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Subsequent Identification (subsequent + identification)
Selected AbstractsAn integrated serum proteomic approach capable of monitoring the low molecular weight proteome with sequencing of intermediate to large peptidesRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 17 2009Karen Merrell The low-abundance, low molecular weight serum proteome has high potential for the discovery of new biomarkers using mass spectrometry (MS). Because the serum proteome is large and complex, defining relative quantitative differences for a molecular species between comparison groups requires an approach with robust separation capability, high sensitivity, as well as high mass resolution. Capillary liquid chromatography (cLC)/MS provides both the necessary separation technique and the sensitivity to observe many low-abundance peptides. Subsequent identification of potential serum peptide biomarkers observed in the cLC/MS step can in principle be accomplished by in series cLC/MS/MS without further sample preparation or additional instrumentation. In this report a novel cLC/MS/MS method for peptide sequencing is described that surpasses previously reported size limits for amino acid sequencing accomplished by collisional fragmentation using a tandem time-of-flight MS instrument. As a demonstration of the approach, two low-abundance peptides with masses of ,4000,5000,Da were selected for MS/MS sequencing. The multi-channel analyzer (MCA) was used in a novel way that allowed for summation of 120 fragmentation spectra for each of several customized collision energies, providing more thorough fragmentation coverage of each peptide with improved signal to noise. The peak list from this composite analysis was submitted to Mascot for identification. The two index peptides, 4279,Da and 5061,Da, were successfully identified. The peptides were a 39 amino acid immunoglobulin G heavy chain variable region fragment and a 47 amino acid fibrin alpha isoform C-terminal fragment. The method described here provides the ability both to survey thousands of serum molecules and to couple that with markedly enhanced cLC/MS/MS peptide sequencing capabilities, providing a promising technique for serum biomarker discovery. Copyright © 2009 John Wiley & Sons, Ltd. [source] The influence of HIV/AIDS on the practice of primary care nurses in Jordan: Rhetoric and realityINTERNATIONAL JOURNAL OF NURSING PRACTICE, Issue 5 2005Hani Nawafleh PhD(Cand) The role of nurses in raising community awareness about HIV/AIDS is well-reported. However, little is known about the practice of Jordanian nurses and the role they play in the prevention and control of HIV/AIDS. This interpretive ethnographic study sought to illuminate the role of primary care nurses and examine the influence of HIV/AIDS on their practice. The study was undertaken in Jordan in three rural and three urban primary health-care centres. Data collection included participant observation, key informant interviews and document analysis. These data informed the development of descriptive ethnographic accounts that allowed for the subsequent identification of common and divergent themes reflective of factors recognized as influencing the practice of the nurse participants. The findings indicate that the rhetoric offered by all levels of administration and endorsed in policy is not reflective of the reality of practice. Poor resources and educational preparation, a limited nursing skill mix and access to professional development, lack of nursing leadership and role models, cultural beliefs and geographic isolation are factors that reduced the capacity of the primary care nurses to raise awareness and, therefore, influence the prevention and control of HIV/AIDS. [source] Input design for model order determination in subspace identificationAICHE JOURNAL, Issue 8 2003Pratik Misra Subspace identification methods require that the inputs to the process being identified be persistently exciting. This may be inadequate for subspace identification of ill-conditioned multivariable processes, because the process model order may be underestimated, leading to subsequent identification of poor models. To remedy the problem, it is proposed that inputs must be used that excite a process to be identified in a way that produces as uncorrelated process outputs as possible. This can be accomplished either in open or in closed-loop fashion. Simulations on a high-purity distillation column and on a heat exchanger illustrate the merit of the proposed approach. [source] Pulsed field gradient (PFG) NMR spectroscopy: An effective tool for the analysis of mixtures of lubricating oil componentsLUBRICATION SCIENCE, Issue 4 2000G. S. Kapur Abstract In the presently reported work, the multinuclear two-dimensional (2D) diffusionordered nuclear magnetic resonance (NMR) spectroscopy (DOSY) technique based on the pulsed field gradient (PFG) has been used in experiments to analyse mixtures of lubricating oil components. One-dimensional (1D) PFG experiments have also been used to simplify and edit the NMR spectra of the mixtures. Such experiments provide a clean spectrum of the highest molecular weight (slower diffusing) component by eliminating the signals of lower molecular weight (faster diffusing) components, without any prior physical separation. These pulsed field gradient experiments not only facilitate the separation of resonance signals of different components, but also lead to their subsequent identification, and provide information about the number and structure of components in a mixture. Some examples of our initial efforts to establish 1D and 2D PFG-based NMR experiments for the analysis of mixtures of lubricating oil components are given and assessed to illustrate the potential applications of such techniques in the field of lubricating oils. [source] Evaluation of the SELDI-TOF MS technique for protein profiling of pancreatic islets exposed to glucose and oleatePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 17 2007Henrik Ortsäter Dr. Abstract The aim of the study was to evaluate the SELDI-TOF MS technique for pancreatic islet research. Mouse islets were cultured at low or high glucose levels in the absence or presence of oleate and characterized by measuring insulin secretion and oxygen tension. Subsequently, the islets were protein profiled. Up to 200 different peaks could be detected in a single experiment with the majority of peaks corresponding to proteins with masses below 30,kDa. By combining different protein arrays, the number of detected peaks could be increased further. The optimal binding of islet proteins was achieved using the anionic exchange array and phosphate buffer (pH,6) when the binding of insulin was low, which allowed other less abundant proteins to be captured. When islets from different culture conditions were profiled and analyzed, in total 25 proteins were found to be oleate/glucose-regulated. An oleate-regulated protein was chosen for identification work, which was conducted by passive elution from SDS-PAGE gels and subsequent in-gel trypsin digestion and MALDI-TOF MS. The protein was identified as peptidyl-prolyl isomerase B (PPI-B). In conclusion, the study demonstrates that SELDI-technique can be used not only to obtain islet protein patterns but is also helpful in the subsequent identification of differentially expressed proteins. [source] Characterisation of organellar proteomes: A guide to subcellular proteomic fractionation and analysisPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 21 2006Edwin Ho Abstract Subcellular fractionation is being widely used to increase our understanding of the proteome. Fractionation is often coupled with 2-DE, thus allowing the visualisation of proteins and their subsequent identification and characterisation by MS. Whilst this strategy should be effective, to date, there has been little or no consideration given to differences in the mass, pI, hydropathy or abundance of proteins in the organelles and how analytical strategies can be tailored to match the idiosyncrasies of proteins in each particular compartment. To address this, we analysed 3962 Saccharomyces cerevisiae proteins, previously localised to one or more of 22 subcellular compartments. Different compartments showed significantly different distributions of protein pI and hydropathy. Mitochondrial and ER proteins showed the most dramatic differences to other organelles, in their protein pIs and hydropathy, respectively. We show that organelles can be clustered by similarities in these physicochemical protein characteristics. Interestingly, the distribution of protein abundance was also significantly different between many organelles. Our results show that to fully explore subcellular fractions of the proteome, specific analytical strategies should be employed. We outline strategies for all 22 subcellular compartments. [source] Proteomic analysis of factors released from p21-overexpressing tumour cellsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 13 2006Caroline A. Currid Abstract The p21Waf1/Cip1/Sdi1 cyclin-dependent kinase inhibitor is a key regulator of cell cycle progression and has also been observed to influence the expression of genes associated with several age-related disorders. Previous work has shown that expression of p21 in tumour cells mediates an antiapoptotic and mitogenic paracrine effect, which is in contrast to the arrested state of p21-expressing cells. Here, we have employed SELDI-MS technology to characterise, at a proteomic level, factors released from HT-1080 human fibrosarcoma cells displaying inducible p21,expression. Conditioned media from induced and noninduced cells were profiled on a range of diverse ProteinChip arrays and subjected to SELDI-MS analysis. Evaluation of proteins binding onto IMAC, Q10 or CM10 surfaces led to the discovery of a number of putative p21-regulated factors. We further validated three p21-regulated proteins observed at 10.2, 11.7 and 13.4,kDa. Using Q,Ceramic HyperD fractionation columns, we were able to selectively enrich for each of these three proteins. Subsequent SDS-PAGE and MS analysis of tryptic digests identified the 13.4,kDa protein as cystatin C and the 10.2,kDa protein as pro-platelet basic protein (PPBP). Judging by the apparent MW and the pI of the 11.7,kDa protein, we reasoned that it may be ,-2-microglobulin, which was confirmed by subsequent identification. Increased levels of cystatin,C and ,-2-microglobulin in conditioned media from p21-expressing cells was confirmed by antibody capture experiments using anticystatin,C and anti-,-2-microglobulin antibodies on preactivated PS-20 arrays. Western blot analysis demonstrated increased expression of intracellular and extracellular cystatin,C and ,-2-microglobulin in p21-expressing cells, compared to noninduced controls. Increased levels of PPBP were validated in cell lysates from p21-expressing cells. The three secreted factors that we have identified in this study, have all been shown previously to have growth modulating effects and, as such, may contribute to the observed mitogenic and anti-apoptotic paracrine activity of p22-expressing cells. [source] Separation with zwitterionic hydrophilic interaction liquid chromatography improves protein identification by matrix-assisted laser desorption/ionization-based proteomic analysisBIOMEDICAL CHROMATOGRAPHY, Issue 6 2009Atsushi Intoh Abstract Comprehensive proteomic analyses necessitate efficient separation of peptide mixtures for the subsequent identification of proteins by mass spectrometry (MS). However, digestion of proteins extracted from cells and tissues often yields complex peptide mixtures that confound direct comprehensive MS analysis. This study investigated a zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) technique for the peptide separation step, which was verified by subsequent MS analysis. Human serum albumin (HSA) was the model protein used for this analysis. HSA was digested with trypsin and resolved by ZIC-HILIC or conventional strong cation exchange (SCX) prior to MS analysis for peptide identification. Separation with ZIC-HILIC significantly improved the identification of HSA peptides over SCX chromatography. Detailed analyses of the identified peptides revealed that the ZIC-HILIC has better peptide fractionation ability. We further demonstrated that ZIC-HILIC is useful for quantitatively surveying cell surface markers specifically expressed in undifferentiated embryonic stem cells. These results suggested the value of ZIC-HILIC as a novel and efficient separation method for comprehensive and quantitative proteomic analyses. Copyright © 2009 John Wiley & Sons, Ltd. [source] | ||||||||||