, focusing on low-molecular-weight protein species. The 38 plasma samples analyzed in this study are listed in Table?1. Open in a separate window Fig. 1 Overview of sample handling. Plasma was collected from hibernating and active Scandinavian brown bears. In triplicates, the plasma proteins were unfolded, reduced, and alkylated. To increase the chance of detecting low-molecular-weight proteins and peptides, we filtered samples using a 50?K MWCO filter. The proteins in the permeate were then tryptically digested, desalted, and analyzed with LC-MS/MS. The figure was created with BioRender.com. Open in a separate window Fig. 2 SDS-PAGE analysis (Coomassie Brilliant Blue R\250 stained) showing the efficiency of sample fractionation using a 50?K MWCO filter to deplete abundant, high-molecular-weight proteins such as albumin (ALB) and immunoglobulins (Ig) which constitute over 75% of the total protein in plasma. The protein content in the samples was analyzed by SDS-PAGE before fractionation (Non-frac) and in the retentate (Ret) and permeate (Per) following fractionation. Additionally, samples were analyzed before (Int) and after tryptic digestion (Dig). Table 1 List of plasma samples analyzed in this study. The internal bear IDs applied in the Scandinavian Brown Bear Research Project are stated in the first column (Wxxxx). The numbers in columns 3C6 indicate the sampling year, e.g., W17 is a winter (February) sample from 2017 and S17 is the summer (June) sample from 2017. protein database from NCBI and a peptide hormone database generated by manual extraction of the mature sequences according to the human annotations in UniProt (available via ProteomeXchange). The MaxQuant output files proteinGroups.txt and peptides.txt are available via ProteomeXchange. The proteinGroups.txt file contains all identified proteins, the number of peptides identified for each protein, sequence coverage, LFQ intensities in each sample, etc., which can be used to investigate protein expression levels. The peptides.txt file contains amino acid sequences for all identified peptides, which can be used to investigate e.g. splice variants. BML-190 2.?Experimental Design, Materials, and Methods 2.1. Sample collection Arterial blood was collected in EDTA tubes from free-ranging (wild) Scandinavian Brown Bears in Dalarna, NCAM1 Central Sweden during hibernation (February) and activity (June). Captures were performed by The Scandinavian Brown Bear Research Project (http://bearproject.info/). Details on the methods of bear capture, anesthesia, and blood sample collection have been published previously . In brief, bears were marked with GPS collars and VHF transmitters to enable localization of the bears. During hibernation, bears were located BML-190 in their dens and anaesthetized with a mixture of medetomidine, zolazepam, tiletamine, and ketamine. During active state, the same BML-190 bears were located in their habitat and darted from a helicopter with a mixture of medetomidine, zolazepam, and tiletamine. Anesthesia was antagonized by atipamezole. In winter, the bears were placed back into their dens. Samples from 14 subadult 2-.