Proteomics for biomarker validation needs high throughput instrumentation to analyze huge set of clinical samplesfor quantitative and reproducible analysis at a minimum time without manual experimental errors. Sample preparation, a vitalstep in proteomics plays a major role in identification and quantification of proteins from biological samples. Tryptic digestion amajor check point in sample preparation for mass spectrometry based proteomics needs to be more accurate with rapid processingtime. The present study focuses on establishing a high throughput automated online system for proteolytic digestion anddesalting of proteins from biological samples quantitatively and qualitatively in a reproducible manner. The present study comparesonline protein digestion and desalting of BSA with conventional off-line (in-solution) method and validated for real timesample for reproducibility. Proteins were identified using SEQUEST data base search engine and the data were quantified usingIDEALQ software. The present study shows that the online system capable of handling high throughput samples in 96 well formatscarries out protein digestion and peptide desalting efficiently in a reproducible and quantitative manner. Label free quantificationshowed clear increase of peptide quantities with increase in concentration with much linearity compared to off linemethod. Hence we would like to suggest that inclusion of this online system in proteomic pipeline will be effective in quantificationof proteins in comparative proteomics were the quantification is really very crucial.
Chalcones are naturally occurring, biologically active molecules generating interest from a wide range of researchapplications including synthetic methodology development, biological activity investigation and studying fragmentation patterns. Inthis article, a series of chalcones has been synthesized and their fragmentation behavior was studied using modern ambient ionizationtechnique Direct Analysis in Real Time (DART). DART ion source connected with an ion trap mass spectrometer wasused for the fragmentation of various substituted chalcones. The chalcones were introduced to the DART source using a glasscapillary without sample preparation step. All the chalcones showed prominent molecular ion peaks [M]•+ corresponding to thestructures. Multistage mass spectral data MSn (MS2 and MS3) were collected for all the chalcones studied. The chalcones withsubstitutions at 3, 4 or 5 positions gave product ion peaks with the loss of a phenyl radical (Ph•) by radical initiated α-cleavage,while substitution at 2 position of chalcone in the A-ring gave a product ion peak with the loss of substituted styryl radical(PhCH = CH•). In case of the chalcones with the substituent at 4 positions in A and B rings gave both types of fragmentation patterns. In conclusion, chalcones can be easily characterized using modern DART interface in very short time and efficientlywithout any cumbersome sample pretreatment.
Honokiol and magnolol, the major bioactive neolignans of magnolia officinalis, are the most important constituents ofthe crude drug prescriptions that are used in the therapy of neuroses and various nervous disorders. There have been limitedreports on the effects of neolignoid compounds on human cytochrome P450 activity. Therefore, the inhibitory effects of honokioland magnolol on seven human cytochrome P450 s were evaluated in human liver microsomes. Honokiol and magnololshowed the most potent inhibition of CYP1A2-mediated phenacetin O-deethylase activity (IC50 values of 3.5 and 5.4 mM,respectively) among the seven P450s tested. These in vitro data indicate that neolignan compounds can inhibit the activity ofCYP1A2 and suggest that these compounds should be examined for potential pharmacokinetic drug interactions in vivo.
Glycans released from ovalbumin by PNGase F were analyzed by matrix-assisted laser desorption/ionization time-offlight(MALDI-TOF) mass spectrometry using three different dihydroxybenzoic acid (DHB) matrix systems: 2,5-DHB, 2,6-DHB, and a 2,5-DHB/2,6-DHB binary matrix. Relative to the results obtained with the single-component matrices (2,5-DHB or2,6-DHB), the 2,5-DHB/2,6-DHB binary matrix boasted lower background noise and higher sensitivity. A total of 16 glycanpeaks were observed using the 2,5-DHB/2,6-DHB binary matrix, while only 10 and 9 glycan peaks were observed using the 2,5-DHB and 2,6-DHB matrices, respectively.