The analysis of phosphopeptides by mass spectrometry (MS) is one of the most challenging tasks in proteomics. This is due to the lower isoelectric point (pI) of phosphopeptides, which leads to inefficient sample ionization in MS, particularly when competing with other peptides. The problem is compounded by the typical low abundance of phosphopeptides in biological samples. We describe here a simple nonsorptive method to isolate phosphopeptides based on their pI. A voltage is applied to selectively migrate the phosphopeptides into a capillary, which are negatively charged at acidic pH. The selectively sampled fraction is directly deposited onto MALDI sample target in nanoliter volumes (7−35 nL) for highly sensitive MS detection. No significant sample loss is evident in this procedure; hence, the MS was able to detect the isolated phosphopeptides at trace quantity. In this case, attomole-level detection limit is achieved for synthetic phosphopeptides (nM concentration and nL volume), from a mixture containing other peptides at up to 1 million times higher in concentration. Selective sampling was also applied to the tryptic digest of β- and α-caseins to reveal the multiple phosphorylated peptides at the low-femtomole level using MALDI MS. Knowledge of pI based on the rejection/injection of peptides was found to be useful in peak assignment. To confirm the sequence of the selectively sampled peptides, fraction collection was performed for offline ESI MS/MS analysis.
Pyrolysis converts biomass such as agricultural and forestry waste into bio-oil, preserving some chemicals while creating other, new ones. Nicotine, a chemical present in tobacco leaves and a known pesticide, was found to remain intact during pyrolysis. As expected, insecticidal properties were observed for tobacco bio-oil. Pesticide characteristics of tobacco bio-oil have been observed on the Colorado potato beetle (CPB), a pest currently resistant to all major insecticides, as well as a few bacteria and fungi that do not currently respond well to chemical treatment. Unexpectedly, nicotine-free fractions of the bio-oil were also found to be highly lethal to the beetles and successful at inhibiting the growth of select microorganisms. Through GC-MS, it was found that the active, nicotine-free fractions were rich in phenolics, chemicals likely created from lignin during pyrolysis. While bio-oils in general are known to contain phenolic chemicals, such as cresols, to our best knowledge, quantitative analysis has not been performed to determine if these chemicals are solely responsible for the observed pesticide activities. Based on GC-MS results, ten of the most abundant chemicals, eight of which were phenolic chemicals, were identified and examined through bio-assays. A mixture of these chemicals at the concentration levels found in the bio-oil did not account for the bio-oil activity towards the microorganisms. Tobacco bio-oil may have potential as a pesticide, however, further analyses using liquid chromatography is necessary to identify the remaining active chemicals.
The Colorado potato beetle (CPB) is one of the most adaptable insect pests to both plant toxins and synthetic insecticides. Resistance in CPB is reported for over 50 classes of insecticides, and mechanisms of insecticide-resistance include enhanced detoxification enzymes, ABC transporters and target site mutations. Adaptation to insecticides is also associated with changes in behaviour, energy metabolism and other physiological processes seemingly unrelated to resistance but partially explained through genomic analyses. In the present study, in place of genomics, we applied 2-dimensional (2-D) gel and mass spectrometry to investigate protein differences in abdominal and midgut tissue of insecticide-susceptible (S) and -resistant (R) CPB. The proteomic analyses measured constitutive differences in several proteins, but the highest match was identified as a C-type lectin (CTL), a component of innate immunity in insects. The constitutive expression of the CTL was greater in the multi-resistant (LI) strain, and the same spot was measured in both midgut and abdominal tissue. Exposure to the neonicotinoid insecticide, imidacloprid, increased the CTL spot found in the midgut but not in the abdominal tissue of the laboratory (Lab) strain. No increase in protein levels in the midgut tissue was observed in the LI or a field strain (NB) tolerant to neonicotinoids. With the exception of biopesticides, such as Bacillus thuringiensis (Bt), no previous studies have documented differences in the immune response by CTLs in insects exposed to synthetic insecticides or the fitness costs associated with expression levels of immune-related genes in insecticide-resistant strains. This study demonstrates again how CPB has been successful at adapting to insecticides, plant defenses as well as pathogens.
Ischemic stroke is a complex and devastating event characterized by cell death resulting from a transient or permanent arterial occlusion. Astrocytic connexin43 (Cx43) gap junction (GJ) proteins have been reported to impact neuronal survival in ischemic conditions. Consequently, Cx43 could be a potential target for therapeutic approaches to stroke. We examined the effect of danegaptide (ZP1609), an antiarrhythmic dipeptide that specifically enhances GJ conductance, in two different rodent stroke models. In this study, danegaptide increased astrocytic Cx43 coupling with no significant effects on Cx43 hemichannel activity, in vitro. Using matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI IMS) the presence of danegaptide within brain tissue sections were detected one hour after reperfusion indicating successful transport of the dipeptide across the blood brain barrier. Furthermore, administration of danegaptide in a novel mouse brain ischemia/reperfusion model showed significant decrease in infarct volume. Taken together, this study provides evidence for the therapeutic potential of danegaptide in ischemia/reperfusion stroke.
Phosphatidylcholine (PC) is one of the major phospholipids that make up the biological cell membrane. It was previously reported to form capillary inner wall coatings for CE. The zwitterionic head group of PC produced a neutral net-charged bilayer, which was found effective in preventing wall adsorption of both cationic and anionic proteins [J. M. Cunliffe et al. Anal. Chem. 2002, 74, 776-783]. Another major membrane phospholipid that possesses a zwitterionic head group is sphingomyelin (SM). In this work, the novel characterization of SM on its effectiveness in capillary coating formation for CE separations of proteins and peptides was presented. Similar properties were observed between PC and SM, including their effects on the EOF, peak efficiencies, and migration time reproducibilities. SM appeared to be more readily soluble in aqueous solutions, and it was found equally effective as PC in facilitating protein separation. The main difference observed was their performances in delivering a peptide mixture for off-line analysis with MALDI-MS. Superior sample recovery was evident from the capillary coated with SM compared with that with PC. The number of peptides identified from a 1 ng/microL myglobin tryptic digest sample increased from 5 to 16 (42 and 69%, respectively in protein sequence coverage).
