Assessment of sample preparation bias in mass spectrometry-based proteomics

For mass spectrometry-based proteomics, the selected sample preparation strategy is a key determinant for information that will be obtained. However, the corresponding selection is often not based on a fit-for-purpose evaluation. Here we report a comparison of in-gel (IGD), in-solution (ISD), on-filter (OFD), and on-pellet digestion (OPD) workflows on the basis of targeted (QconCAT-multiple reaction monitoring (MRM) method for mitochondrial proteins) and discovery proteomics (data dependent acquisition, DDA) analyses using three different human head and neck tissues (i.e. nasal polyps, parotid gland, and palatine tonsils). Our study reveals differences between the sample preparation methods, for example with respect to protein and peptide losses, quantification variability, protocol-induced methionine oxidation and asparagine/glutamine deamidation as well as identification of cysteine containing peptides. However, none of the methods performed best for all types of tissues, which argues against the existence of a universal sample preparation method for proteome analysis

Association of HPA axis-related genetic variation with stress reactivity and aggressive behaviour in pigs

<p>Abstract</p> <p>Background</p> <p>Stress, elicited for example by aggressive interactions, has negative effects on various biological functions including immune defence, reproduction, growth, and, in livestock, on product quality. Stress response and aggressiveness are mutually interrelated and show large interindividual variation, partly attributable to genetic factors. In the pig little is known about the molecular-genetic background of the variation in stress responsiveness and aggressiveness. To identify candidate genes we analyzed association of DNA markers in each of ten genes (<it>CRH </it>g.233C>T, <it>CRHR1 </it>c.*866_867insA, <it>CRHBP </it>c.51G>A, <it>POMC </it>c.293_298del, <it>MC2R </it>c.306T>G, <it>NR3C1 </it>c.*2122A>G, <it>AVP </it>c.207A>G, <it>AVPR1B </it>c.1084A>G, <it>UCN </it>g.1329T>C, <it>CRHR2 </it>c.*13T>C) related to the hypothalamic-pituitary-adrenocortical (HPA) axis, one of the main stress-response systems, with various stress- and aggression-related parameters at slaughter. These parameters were: physiological measures of the stress response (plasma concentrations of cortisol, creatine kinase, glucose, and lactate), adrenal weight (which is a parameter reflecting activity of the central branch of the HPA axis over time) and aggressive behaviour (measured by means of lesion scoring) in the context of psychosocial stress of mixing individuals with different aggressive temperament.</p> <p>Results</p> <p>The SNP <it>NR3C1 </it>c.*2122A>G showed association with cortisol concentration (p = 0.024), adrenal weight (p = 0.003) and aggressive behaviour (front lesion score, p = 0.012; total lesion score p = 0.045). The SNP <it>AVPR1B </it>c.1084A>G showed a highly significant association with aggressive behaviour (middle lesion score, p = 0.007; total lesion score p = 0.003). The SNP <it>UCN </it>g.1329T>C showed association with adrenal weight (p = 0.019) and aggressive behaviour (front lesion score, p = 0.029). The SNP <it>CRH </it>g.233C>T showed a significant association with glucose concentration (p = 0.002), and the polymorphisms <it>POMC </it>c.293_298del and <it>MC2R </it>c.306T>G with adrenal weight (p = 0.027 and p < 0.0001 respectively).</p> <p>Conclusions</p> <p>The multiple and consistent associations shown by SNP in <it>NR3C1 </it>and <it>AVPR1B </it>provide convincing evidence for genuine effects of their DNA sequence variation on stress responsiveness and aggressive behaviour. Identification of the causal functional molecular polymorphisms would not only provide markers useful for pig breeding but also insight into the molecular bases of the stress response and aggressive behaviour in general.</p

Biochemical and organoleptic characteristics of muscle from early and late maturing bulls in different production systems

In grass-based beef production systems (PS), early maturing (EM) breed types may be preferable to late maturing (LM) breed types in achieving adequate carcass fat cover. Biochemical and organoleptic characteristics of muscle from suckler bulls were investigated in EM and LM (n = 28/breed) assigned to one of two PS (ad libitum concentrates and grass silage to slaughter (C) or ad libitum silage plus 2 kg concentrate daily during winter followed by 99 days at pasture and then an indoor finishing period on C (GSPC)) in a 2 breed type x 2 PS factorial arrangement of treatments. Bulls were managed to have a common target carcass weight of 380 kg. Intramuscular fat (IMF) content was higher (P < 0.05) for EM than LM, and for C than GSPC bulls. Collagen solubility was higher (P < 0.05) for C than GSPC bulls. Lactate dehydrogenase (LDH) and phosphofructokinase activities were higher (P < 0.05) for LM than EM. Isocitrate dehydrogenase activity and the Type I myosin heavy chain (MyHC) proportion were higher (P < 0.05) for EM than LM. The LDH activity and the Type IIX MyHC proportion were higher (P < 0.05) for C than GSPC bulls. Sensory ratings for tenderness and juiciness were higher (P < 0.01) for beef from EM than LM while sensory ratings for tenderness, flavour liking and overall liking were higher (P < 0.001) for C than for GSPC bulls. Differences in sensory quality were largely eliminated when adjusted for IMF. Overall, carcass fat scores, IMF and sensory scores were higher in EM than LM and in C than GSPC bulls but most differences in sensory quality could be attributed to differences in IMF