Application of proteomics to the areas of milk production,processing and quality control – A review

Milk contains a complex mixture of proteins that undergo several qualitative and quantitative changes during processing and storage. Elucidation of these changes is important to optimise milk processing and storage parameters. Proteomics has become widely used in the field of research on milk proteins due to improvements in the tools and diversified ways in which the methodologies of protein analysis have been developed. This review describes the use of proteomics in studies on milk proteins in relation to species–species differences, post‐translational modification, effect of lactation, mastitis, changes in heated milk and during cheese ripening, and the detection of milk adulteration.     

Proteome changes during pork meat ageing following use of two different pre-slaughter handling procedures

The influence of postmortem storage time and pre-slaughter conditions (transport the day before slaughter or immediately before slaughter) on proteome changes of pork meat was investigated over a 72 h ageing period. Intensities of 37 spots varied significantly (p<0.05) with ageing time. Changes indicated proteolysis of troponin T, actin, -crystallin, myokinase, creatine kinase and mitochondrial ATPase, but also of proteins constitutive of the Z-lines, namely cypher proteins and myozenin. Other modifications were the intensity increase of a full-length protein of the sarcoplasmic reticulum, which may be linked to its increased extractibility after membrane disruption, and a gradual shift in pHi towards alkaline values of some forms of myosin light chains (MLC) 2 and 3. The pre-slaughter conditions affected significantly (p<0.05) 8 spots. Mitochondrial ATPase was over-expressed in the group transported immediately before slaughter, also characterised by a faster pH fall, and the shift in pHi of MLC 2 was more pronounced. The pre-slaughter conditions had no significant effect on the above proteolytic events.     

Prolonged cold stress response of Escherichia coli O157 and the role of rpoS

Phenotypic analyses were performed using an enterohemorrhagic Escherichia coli O157 (EHEC) strain (B-1) and a commensal E. coli K-12 strain, exposed to prolonged cold stress. The EHEC E. coli O157 showed significantly (P < 0.05) higher resistance to cold stress compared to non-pathogenic E. coli K-12 DH5α. Further, it was found that RpoS sigma factor plays a significant (P < 0.05) role in the cold stress physiology of the enterohemorrhagic E. coli strain. Using comparative proteomic analysis of hypo-thermally adapted E. coli O157 wild-type and rpoS mutant strains, we identified 21 proteins that were differentially expressed upon cold temperature shifts or rpoS mutation. All identified proteins of cold post-acclimation stimulons fell into two large sub-groups: (i) stress proteins, and (ii) housekeeping proteins. This prolonged cold stress response included proteins involved in mRNA turnover, cell replication efficiency, conditional and post-synthetic modification of membrane lipid bilayers, biosynthetic processes, and the uptake of different sugars. The RpoS sigma factor had no control over the key stress proteins, polynucleotide phosphorylase and elongation factor G, in prolonged stress stimulon. However, RpoS was shown to regulate the expression of proteins involved in homeoviscous adaptation during cold shock, as well as various proteins involved in central metabolic pathways of this food-borne pathogen.     

Characterization of Cauxin in the Urine of Domestic and Big Cats

Cauxin is an abundant protein in feline urine. We have used proteomics strategies to characterize cauxin from the urine of domestic cats and a number of big cat species. Proteins were resolved by gel-based electrophoretic purification and subjected to in-gel digestion with trypsin. The resultant tryptic peptides were mass-measured by matrix-assisted laser desorption ionization time of flight mass spectrometry. Peptides were also resolved by liquid chromatography and analyzed by electrospray ionization and tandem mass spectrometry to generate fragment ion data to infer the amino acid sequence. We identified cauxin polymorphisms and corrected a sequencing artifact in cauxin from the domestic cat. The proteomics data also provided positive evidence for the presence of a cauxin homolog in the urine of big cats (Pantherinae), including the Sumatran tiger, Asiatic lion, clouded leopard, Persian leopard, and jaguar. The levels of cauxin in the urine of all big cats were substantially lower than that in the urine of intact male domestic cats.     

Isolation and characterization of Streptococcus parauberis from vacuum-packaging refrigerated seafood products

Streptococcus parauberis is known as an etiological agent of mastitis in cows and for producing streptococcosis in farmed fish, although its presence in foods has seldom been reported. In this work, two bacterial isolates were recovered from a spoiled vacuum-packaged refrigerated seafood product. Both isolates were identified by 16S rRNA gene sequencing, exhibiting 99% homology with respect to S. parauberis. Both isolates were also characterized by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). Genetic analysis revealed the clonal homogeneity of the isolates and their grouping together with other S. parauberis strains in a different cluster with respect to Streptococcus uberis strains. Proteomic analysis by MALDI-TOF MS allowed for the identification of five mass peaks in the range of 2200–6000 m/z that resulted to be specific to the species S. parauberis and allowed its rapid and direct identification with respect to other pathogenic and spoilage bacteria potentially present in seafood and other food products. This study represents, to our knowledge, the first report of S. parauberis in seafood in general and in vacuum-packed food products in particular. Moreover, it provides a rapid method based on MALDI-TOF MS for the identification of S. parauberis.     

基于iTRAQ技术的天然富硒与非富硒水稻差异蛋白分析

为解析不同基因型水稻次生代谢差异成因及其富硒品质形成的蛋白质机制提供参考。采用同位素标记相对和绝对定量(iTRAQ)技术对天然富硒与非富硒水稻进行定量蛋白质组学研究,探讨两种基因型水稻蛋白质组表达水平的差异。结果共鉴定到定量蛋白质有3 161个,差异蛋白为401个,其中上调差异蛋白193个,下调蛋白208个,生物信息学分析得出77个目标差异蛋白,主要分成10类:氧化还原酶、转移酶、异构酶、热休克蛋白、裂解酶、水解酶、连接酶、合成酶、微管蛋白、肌动蛋白。分析表明天然富硒水稻的抗应激、抗氧化、活性氧代谢、碳水化合物和氨基酸代谢较非富硒水稻强,但参与淀粉合成途径作用较非富硒水稻弱。CYS和metE可能是导致两种类型水稻氨基酸差异的2种关键蛋白,OsAPx02、CatC、riPHGPX、HSP70和HSP90可能是调节两种类型水稻抗氧化和抗应激作用的蛋白基因。     

Analysis of major proteins and fat fractions associated with mare's milk fat globules

Although several studies have aimed to identify mare's milk proteins, only the major whey proteins and some caseins have yet been characterized. Incomplete sequencing of the equine genome and the difficulty of recovering highly hydrophobic proteins mean that little is known to date about the proteins associated with milk fat globules, which have been shown to play an important role in newborns' defense mechanisms. The fat fraction, in particular the distribution of unsaturated fatty acids, has been more extensively studied, but complex lipids are only partially elucidated. This study reports a 2-DE approach combined with a powerful method for de novo protein sequencing, and quali-quantitative data on complex lipid composition determined by high performance TLC (HPTLC) and GC. The presence in mare's milk of long-chain highly unsaturated fatty acids, and the evidence of close similarity between equine and human milk fat globule membrane proteins, support the use of mare's milk for human nutrition.     

Improved parametric time warping for proteomics

We present an improved version of parametric time warping, which enables the method to be used in LC-MS measurements in proteomics. The new features include a new similarity measure for comparing warped chromatograms, an insurance against peaks at the extremes of the chromatograms disappearing because of the warping, and the possibility to select and use multiple traces in searching the optimal alignment. Moreover, we present an alignment strategy combining global and individual alignments for aligning LC-MS data that exhibit different shifts within the same sample, at the same retention time. Using an LC-MS data set consisting of E. coli homogenates that were measured in several batches over a period of six months, we show the benefits of the improved algorithm and the merits of the new strategy. The algorithm is publicly available as the R package ptw.     

功能基因组学及其在辐射生物效应研究中的应用

功能基因组学是基因组学的重要组成部分,近年来已应用到辐射生物效应研究领域。本文介绍了功能基因组学的基本概念,主要研究内容、研究方法,以及在辐射生物效应研究领域中的应用。     

Analysis of mass spectrometry data of cerebral stroke samples: an evolutionary computation approach to resolve and quantify peptide peaks

A preliminary investigation of cerebral stroke samples injected into a mass spectrometer is performed from an evolutionary computation perspective. The detection and resolution of peptide peaks is pursued for the purpose of automatically and accurately determining unlabeled peptide quantities. A theoretical peptide peak model is proposed and a series of experiments are then pursued (most within a distributed computing environment) along with a data preprocessing strategy that includes (i) a deisotoping step followed by (ii) a peak picking procedure, followed by (iii) a series of evolutionary computation experiments oriented towards the investigation of their capability for achieving the aforementioned goal. Results from four different genetic algorithms (GA) and one differential evolution (DE) algorithm are reported with respect to their ability to find solutions that fit within the framework of the presented theoretical peptide peak model. Both unconstrained and constrained (as determined by a course grained preprocessing stage) solution space experiments are performed for both types of evolutionary algorithms. Good preliminary results are obtained.