Project PXD004908

PRIDE Assigned Tags:
Biological Dataset



Brown bear skeletal muscle quantitative LC-MSMS


Muscle atrophy is one of the main deleterious consequences of ageing and physical inactivity. Although basic knowledge regarding the underlying mechanisms of muscle atrophy is continuously growing, there are still no efficient therapeutic strategies for its prevention and treatment. Hibernating bears exhibit a strong and unique ability to preserve muscle mass in conditions where muscle atrophy is observed in humans. However, underlying mechanisms have not been understood yet. To fill this gap, the aim of this study was to characterize changes in the bear muscle proteome during hibernation versus the active period. Muscle biopsies were obtained from Ursus arctos bears.

Sample Processing Protocol

Seven brown bears from Dalarna and Gävleborg counties (Sweden) were captured twice in 2013, during hibernation (February) and then during their active period (June). Biopsies of Vastus lateralis muscle were collected and immediately frozen on dry ice until storage at -80°C. After extraction, protein samples were electrophoresed (SDS-PAGE gels) to obtain five separated protein bands. After colloidal Coomassie blue staining, protein bands were excised then proteins were in-gel reduced and alkylated and digested with trypsin. Peptides were extracted from the gels, and analyzed on a UPLC-system (nanoAcquity, Waters) coupled to a quadrupole-Tof hybrid mass spectrometer (maXis 4G; Bruker Daltonik GmbH, Bremen, Germany). the nanoAcquity-Q-Exactive/maXis 4G was controlled by Bruker compass Hystar (v3.2) and OtofControl (Rev3.2). The instrument was controlled by Bruker compass Hystar (v3.2) and OtofControl (Rev3.2). The solvent system consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Each sample was first concentrated/desalted on a trap column (Symmetry C18, 180 µm x 20 mm, 5 µm; Waters) at 1% B at a flow rate of 5 µl/min for 3 min. Afterwards, peptides were eluted from the separation column (BEH130 C18, 75 µm x 250 mm, 1.7 µm; Waters) maintained at 60°C using a 60 min gradient from 8-35% B at a flow rate of 450 nl/min. The mass spectrometer was operated in positive mode, with automatic switching between MS and MS/MS scans. The source temperature was set to 160°C with a spray voltage of -4.5kV and dry gas flow rate of 5 l/min. External mass calibration of the Tof (MaXis 4G) was achieved before each set of analyses using Tuning Mix (Agilent Technologies, Paolo Alto, USA) in the mass range of 322-2722m/z, and mass correction was achieved by recalibration of acquired spectra to the applied lock mass using hexakis (2,2,3,3,-tetrafluoropropoxy)phosphazine ([M+H]+ 922.0098m/z). The MS acquisition time was set to 0.4 sec, and MS/MS acquisition time to a range from 0.05 sec (intensity > 250000) to 1.25 sec (intensity < 5000), and ions were excluded after acquisition of one MS/MS spectrum with release of exclusion after 0.2 min. Up to 10 most intense multiply charged precursors per MS scan were isolated, using an isolation window adapted to the isolated m/z (2-5m/z), then fragmented using energy collisional dissociation.

Data Processing Protocol

MS raw data were processed using MaxQuant (v1.5.3.30). Peak lists were searched using the decoy mode (revert) of the Andromeda search engine implemented in MaxQuant. The database contained bear protein sequences (UniprotKb; Taxonomy ID: 9632; downloaded in September 2017) to which sequences of common contaminants (e.g. trypsin and keratins) were automatically added using the MSDA software suite (36931 entries). Sequences of common contaminants like keratins and trypsin (247 entries) were afterwards automatically added by MaxQuant. Regarding search parameters, MS tolerance was set to 0.07 Da for the first search and 0.006 Da for the main search. MS/MS tolerance was set to 40 ppm. A maximum number of two missed cleavages was accepted, and carbamidomethylation of cysteine residues was set as fixed modification, while acetylation of protein N-termini and oxidation of methionine residues were set as variable modifications. False discovery rates (FDR) were set to 1% for both peptide spectrum matches (minimum length of 7 amino acids) and proteins. The final list of identified proteins did not consider common contaminants, which were removed. Regarding quantification, data normalisation and estimation of protein abundance was performed using the MaxLFQ (label free quantification) option implemented in MaxQuant. MaxLFQ quantification was applied using a minimal ratio count of two. Both unmodified and modified (acetylation of protein N-termini and oxidation of methionine residues) peptides were considered for quantification. All other MaxQuant parameters were set as default. Then, only proteins with at least five of seven values per season (i.e. accepting a maximum of 2 missing values) were retained for further analysis, as well as “present/absent” cases (i.e. 0 values in the samples from one of the two seasons).


Sarah Cianférani, Laboratoire de Spectrométrie de Masse BioOrganique, Analytical Science department, IPHC, CNRS, Strasbourg University, UMR7178, Strasbourg, France ( lab head )

Submission Date


Publication Date



    Chazarin B, Storey KB, Ziemianin A, Chanon S, Plumel M, Chery I, Durand C, Evans AL, Arnemo JM, Zedrosser A, Swenson JE, Gauquelin-Koch G, Simon C, Blanc S, Lefai E, Bertile F. Metabolic reprogramming involving glycolysis in the hibernating brown bear skeletal muscle. Front Zool. 2019 16:12 PubMed: 31080489