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Bovine sarcoplasmic sub-proteome was studied through a straightforward gel-free pipeline supported by liquid isoelectric focusing (OFFGEL) protein fractionation coupled to liquid chromatography-mass spectrometry (LC-MS) analysis. Full-MS and data-dependent MS/MS analyses were simultaneously performed by a conventional three-dimensional ion-trap addressing targeted quantitative and untargeted qualitative research, respectively. There were unambiguously identified 47 proteins distributed along 12 OFFGEL fractions assayed. Regarding intermediate- and high-abundant peptides, bulky quantitative data processing performed by MZmine 2 freeware yielded a satisfactory linearity and coefficient of variation with r2 in the 0.95–0.99 range and about 25%, respectively. Up to 41 peptides from 20 identified proteins were relatively quantified throughout OFFGEL fractions. This reliable, flexible and affordable gel-free proteomic approach could be readily implemented by industry to improve quality assessment of protein-based food products., This work was funded by the Spanish Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (RTA2014-00034-C04).

In this work, we explored the “deep” seed peanut proteome by using both two dimensional electrophoresis (2- DE)-based analysis run under reducing and non-reducing condition (protein-centric) and LC-MS/MS gel-free proteomic (peptide-centric). The former approach allowed to identify high molecular weight disulfide-linked Ara h 1 and Ara h 3 heteroligomers and Ara h 1 homoligomers linked through covalent bonds other than disulfides. The occurrence of these protein complexes revealed natural interactions between Ara(s) subunits with a possible involvement in the allergenic potential of peanut. The second approach, also referred to as shot-gun proteomics, allowed the identification of 149 gene products, including low-abundance proteins escaped the 2-DE detection. Interestingly, we identified 60 proteins never catalogued previously. The complementary exploitation of two proteomic approaches enabled the access to new relevant information about the complexity of the peanut proteome, with special emphasis to the complement of allergens (allergome).

The objective of this study was to use the Tandem Mass Tag (TMT) isobaric label-based proteomic approach, in order to identify new potential biomarkers for the treatment monitoring of canine leishmaniosis that could not be identified by the use of gel-based techniques. For this purpose serum samples were obtained from 5 clinically diseased dogs before and one month after the treatment of canine leishmaniosis. The non-depleted serum samples were subjected to reduction, alkylation and trypsin digestion, and the resulting peptides were labeled using 6-plex TMT reagents. To obtain information about protein identities and relative quantification, liquid chromatography-MS analysis of multiplexed TMT-labeled peptides was employed. This gel-free, label-based quantitative proteomic approach enabled identification of 117 canine proteins. Among these, 23 showed significant difference (p < 0.05) in expression (two downregulated and 21 upregulated ranging from 1.25 to 2.5 fold change). Comparison of gel-free TMT-based quantification and a gel-based approach previously applied to the same samples resulted in the identification of some common markers (Apo-A1, vitamin D binding protein and RBP4). However, 20 additional differentially represented proteins were highlighted by the gel-free approach, 13 of which have not been previously reported in canine leishmaniosis. In conclusion, the TMT-based proteomic approach allowed identification of new serum proteins that significantly change in concentration after canine leishmaniosis treatment. These proteins are involved in various physiopathological processes such as inflammatory, coagulation or defense mechanisms, and could potentially be suitable biomarkers for treatment monitoring of this parasitic disease. Fundacion Seneca - 19894/GERM/15 ERA Chair initiative (VetMedZg) - 621394 Robles Chillida foundation European Commission European Commission Joint Research Centre

Mass spectrometry (MS)-based proteomics is nowadays considered as a mainstream analytical tool for life sciences. This success is mainly to be attributed to the tremendous improvements in the core technology of mass spectrometry coupled with recent developments in bioinformatics and separation sciences. The level of information that is henceforth accessible through MS techniques is broad and encompasses the protein sequence as well as its quantity, modification(s), structure, and implication in macromolecular assemblies. The current chapter focuses on the existing gel-free MS-based quantitative approaches that are applicable in the field of animal sciences and summarizes their principal features together with their respective advantages and drawbacks. In this context, we will discuss both the label-free and label-based relative quantitation strategies as well as the existing alternatives for absolute quantitation. The specific case of posttranslational modifications, whose extensive characterization is becoming a crucial topic in MS-based proteome studies, is next addressed under the quantitative scope of phosphorylation and glycosylation. Lastly, other promising nonmainstream techniques are presented, emphasizing their potent use to solve major proteomics challenges of tomorrow.

A proteome map of the foodborne pathogen Campylobacter jejuni NCTC11168 was analyzed using a state-of-the-art gel-free proteomic approach for the first time . A whole cell protein extract was prepared from the C. jejuni strain NCTC11168 grown in brain heart infusion (BHI) broth at 42°C under microaerobic conditions. A gel-free technique using isobaric tags for relative and absolute quantitation (iTRAQ) was employed to create a protein expression profile of the strain. Liquid chromatography-mass spectrometry (LC-MS/MS) was used to identify the proteins. Protein functionalities were searched to classify them. A total of 235 proteins were identified in the whole cell protein fraction of C. jejuni NCTC11168 cells using iTRAQ analysis. Functional grouping of the identified proteins showed that forty percent of these proteins were associated with energy metabolism, protein synthesis and genetic information processing. iTRAQ was faster, easier and proved more sensitive than two-dimensional gel-based proteomics approaches previously applied to C. jejuni , making it an attractive tool for further studies of cellular physiological response. DOI: http://dx.doi.org/10.3126/ijasbt.v2i4.11253 Int J Appl Sci Biotechnol, Vol. 2(4): 464-477

Proteomic analysis of human vitreous humor (VH) may elucidate the pathogenesis of retinal ocular diseases and may provide information for the development of potential therapeutic targets due to its pivotal location near lens and retina. The discovery of whole VH proteome involves a complex analysis of thousands of proteins simultaneously. Therefore, in proteomic studies the protein fractionation is important for reducing sample complexity, facilitating the access to the low-abundant proteins, and recognizing them as biotargets for clinical research. Although several separation methods have been used, gel-based proteomics are the most popular and versatile ones applied for global protein separation. However, chromatographic methods and its combination with other separation techniques are now beginning to be used as promising set-ups for VH protein identification. This review attempts to offer an overview of the techniques currently used with VH, exploring its methodological demands, exposing its advantages, and helping the reader to plan future experiences. Moreover, this review shows the relevance of VH proteomic analysis as a tool for the study of the mechanisms underlying some ocular diseases and for the development of new therapeutic approaches.

Alireza Ghassempour, PhDDepartment of PhytochemistryMedicinal Plants and Drugs Research InstituteShahid Beheshti UniversityDaneshjoo Boulevard., 19835-389 Tehran, Iranaghassempour@scientist.com© 2014 S. Karger GmbH, Freiburg1661-4119/14/0214-••••$39.50/0Accessible online at: www.karger.com/fokFax +49 761 4 52 07 14Information@Karger.comwww.karger.com

Absolute protein quantification is an essential tool for system biology approaches and elucidation of stoichiometry of multi-protein complexes. In this chapter, a universal protocol for gel free absolute protein quantification in bacterial systems is described, which can be used for sample preparation prior to miscellaneous mass-spectrometry-based quantification workflows like AQUA, Hi3, and emPAI. In addition, a focus has been set to the specific challenges in antibiotic stress research.

In more than 20 years of the proteomics era, considerable technical developments and scientific discoveries have contributed to the advancement of this field of research. Gel-based proteomics has been popular for studying the proteomic changes during growth and development of plants as well as for the analysis of responses to different biotic and abiotic stimuli. The most widely used gel-based technique is two dimensional (2D) gel electrophoresis, in which around 2000 protein spots can be clearly visualized and processed prior to identification by mass spectrometry. Although many techniques have been developed to improve the quality and number of spots in a 2D gel, these enhancements are still not good enough to study the entire cellular proteome. It is important to also consider low abundant proteins, many of which could play critical roles in particular biological processes. Considering that thousands of proteins are expressed at a given time and each protein can undergo one or more post-translational modifications—are we able to capture all this information and the entire proteome using gel-based proteomic techniques? Are gel-free alternatives capable of addressing these challenges? The answer is no for both approaches. So, how long are we going to run traditional 1D and 2D gels in our research? Though we have been successful in developing many advanced gel-free proteomic techniques to study the cellular proteome, we still lack advances that enable the examination of each and every protein expressed in a system as well as any post-translational modification (PTM) at a given time. Gel-free and gel-based techniques may complement each other, however there needs to be synergy or a “technical fusion” in which researchers can find a balance between quick and efficient methods for studying whole cell proteomes in plants.

Understanding the activity and viability of bacterial populations provides an insight into their physiology, reactions to stress and response to environmental change. As the key goals in food safety involve the surveillance, control and risk reduction of foodborne pathogens, physiologically oriented studies are of great relevance, allowing for detection and prediction of growth and activity. This review examines the progress, utilization and pitfalls of innovative and emerging non-nucleic acid based methods that can be linked with data generated through genome sequencing projects to provide an enhanced understanding of foodborne pathogen physiology and activity.