Differential peptidomics highlights adipokinetic hormone as key player in regulating digestion in anautogenous flesh fly, Sarcophaga crassipalpis.

Females of anautogenous flesh flies, Sarcophaga crassipalpis, need a protein meal in order to produce their first batch of eggs. This protein meal elicits an increase in midgut proteolytic activity that is under neuropeptidergic regulation. Time series of decapitation and rescue experiments of liver fed flies evidenced the need of a peptide factor released by corpora cardiaca (CC) within 4h post protein feeding in order to assure complete protein digestion. Q-Exactive quantitative differential peptidomics analysis on CC of sugar fed flies and flies 5h post protein feeding respectively, showed a unique consistent decrease in the stored amount of adipokinetic hormone (AKH) ranging between 16% up to 63%. Injection of AKH into liver fed decapitated flies as well as sugar fed intact flies resulted in dose dependent enhanced midgut proteolytic activity up to the level of intact protein fed flies. This suggests a key role of AKH in food depended reproduction.

Analysis of the formalin-fixed paraffin-embedded tissue proteome: pitfalls, challenges, and future prospectives.

Formalin-fixed paraffin-embedded (FFPE) tissues are a real treasure for retrospective analysis considering the amount of samples present in hospital archives, combined with pathological, clinical, and outcome information available for every sample. Although unlocking the proteome of these tissues is still a challenge, new approaches are being developed. In this review, we summarize the different mass spectrometry platforms that are used in human clinical studies to unravel the FFPE proteome. The different ways of extracting crosslinked proteins and the analytical strategies are pointed out. Also, the pitfalls and challenges concerning the quality of FFPE proteomic approaches are depicted. We also evaluated the potential of these analytical methods for future clinical FFPE proteomics applications.

Abundant plasma protein depletion using ammonium sulfate precipitation and Protein A affinity chromatography.

Plasma is a highly valuable resource for biomarker research since it is easy obtainable and contains a high amount of information on patient health status. Although advancements in the field of proteomics enabled analysis of the plasma proteome, identification of low abundant proteins remains challenging due to high complexity and large dynamic range. In order to reduce the dynamic range of protein concentrations, a tandem depletion technique consisting of ammonium sulfate precipitation and Protein A affinity chromatography was developed. Using this method, 50% of albumin, together with other high abundant proteins such as alpha-1-antitrypsin, was depleted from the plasma sample at 20% to 40% ammonium sulfate saturation levels. In combination with immunoglobulin removal using a Protein A column, this technique delivered up to 40 new low- to medium abundance protein identifications when performing a shotgun mass spectrometry analysis. Compared to non-depleted plasma, 270 additional protein spots were observed during 2D-PAGE analysis. These results illustrate that this tandem depletion method is equivalent to commercial kits which are based on immune-affinity chromatography. Moreover, this method using Protein A immunoglobulin depletion was shown to be highly reproducible and a minimal amount of non-target proteins was depleted. The combination of ammonium sulfate precipitation and Protein A affinity chromatography offers a low cost, efficient, straightforward and reproducible alternative to commercial kits, with proteins remaining in native conformation, allowing protein activity and protein interaction studies.

Comparison of multiple protein extraction buffers for GeLC-MS/MS proteomic analysis of liver and colon formalin-fixed, paraffin-embedded tissues.

Formalin-fixed paraffin-embedded (FFPE) tissue specimens represent a potential valuable source of samples for clinical research. Since these specimens are banked in hospital archives, large cohorts of samples can be collected in short periods of time which can all be linked with a patients' clinical history. Therefore, the use of FFPE tissue in protein biomarker discovery studies gains interest. However, despite the growing number of FFPE proteome studies in the literature, there is a lack of a FFPE proteomics standard operating procedure (SOP). One of the challenging steps in the development of such a SOP is the ability to obtain an efficient and repeatable extraction of full length FFPE proteins. In this study, the protein extraction efficiency of eight protein extraction buffers is critically compared with GeLC-MS/MS (1D gel electrophoresis followed by in-gel digestion and LC-MS/MS). The data variation caused by using these extraction buffers was investigated since the variation is a very important aspect when using FFPE tissue as a source for biomarker detection. In addition, a qualitative comparison was made between the protein extraction efficiency and repeatability for FFPE tissue and fresh frozen tissue.

Characterization and pharmacological analysis of two adipokinetic hormone receptor variants of the tsetse fly, Glossina morsitans morsitans.

Adipokinetic hormones (AKH) are well known regulators of energy metabolism in insects. These neuropeptides are produced in the corpora cardiaca and perform their hormonal function by interacting with specific G protein-coupled receptors (GPCRs) at the cell membranes of target tissues, mainly the fat body. Here, we investigated the sequences, spatial and temporal distributions, and pharmacology of AKH neuropeptides and receptors in the tsetse fly, Glossina morsitans morsitans. The open reading frames of two splice variants of the Glomo-akh receptor (Glomo-akhr) gene and of the AKH neuropeptide encoding genes, gmmhrth and gmmakh, were cloned. Both tsetse AKHR isoforms show strong sequence conservation when compared to other insect AKHRs. Glomo-AKH prepropeptides also have the typical architecture of AKH precursors. In an in vitro Ca(2+) mobilization assay, Glomo-AKH neuropeptides activated each receptor isoform up to nanomolar concentrations. We identified structural features of tsetse AKH neuropeptides essential for receptor activation in vitro. Gene expression profiles suggest a function for AKH signaling in regulating Glossina energy metabolism, where AKH peptides are released from the corpora cardiaca and activate receptors mainly expressed in the fat body. This analysis of the ligand-receptor coupling, expression, and pharmacology of the two Glomo-AKHR variants facilitates further elucidation of the function of AKH in G. m. morsitans.

Integrating -Omics: Systems Biology as Explored Through C. elegans Research.

-Omics data have become indispensable to systems biology, which aims to describe the full complexity of functional cells, tissues, organs and organisms. Generating vast amounts of data via such methods, researchers have invested in ways of handling and interpreting these. From the large volumes of -omics data that have been gathered over the years, it is clear that the information derived from one -ome is usually far from complete. Now, individual techniques and methods for integration are maturing to the point that researchers can focus on network-based integration rather than simply interpreting single -ome studies. This review evaluates the application of integrated -omics approaches with a focus on Caenorhabditis elegans studies, intending to direct researchers in this field to useful databases and inspiring examples.

Formalin-fixed paraffin-embedded tissue: The holy grail of clinical proteomics.

Tissue is the most relevant biological material to gather insight in disease mechanisms by means of omics technologies. However, fresh frozen tissue, which is generally regarded as the best imaginable source for such studies, is often not available. In case it is available, the different ways of storage (e.g. -20°C, -80°C, liquid nitrogen, etc.) hamper the conduction of reproducible multicenter studies because of different protein degradation rates. Formalin-fixed paraffin-embedded (FFPE) tissue on the contrary is considered as a valuable alternative for fresh frozen tissue, because only a few standard operation procedures are applied worldwide for the preparation of these tissues and because they are all stored in the same way. However, a study on the impact of the different preparation protocols for FFPE tissue was still lacking. Therefore, Bronsert et al. in this issue [Bronsert, P., Weißer, J., Biniossek, M. L., Kuehs, M. et al., Proteomics Clin. Appl. 2014, 8 786-804] conducted such a study that provides proof that there is no significant effect between these sample preparations procedures, and thereby they further open the gate for FFPE tissues to enter the field of clinical proteomics.

Proteomic analysis of formalin-fixed paraffin-embedded colorectal cancer tissue using tandem mass tag protein labeling.

In clinical research, repositories of biological samples form a rich source of clinical material for biomarker studies. Banked material, however, is often not stored in optimal conditions regarding the technology used for biomarker research. A case in point is formalin-fixed paraffin-embedded (FFPE) tissue that could be used to obtain large cohorts of samples over a short period of time, as these tissues are routinely prepared for pathological analysis. However, in the context of mass spectrometry based peptide-centric proteomics, protein extraction and identification can be hampered by formalin-induced crosslinking. Furthermore, the molecular formalin crosslinks might be entangled differently across various samples, making it more difficult to reproducibly extract the same proteins from different samples. In this study, we establish the crosslink variability using Tandem Mass Tag (TMT) protein labeling followed by digestion, separation, identification and quantification of proteins extracted from FFPE colorectal cancer and paired healthy tissues. Moreover, by applying de novo interpretation of tandem mass spectra and subsequent analysis by Peaks PTM, unspecified modifications could be elucidated, leading to increased protein and proteome coverage. This approach might be useful for future FFPE proteomics studies.