Proteome alterations associated with the oleic acid and cis-9, trans-11 conjugated linoleic acid content in bovine skeletal muscle.

Oleic acid (OA) and cis-9, trans-11 conjugated linoleic acid (c9t11-CLA) are fatty acids found in beef with beneficial effects in human health. This study investigated differentially abundant proteins (DAPs) in skeletal muscle of bovines with extreme values of OA, and c9t11-CLA. For each one of the fatty acids, twenty muscle samples were divided into two groups (N = 10_High; N = 10_Low) and analyzed by high definition mass spectrometry. We identified 103 and 133 DAPs between the groups for each fatty acid. We found 64 and 45 up-regulated and 39 and 68 down-regulated proteins for OA and c9t11-CLA, respectively. Comparative analysis between proteomic and transcriptomic data revealed eight and ten genes with a consistent between mRNA expression levels and protein abundance for OA and c9t11-CLA, respectively. Unconventional myosin-Id (MYO1D), mineralocorticoid receptor (NR3C2), geranylgeranyl transferase type-2 subunit-alpha (RABGGTA), and uveal autoantigen with coiled-coil domains and ankyrin repeats (UACA) were found as putative candidate proteins for OA content. Fatty acid synthase (FASN), tubulin alpha-4A chain (TUBA4A), vinculin (VCL), NADH dehydrogenase 1 alpha subcomplex 5 (NDUFA5), and prefoldin subunit 6 (PFDN6) for c9t11-CLA. Our findings contribute to a deeper understanding of the molecular mechanisms behind the regulation of the OA and c9t11-CLA content in cattle skeletal muscle. SIGNIFICANCE: Questions about the association between meat intake and disease incidence in humans has driven animal scientist to pursue a better understanding of the biological processes associated with differences in the intramuscular fat composition. The beneficial effects of oleic acid and conjugated linoleic acid in human health have been demonstrated by improving the immune system and preventing atherosclerosis, different types of cancers, hypertension, and diabetes. Previous genome-wide association and gene expression studies identified genomic regions and differentially expressed genes associated with the fatty acid profile in skeletal muscle. In this work, differences were evaluated at the protein level. The use of a label-free quantitative proteomic approach, compared with muscle transcriptome results obtained by RNA-sequencing, allowed us to earn new insights into the variability in fatty acid deposition in skeletal muscle of farm animals. This study opens new avenues to explore the effect of the fatty acids in the skeletal muscle of livestock animals, which is associated with nutritional values of the meat, and perhaps to understand the mechanisms correlated with metabolic diseases in other species.

Proteomics in quality control: Whey protein-based supplements.

UNLABELLED: The growing consumption of nutritional supplements might represent a problem, given the concern about the quality of these supplements. One of the most used supplements is whey protein (WP); because of its popularity, it has been a target of adulteration with substitute products, such as cheaper proteins with lower biological value. To investigate this type of adulteration, this study used shotgun proteomics analyses by MS(E) (multiplexed, low- and high-collision energy, data-independent acquisition) of WP-based supplements. Seventeen WP-based supplement samples were evaluated. Chicken, maize, rice, potato, soybean, and wheat proteins were considered as probable sources of bovine whey adulteration. Collectively, 523 proteins were identified across all 16 samples and replicates, with 94% of peptides inside a normal distribution within 10ppm of maximum error. In 10 of the 16 samples analyzed, only proteins from bovine whey could be detected, while in the other samples several other protein sources were detected in high concentrations, especially soybean, wheat, and rice. These results point out a probable adulteration and/or sample contamination during manufacturing that could only be detected using this proteomic approach. SIGNIFICANCE: The present work shows how shotgun proteomics can be used to provide reliable answers in quality control matters, especially focusing on Whey Protein nutritional supplements which are a very popular subject in food and nutrition. In order to achieve an appropriate methodology, careful evaluation was performed applying extremely rigorous quality criteria, established for the proteomic analysis. These criteria and the methodological approach used in this work might serve as a guide for other authors seeking to use proteomics in quality control.

Detection and expression analysis of recombinant proteins in plant-derived complex mixtures using nanoUPLC-MS(E).

The use of mass spectrometry to identify recombinant proteins that are expressed in total soluble proteins (TSPs) from plant extracts is necessary to accelerate further processing steps. For example, the method consists of TSP sample preparation and trypsin digestion prior to the preliminary characterization using nanoUPLC-MS(E) analysis of the recombinant proteins that are expressed in TSP samples of transgenic soybean seeds. A TSP sample as small as 50 µg can be effectively analyzed. In this study, transgenic soybean seeds that expressed recombinant cancer testis antigen (CTAG) were used. The procedure covered 30% of the protein sequence and was quantified at 0.26 ng, which corresponded to 0.1% of the TSP sample. A comparative proteomic profile was generated by the comparison of a negative control and sample that showed a unique expression pattern of CTAG in a transgenic line. The experimental data from the TSP extraction, sample preparation and data analysis are discussed herein.