For label-free expression profiling of tissue proteomes, efficient protein extraction, thorough and quantitative sample cleanup and digestion procedures, as well as sufficient and reproducible chromatographic separation, are highly desirable but remain challenging. However, optimal methodology has remained elusive, especially for proteomes that are rich in membrane proteins, such as the mitochondria. Here we describe a straightforward and reproducible sample preparation procedure, coupled with a highly selective and sensitive nano-LC/Orbitrap analysis, which enables reliable and comprehensive expression profiling of tissue mitochondria. The mitochondrial proteome of swine heart was selected as a test system. Efficient protein extraction was accomplished using a strong buffer containing both ionic and non-ionic detergents. Overnight precipitation was used for cleanup of the extract, and the sample was subjected to an optimized 2-step, on-pellet digestion approach. In the first step, the protein pellet was dissolved via a 4 h tryptic digestion under vigorous agitation, which nano-LC/LTQ/ETD showed to produce large and incompletely cleaved tryptic peptides. The mixture was then reduced, alkylated, and digested into its full complement of tryptic peptides with additional trypsin. This solvent precipitation/on-pellet digestion procedure achieved significantly higher and more reproducible peptide recovery of the mitochondrial preparation, than observed using a prevalent alternative procedure for label-free expression profiling, SDS-PAGE/ingel digestion (87% vs. 54%). Furthermore, uneven peptide losses were lower than observed with SDS-PAGE/in-gel digestion. The resulting peptides were sufficiently resolved by a 5 h gradient using a nano-LC configuration that features a low-void-volume, high chromatographic reproducibility, and an LTQ/Orbitrap analyzer for protein identification and quantification. The developed method was employed for label-free comparison of the mitochondrial proteomes of myocardium from healthy animals vs. those with hibernating myocardium. Each experimental group consisted of a relatively large number of animals (n=10), and samples were analyzed in random order to minimize quantitative false-positives. Using this approach, 904 proteins were identified and quantified with high confidence, and those mitochondrial proteins that were altered significantly between groups were

Datura metel is a plant that contains several different neurologically active phytochemicals which affect human health. On-going research has examined the potential role of the human neuroindoles, melatonin and serotonin, in medicinal plants with neurological efficacy. In this report, we describe the quantification of melatonin and serotonin in flowers and developing fruits of Datura metel and the effects of cold stress on the levels of these neuroindoles in the reproductive tissues of this plant. Melatonin and serotonin were found at the highest levels in the least developed flower buds with decreasing concentrations as the flower buds matured. Cold stress significantly increased the concentration of melatonin in young flower buds. In the developing fruit, melatonin was present at relatively stable, high concentrations for the first 10 days after anthesis. After 10-15 days, the ovule had grown to a sufficient size for excision and analysis and melatonin was found to be at the highest concentrations in the developing ovule with minimal concentrations of the neuroindoles in the fleshy fruit. Together, these data indicate that melatonin may play a role in protecting the reproductive tissues during flower and seed formation in a Datura species.

Liquid chromatography (LC)/mass spectrometry (MS) in selected-reactions-monitoring (SRM) mode provides a powerful tool for targeted protein quantification. However, efficient, high-throughput strategies for proper selection of signature peptides (SP) for protein quantification and accurate optimization of their SRM conditions remain elusive. Here we describe an on-the-fly, orthogonal array optimization (OAO) approach that enables rapid, comprehensive, and reproducible SRM optimization of a large number of candidate peptides in a single nanoflow-LC/MS run. With the optimized conditions, many peptide candidates can be evaluated in biological matrices for selection of the final SP. The OAO strategy employs a systematic experimental design that strategically varies product ions, de-clustering energy and collision energy in a cycle of 25 consecutive SRM trials, which accurately reveals the effects of these factors on the single-to-noise ratio of a candidate peptide, and optimizes each. As proof of concept, we developed a highly sensitive, accurate, and reproducible method for the quantification of carbonyl reductases CBR1 and CBR3 in human liver. Candidate peptides were identified by nano-LC/LTQ/Orbitrap, filtered using a stringent set of criteria, and subjected to OAO. After evaluating both sensitivity and stability of the candidates, two SP were selected for quantification of each protein. As a result of the accurate OAO of assay conditions, sensitivities of 80 and 110 amol were achieved for CBR1 and CBR3, respectively. The method was validated and used to quantify the CBRs in 33 human liver samples. The mean level of CBR1 was 93.4±49.7 (range: 26.2–241) ppm of total protein, and for CBR3 was 7.69±4.38 (range: 1.26–17.9) ppm. Key observations of this study are that: i) evaluation of peptide stability in the target matrix is essential for final selection of the SP; ii) utilization of two unique SP contributes to high reliability of target protein quantification; and iii) it is beneficial to construct calibration curves using standard proteins of verified concentrations to avoid severe biases that may result if synthesized peptides alone are used. Overall, the OAO method is versatile and adaptable to high-throughput quantification of validated biomarkers identified by proteomic discovery experiments.

Oxyntomodulin (OXM) is a peptide secreted postprandially from the L-cells of the gut that has a weak affinity for both the glucagon-like peptide-1 receptor (GLP1R) and the glucagon receptor (GCGR). Peripheral administration of OXM in humans and rodents causes weight loss reducing food intake and increasing energy expenditure. It has been suggested that OXM modulates energy intake solely through GLP1R agonism. Because glucagon decreases food intake in rodents and humans, we examined whether activation of the GCGR is involved in the body weight-lowering effects of OXM. We identified an equipotent GLP1R-selective peptide agonist that differs from OXM by only one residue (Q3→E, OXMQ3E), but has no significant GCGR agonist activity in vitro and ~100-fold reduced ability to stimulate liver glycogenolysis. Chronic treatment of obese mice with OXM and OXMQ3E demonstrated that OXM exhibits superior weight loss and lipid-lowering efficacy, and antihyperglycemic activity that is comparable to the corresponding GLP1R-selective agonist. Studies in Glp1r−/− mice and coadministration of OXM and a GCGR antagonist revealed that the antiobesity effect of OXM requires activation of both GLP1R and GCGR. Our data provide new insight into the mechanism of action of OXM and suggest that activation of GCGR is involved in the body weight-lowering action of OXM.

Protein arginine (Arg) methylation serves an important functional role in eukaryotic cells, and typically occurs in domains consisting of multiple Arg in close proximity. Localization of methylarginine (MA) within Arg-rich domains poses a challenge for mass spectrometry (MS)-based methods; the peptides are highly-charged under electrospray ionization (ESI), which limits the number of sequence-informative products produced by collision induced dissociation (CID), and loss of the labile methylation moieties during CID precludes effective fragmentation of the peptide backbone. Here the fragmentation behavior of Arg-rich peptides was investigated comprehensively using electron transfer dissociation (ETD) and CID for both methylated and unmodified glycine-/Arg-rich peptides (GAR), derived from residues 679-695 of human nucleolin, which contains methylation motifs that are widely-represented in biological systems. ETD produced abundant information for sequencing and MA localization, whereas CID failed to provide credible identification for any available charge state (z=2-4). Nevertheless, CID produced characteristic neutral losses that can be employed to distinguish among different types of MA, as suggested by previous works and confirmed here with product ion scans of high accuracy/resolution by an LTQ/Orbitrap. To analyze MA-peptides in relatively complex mixtures, a method was developed that employs nano-LC coupled to alternating CID/ETD for peptide sequencing and MA localization/characterization, and an Orbitrap for accurate precursor measurement and relative quantification of MA-peptide stoichiometries. As proof of concept, GAR-peptides methylated in vitro by protein arginine N-methyltransferases PRMT1 and PRMT7 were analyzed. It was observed that PRMT1 generated a number of monomethylated (MMA) and asymmetric-dimethylated peptides, while PRMT7 produced predominantly MMA peptides and some symmetric-dimethylated peptides. This approach and the results may advance understanding of the actions of PRMTs and the functional significance of Arg methylation patterns.