Phosphoproteome Analysis Using Two-Dimensional Electrophoresis Coupled with Chemical Dephosphorylation.

Protein phosphorylation is a reversible post-translational modification (PTM) with major regulatory roles in many cellular processes. However, the analysis of phosphoproteins remains the most challenging barrier in the prevailing proteome research. Recent technological advances in two-dimensional electrophoresis (2-DE) coupled to mass spectrometry (MS) have enabled the identification, characterization, and quantification of protein phosphorylation on a global scale. Most research on phosphoproteins with 2-DE has been conducted using phosphostains. Nevertheless, low-abundant and low-phosphorylated phosphoproteins are not necessarily detected using phosphostains and/or MS. In this study, we report a comparative analysis of 2-DE phosphoproteome profiles using Pro-Q Diamond phosphoprotein stain (Pro-Q DPS) and chemical dephosphorylation of proteins with HF-P from longissimus thoracis (LT) muscle samples of the Rubia Gallega cattle breed. We found statistically significant differences in the number of identified phosphoproteins between methods. More specifically, we found a three-fold increase in phosphoprotein detection with the HF-P method. Unlike Pro-Q DPS, phosphoprotein spots with low volume and phosphorylation rate were identified by HF-P technique. This is the first approach to assess meat phosphoproteome maps using HF-P at a global scale. The results open a new window for 2-DE gel-based phosphoproteome analysis.

Comparative Proteomics of Potato Cultivars with a Variable Dormancy Period.

The control of the duration of the dormancy phase is a significant challenge in the potato industry and for seed producers. However, the proteome landscape involved in the regulation of the length of the dormancy period over potato cultivars remains largely unexplored. In this study, we performed for the first time a comparative proteome profiling of potato cultivars with differential duration of tuber dormancy. More specifically, the proteome profiling of Agata, Kennebec and Agria commercial potato varieties with short, medium and medium-long dormancy, respectively, was assessed at the endodormancy stage using high-resolution two-dimensional electrophoresis (2-DE) coupled to reversed-phase liquid chromatography-tandem mass spectrometry (LC-TripleTOF MS/MS). A total of 11 proteins/isoforms with statistically significant differential abundance among cultivars were detected on 2-DE gels and confidently identified by LC-TripleTOF MS/MS. Identified proteins have known functions related to tuber development, sprouting and the oxylipins biosynthesis pathway. Fructokinase, a mitochondrial ADP/ATP carrier, catalase isozyme 2 and heat shock 70 kDa were the proteins with the strongest response to dormancy variations. To the best of our knowledge, this study reports the first candidate proteins underlying variable dormancy length in potato cultivars.

Sensory and Physicochemical Analysis of Meat from Bovine Breeds in Different Livestock Production Systems, Pre-Slaughter Handling Conditions and Ageing Time.

Different bovine breeds and production systems are used worldwide, giving rise to differences in intrinsic and extrinsic characteristics of beef. In order to meet the consumer requirements, new approaches are currently being developed to guarantee tenderness, taste, and juiciness of beef. However, the final consumer perception is complex, and it is also affected by several interrelated variables. This study aimed to evaluate the physicochemical parameters and sensory profile of three Spanish cattle breeds under different livestock production systems (extensive and intensive) and pre-slaughter handling conditions (mixing and not mixing with unfamiliar individuals at pre-mortem time). Meat samples from each group were also studied at different ageing times (7 and 14 days). Regarding sensory attributes, twelve panelists assessed meat samples and an exhaustive statistical analysis was carried out. The most evident and strongest effect was the breed type, allowing a great differentiation among them using principal components and discriminant analysis. The livestock production system was the second most important parameter, significantly affecting odor, flavor, and textural profile (fibrousness). It can be concluded that there were marked differences in the traits of these beef that could be modified by other factors in order to fulfill consumer tastes.

The first evidence of global meat phosphoproteome changes in response to pre-slaughter stress.

BACKGROUND: Pre-slaughter stress (PSS) impairs animal welfare and meat quality. Dark, firm and dry (DFD) are terms used to designate poor quality meats induced by PSS. Protein phosphorylation can be a potentially significant mechanism to explain rapid and multiple physiological and biochemical changes linked to PSS-dependent muscle-to-meat conversion. However, the role of reversible phosphorylation in the response to PSS is still little known. In this study, we report a comparative phosphoproteomic analysis of DFD and normal meats at 24 h post-mortem from the longissimus thoracis (LT) bovine muscle of male calves of the Rubia Gallega breed. For this purpose, two-dimensional gel electrophoresis (2-DE), in-gel multiplex identification of phosphoproteins with PRO-Q Diamond phosphoprotein-specific stain, tandem (MALDI-TOF/TOF) mass spectrometry (MS), novel quantitative phosphoproteomic statistics and bioinformatic tools were used. RESULTS: Noticeable and statistically significant differences in the extent of protein phosphorylation were detected between sample groups at the qualitative and quantitative levels. Overall phosphorylation rates across significantly changed phosphoproteins were about three times higher in DFD than in normal meat. Significantly changed phosphoproteins involved a variable number of isoforms of 13 myofibrillar and sarcoplasmic nonredundant proteins. However, fast skeletal myosin light chain 2 followed by troponin T, F-actin-capping and small heat shock proteins showed the greatest phosphorylation change, and therefore they were the most important phosphoproteins underlying LT muscle conversion to DFD meat in the Rubia Gallega breed. CONCLUSIONS: This is the first study reporting global meat phosphoproteome changes in response to PSS. The results show that reversible phosphorylation is a relevant mechanism underlying PSS response and downstream effects on meat quality. This research opens up novel horizons to unravel the complex molecular puzzle underlying muscle-to-meat conversion in response to PSS.