Day 2 :
Albert Einstein College of Medicine, USA
Time : 09:00-09:25
Rogatsky is a senior faculty member at Albert Einstein College of Medicine (NY, Bronx) and director of mass spectrometry at Biomarker Analytical Resource Core as part of the Harold and Muriel Block Institute for Clinical and Translational Research at Einstein and Montefiore . He has worked in the field of chromatography more than 20 years. Since 2001 his work has been within the service of the field of clinical mass spectrometry. During last 10 years (from 2004) Dr Rogatsky published 25 scientific papers in per-reviewed journals (mostly as the first author) and presented over 50 posters and lectures. Currently Dr Rogatsky serve as the Editor-in-Chief for the Journal of Chromatography and Separation Techniques (OMICS publishing group). Eduard Rogatsky completed his M.Sc (physical chemistry) in Belarus State University (former USSR) in 1990. In 1998 has completed PhD in bioanalytical chemistry (Bar-Ilan University, Israel). At the end of 1999 he started post-doctorate at Albert Einstein College of medicine and since 2001 joined faculty.
Vitamin D deficiency is a widespread clinical problem and has been associated with many adverse health outcomes. Analysis of vitamin D2 (ergocalciferol) and D3 (cholecalciferol) and their major metabolites 25 (OH) D2 and 25 (OH) D3 has become a high priority topic in clinical analysis. Currently a variety of LC/MS methods have been developed to support vitamin D analysis. These LC/MS methods utilize different transitions, ionization modes, sample preparation strategies, mobile phases and columns. In LC/MS analysis of 25 OH Vitamin D, dehydration (water loss) is the major side reaction. Comparing acetonitrile to methanol, which are typically used as mobile phases for LC separation, acetonitrile does not support hydrogen bond formation; therefore, proton-induced water elimination in-source becomes a major side-reaction, especially given the low pH of the mobile phase and positive mode electrospray and APCI ionization. MeOH, in contrast, supports hydrogen bond formation with the 25 (OH) D2 and 25 (OH) D3 hydroxyl groups. This efficiently “shields” most of hydroxyl groups by hydrogen bonding, and protects against protonation and resultant water elimination. We found that quantitation of the 25 (OH) D from its [M+H]+, “intact” precursor ion, is temperature invariant. In contrast, quantitation using the in-source dehydrated precursor (parent) ion, leads to increased sensitivity with a rise in temperature, due to its better ionization efficiency at higher temperatures. Since actual temperature of droplets can vary with mass spectrometer hardware, flow rate, and mobile phase composition, fluctuations of these factors may contribute additional variability to the assay.
University of Warsaw, Poland
Keynote: Approach to structures of difficult protein targets by means of monitoring hydrogen-deuterium exchange with mass spectrometry – New clothes for the old emperor
Time : 09:25-09:50
Michał Dadlez has completed his PhD in 1992 from the Institute of Biochemistry and Biophysics, Pol. Acad. Sci. and postdoctoral studies from Whitehead Institute, MIT, Cambridge, MA, supervised by Prof. Peter S. Kim. He organized Mass Spectrometry Lab, IBB PAS in 2001, being its head since then. In 2006 he became a Professor of Biophysics. He has published more than 90 papers in peer-reviewed journals. His major field of interest are protein structure studies with use of MS-based methods (HDex, X-linking, Ion Mobility separation, etc.)
It is estimated that a stunning 25–30% of eukaryotic proteins are mostly disordered, while more than half of eukaryotic proteins and more than 70% of signaling proteins have long regions of disorder – IDRs. Due to a variety of reasons access by classic methods (crystallography or NMR) to this vast part of structural proteome including IDRs and other difficult protein targets, is severely limited. A major breakthrough in the methodologies available to study the structures of this class of proteins is a must. In this respect recent developments of mass spectrometry-based approaches to protein structure analysis are the most promising path. Both, new approaches, like ion mobility separation (IM), electron transfer dissociation (ETD) or technical improvements in the methods already known (like measurements of the hydrogen-deuterium exchange kinetics - HDex) or combination of both (solution/gas phase HDex combined with IM and/or ETD) represent such a major breakthrough, providing new types of experimental constraints and unique access to structural properties of difficult protein assemblies which in combination with classic methods speed up their structural characterization. Using HDex-based multipole approaches we have successfully characterized several protein assemblies (vimentin oligomers, kinetochore proteins, histone pre-mRNA processing assembly, perfringolysin toxin membrane pores and alike). These results will be presented to illustrate recent breakthrough developments in approach to structural characterization of difficult protein targets.