Steve completed his PhD (Pharmacology) at the University of California, Davis in 2005 followed by Postdoctoral studies in the labs of Dr Julie Andersen and Dr Brad Gibson at the Buck Institute in Novato, CA where he published several papers on mass spectrometry techniques to study Parkinson’s disease. He is currently a Demonstration Chemist focusing on LCMS proteomic applications for Thermo Fisher Scientific in San Jose CA.
Quantitation of intact proteins provides information unavailable at the peptide level by preserving the molecular integrity of the protein state. Specific proteoforms can be monitored such as unmodified vs. modified forms (post-translational modification and truncation) these different forms may have functional consequences for protein activity. However there are several challenges to intact protein quantitation including loss of sensitivity and dynamic range due to distribution of signal among many charge states, complex highly charged isotope patterns that require high resolution mass spectrometry (MS) beyond the range of most MS instruments to resolve and a lack of MS scan speed to target multiple proteoforms on a chromatographic timescale. Here we describe a method for improved intact protein quantitation using recent advancements in chromatography and detection utilizing the Thermo ScientificTM Orbitrap Fusion TribridTM Mass Spectrometer. This very sensitive, fast, accurate mass instrument offers ultra high resolution (450,000 @ 200 m/z), this in combination with its ability to optimize pressure of the HCD collision cell results in improved sensitivity and baseline resolved isotopic peaks for intact proteins <50 kD. Additionally the Orbitrap Fusion mass spectrometer can multiplex (accumulation of multiple targets including the same species with different charge states in the HCD collision cell) either precursors through single ion monitoring (SIM) scans or by using parallel reaction monitoring (PRM) which targets specific fragment ions. The combination of these enhancements improved the sensitivity and reproducibility of intact proteins quantified including carbonic anydrase II (2-172 pmol) and other plasma proteins at a flow rate of 0.5 mL/min.
Marwa Eltoweissy has completed her PhD at the age of 30 through a scholarship and cooperation work between faculty of Science, Alexandria University, Egypt and Rheinische Friedrich-Wilhelms-University Medical Center Bonn, Institute for Physiology II, Germany. She achieved postdoctoral studies at the Gastroenterology and Endocrinology department, Georg-August University Medical Center, Göttingen, Germany. She received the Doctor of Natural Sciences (Dr. rer. nat.) degree through her work at the Nephrology and Rheumatology department, Georg-August University Medical Center, Göttingen, Germany. Works as a Major scientific researcher at the later department and as an Assistant Professor of Physiology at the Zoology department, Alexandria University, Egypt. She has published more than 15 papers in reputed journals and serving as a reviewer for privileged journals.
Secreted proteins are major factors in cell invasion, migration, motility, growth control, matrix degradation and adhesion. Inflammatory agents (cytokines and growth factors) activation results in impairment of secretome and can result in cell transformation. These aspects play an important role in renal fibrosis onset and progression. In the present study we targeted to investigate the alteration of renal cell secretome upon treatment with pro-fibrotic inflammatory factors and to identify potential key proteins in renal fibrosis progression. For this purpose renal fibroblasts (TK173) and renal epithelial cells (HK2) were cultured in FCS free medium for 24 h and then treated with TGFß-1, PDGF or ANG II for 72 h. Subsequently the secretomes were collected and proteins were enriched. Two-dimensional secretome protein maps were generated using 2D-gel electrophoresis and the proteins were processed and identified using mass spectrometry analysis combined with data base search. Differential secreted proteins were annotated and bioinformatics functional analyses were carried out to investigate the potential role of secreted proteins in renal fibrosis progression. We optimized a protocol, which allowed us to achieve high purity in secretome protein for further analysis. 2-DE combined with peptide sequence analysis and database searches identified sets of 61, 41 and 66 non-redundant protein, which were differently secreted in TK173 cells under TGFß-1, PDGF and ANG II treatment respectively. Among the identified proteins, 15 were shared in all treatments. Treatment of renal epithelial cells HK2 with the same agents resulted in differential secretion of 48, 57 and 49 proteins respectively. In case of HK2 only 8 proteins were shared by all three treatments. Functional analysis of the identified proteins by DAVID Bioinformatics software packages identified 41.8% for TK173 and 38.6% for HK2 as secretory proteins, which could be attributed to classical and non-classical secretory pathways. Further functional classification of proteins ascribed to classical pathway verified the presence of proteins belonging to cytokines, receptors, membrane proteins, lysosomal proteins and proteins associated with specific sub-cellular localizations such as endoplasmic reticulum, mitochondria, nucleus, cytoplasm and ribosome. Our results shied light on a very interesting aspect in renal fibrosis progression. The Investigation of secretome could help to understand the mechanism of acceleration of fibrosis progression and offer new insights in the pathogenesis of this disease.