The iTRAQ (isobaric tags for relative and absolute quantification) technique is widely employed in proteomic workflows requiring relative quantification. Here, we review the iTRAQ literature; in particular, we focus on iTRAQ usage in relation to other commonly used quantitative techniques e.g. stable isotope labelling in culture (SILAC), label-free methods and selected reaction monitoring (SRM). As a result, we identify several issues arising with respect to iTRAQ. Perhaps frustratingly, iTRAQ's attractiveness has been undermined by a number of technical and analytical limitations: it may not be truly quantitative, as the changes in abundance reported will generally be underestimated. We discuss weaknesses and strengths of iTRAQ as a methodology for relative quantification in the light of this and other technical issues. We focus on technical developments targeted at iTRAQ accuracy and precision, use of 4-plex over 8-plex reagents and application of iTRAQ to post-translational modification (PTM) workflows. We also discuss iTRAQ in relation to label-free approaches, to which iTRAQ is losing ground.

We present a predicted passerine genome map consisting of 196 microsatellite markers distributed across 25 chromosomes. The map was constructed by assigning chromosomal locations based on the sequence similarity between 550 publicly available passerine microsatellites and the draft chicken genome sequence published by the International Chicken Genome Sequencing Consortium. We compared this passerine microsatellite map with a recently published great reed warbler (Acrocephalus arundinaceus) linkage map derived from the segregation of marker alleles in a pedigree of a natural population. Twenty-four microsatellite markers were shared between the two maps, distributed across ten chromosomes. Synteny was maintained between the predicted passerine microsatellite map and the great reed warbler linkage map, confirming the validity and accuracy of our approach. Possible applications of the predicted passerine microsatellite map include genome mapping; quantitative trait locus (QTL) discovery; understanding heterozygosity-fitness correlations; investigating avian karyotype evolution; understanding microsatellite mutation processes; and for identifying loci conserved in multiple species, unlinked loci for use in genotyping sets and sex-linked markers.

The commercial reality of bioactive compounds and oil production from microalgal species is constrained by the high cost of production. Downstream processing, which includes harvesting and extraction, can account for 70–80% of the total cost of production. Consequently, from an economic perspective extraction technologies need to be improved. Microalgal cells are difficult to disrupt due to polymers within their cell wall such as algaenan and sporopollenin. Consequently, solvents and disruption devices are required to obtain products of interest from within the cells. Conventional techniques used for cell disruption and extraction are expensive and are often hindered by low efficiencies. Microwave-assisted extraction offers a possibility for extraction of biochemical components including lipids, pigments, carbohydrates, vitamins and proteins, individually and as part of a biorefinery. Microwave technology has advanced since its use in the 1970s. It can cut down working times and result in higher yields and purity of products. In this review, the ability and challenges in using microwave technology are discussed for the extraction of bioactive products individually and as part of a biorefinery approach.

Nitrogen starvation induced changes in carbohydrate and lipid content is described in several algal species. Although these phenotypic changes are desirable, such manipulations also significantly deteriorate culture health, ultimately halting growth. To optimize biofuel production from algae, it is desirable to induce lipid accumulation without compromising cell growth and survival. In this study, we utilized an 8-plex iTRAQ-based proteomic approach to assess the model alga Chlamydomonas reinhardtii CCAP 11/32CW15+ under nitrogen starvation. First-dimension fractionation was conducted using HILIC and SCX. A total of 587 proteins were identified (≥3 peptides) of which 71 and 311 were differentially expressed at significant levels (p<0.05), during nitrogen stress induced carbohydrate and lipid production, respectively. Forty-seven percent more changes with significance were observed with HILIC compared to SCX. Several trends were observed including increase in energy metabolism, decrease in translation machinery, increase in cell wall production and a change of balance between photosystems I and II. These findings point to a severely compromised system where lipid is accumulated at the expense of normal functioning of the organism, suggesting that a more informed and controlled method of lipid induction than gross nutrient manipulation would be needed for development of sustainable processes.

We examine differential protein expression in Euhalothece sp. BAA001, an extremely halotolerant and unsequenced cyanobacterium, under adaptation to low (0% w/v), medium (3% w/v), high (6% w/v) and very high (9% w/v) salt concentrations using cross-species protein identification tools. We combine stable isotope labelling with 1-D SDS-PAGE, and MASCOT protein identification software with MS-driven BLAST searches, to produce an accurate method for protein identification and quantitation. The use of metabolic labelling to improve the confidence in identification of proteins in cross-species proteomics is demonstrated. Three hundred and eighty-three unique proteins were identified, and 72 were deemed to be differentially expressed (average CV for quantitations was 0.10 +/- 0.08), belonging to 24 functional groups. Responses to low salt as well as high salt are discussed in terms of adaptation and evidence shows that Euhalothece cells display 'stress' responses in nonsaline conditions as well as higher salt environments.

Cyanobacteria are ancient life forms and have adapted to a variety of extreme environments, including high salinity. Biochemical, physiological and genetic studies have contributed to uncovering their underlying survival mechanisms, and as recent studies demonstrate, proteomics has the potential to increase our overall understanding further. To date, most salt-related cyanobacterial proteomic studies have utilised gel electrophoresis with the model organism Synechocystis sp. PCC6803. Moreover, focus has been on 2-4% w/v NaCl concentrations within different cellular compartments. Under these conditions, Synechocystis sp. PCC6803 was found to respond and adapt to salt stress through synthesis of general and specific stress proteins, altering the protein composition of extracellular layers, and re-directing control of complex central intermediary pathways. Post-transcriptional control was also predicted through non-correlating transcript level data and identification of protein isoforms.In this paper, we also review technical developments with emphasis on improving the quality and quantity of proteomic data and overcoming the detrimental effects of salt on sample preparation and analysis. Developments in gel-free methods include protein and peptide fractionation workflows, which can increase coverage of the proteome (20% in Synechocystis sp. PCC6803). Quantitative techniques have also improved in accuracy, resulting in confidence in quantitation approaching or even surpassing that seen in transcriptomic techniques (better than 1.5-fold in differential expression). Furthermore, in vivo metabolic labelling and de novo protein sequencing software have improved the ability to apply proteomics to unsequenced environmental isolates. The example used in this review is a cyanobacterium isolated from a Saharan salt lake.

Recently, the prospect of using Escherichia coli as a host for human glycoprotein production has increased due to detailed characterization of the prokaryotic N-glycosylation process and the ability to transfer the system into this bacterium. Although functionality of the native Campylobacter jejuni N-glycosylation system in E. coli has been demonstrated, the efficiency of the process using the well-characterized C. jejuni glycoprotein AcrA, was found to be low at 13.4±0.9% of total extracted protein. A combined approach using isobaric labeling of peptides and probability-based network analysis of metabolic changes was applied to forward engineer E. coli to improve glycosylation efficiency of AcrA. Enhancing flux through the glyoxylate cycle was identified as a potential metabolic manipulation to improve modification efficiency and was achieved by increasing the expression of isocitrate lyase. While the overall recombinant protein titre did not change significantly, the amount of glycosylated protein increased by approximately 300%.

In this study, we conducted biological and technical replicate proteomic experiments using isobaric tags for relative and absolute quantification (iTRAQ), to elucidate the light adaptation strategies of Prochlorococcus marinus MED4. The MED4 strain is adapted to an oceanic environment characterized by low nutrient levels, and ever-changing light intensities. Approximately 11% of the proteome was identified, with an average coefficient of variation of iTRAQ quantification values of 0.15. Fifteen proteins were deemed to be statistically and significantly differentially expressed in changing light intensities, particularly the down-regulation of photosystem-related proteins, and the up-regulation of the stress-related chaperone GroEL in high light compared to low light.