8 - 9 a.m.  

Registration, breakfast, meet and greet

9 - 9:30 a.m.

Isotopocules – Everything, everywhere, all at once?
For a generation or more, the mass spectrometry that developed at the frontier of molecular biology was worlds apart from isotope ratio mass spectrometry, a label-free approach done on optimized gas-source magnetic sector instruments. Recent studies show that mass spectrometers used in the life sciences, including electrospray-ionization Orbitrap instruments, can be optimized for high-precision isotope ratio analysis. Intramolecular isotope measurements offer unique insights into a wide range of research topics, thanks to the ubiquitous nature of isotope patterns. This talk provides an overview of current research topics in life and environmental sciences that may benefit from measuring isotopocules (isotopically substituted molecules) of biomolecules and oxyanions by ESI-Orbitrap MS. We will discuss how soft-ionization mass spectrometry and ultrahigh mass resolution can drive progress in long-envisioned areas of isotope research. We also talk about current limitations and speculate on future directions of this adventure into the overlapping realms of biology, chemistry, and geology.
Speakers: Cajetan Neubauer, University of Colorado Boulder; Kristýna Kantnerová, University of Colorado Boulder

9:30 - 9:45 a.m.

Overview of Thermo Scientific Orbitrap Exploris Isotope Solutions
Speaker: Brett Davidheiser, Thermo Fisher Scientific

9:45 - 10 a.m.

Question and answer session

10 - 10:30 a.m.

Expanding the Orbitrap-IRMS Frontier: Coalescence, Duration, and Interpretation of Large Datasets
Orbitrap-IRMS is becoming a popular technique for stable isotope measurements because it can observe the isotopic structures of a wide variety of molecules at a high level of detail (e.g., site-specific and clumped isotopes, as well as more exotic properties which are harder to interpret). Many challenges to Orbitrap-IRMS application remain, including experimental artifacts associated with observation and the long duration required for many targets. I will examine the frontiers of Orbitrap-IRMS via two sets of experiments. First, I’ll discuss experimental artifacts associated with “space charge” effects in the Orbitrap, i.e., interferences of close-lying ion beams at high ion loads, which suppress peak heights and shift the locations of observed ion beams. This discussion will address the underlying causes and include results from sulfate and methionine demonstrating the effect on observed isotope ratios. Next, I will examine the high dimensional fingerprinting of target molecules via a “M+N” experiment, which mass selects and fragments isotopologues with a cardinal mass of “N” above the unsubstituted isotopologue. This section will describe a general theory for this type of experiment and present results of M+1, M+2, M+3, and M+4 experiments performed on methionine which, combined, yield 146 isotopic observations at precisions of 0.5-3 ‰. These observations can be used to compute the enrichments of various singly-, doubly-, and triply-substituted isotopologues. I will also discuss technical challenges for these measurements, included acquisitions of hours or more, and suggest possible routes for their application. The lessons from each section should be generalizable to new Orbitrap-IRMS targets.
Speaker: Tim Csernica, California Institute of Technology

10:30 - 11:30 a.m.

Coffee Roundtables: Technology and troubleshooting

11:30 a.m. - 12:30 p.m.

Can one Orbitrap do it all?  In vivo measurement of protein synthesis from fast to slow
Scientists have been developing methods to measure in vivo synthesis and breakdown rates of protein since the 1930’s. The early methods used isotope ratio mass spectrometry (IRMS) and stable isotopes as tracers, but these methods were very difficult and tedious to apply. Although IRMS has excellent sensitivity for measuring natural abundance variations in isotopes, it requires purification of the analyte species to be measured. The development of gas chromatography (GC) combustion (C) IRMS dramatically changed how IRMS was used by providing GC separation of analytes before measurement of 13C as CO2 or 15N as N2 by IRMS. Pyrolysis (P) was later added to provide measurement of 2H as H2, 13C and 18O as CO by a modified IRMS. Now if a protein could be isolated and hydrolyzed, its synthetic rate could be determined from incorporation of a stable isotopically labeled amino acid into that protein. More recently, gas chromatography-mass spectrometry (GC-MS) techniques and later liquid chromatography-mass spectrometry (LC-MS) were also developed for measuring very low isotope enrichments while adding the advantages of improved sensitivity, requiring less sample, and the ability to measure multiple isotopes and isotopomers in the same analyte molecule. The problem remained that individual proteins had to be isolated and hydrolyzed for the tracer amino acid enrichments to be measured. The introduction high resolution MS and tandem LC-MS/MS techniques advanced the field of proteomics and added the ability to measure stable isotope enrichments in peptides of multiple individual proteins without requiring separation. Now the synthesis rates of multiple proteins could be determined, if their synthesis rates were relatively rapid and enough stable isotope tracer incorporated. Slow turnover proteins could only be measured by conventional IRMS or GC-MS/MS methods requiring protein isolation and hydrolysis. Recently, several groups have used high resolution LC-MS/MS measurement to determine natural abundance variations in stable isotopic abundances of immonium ions generated from peptides determined in a normal proteomics workflow. This new approach now enables a single, high resolution LC-MS/MS, such as the Orbitrap, to be used to determine protein synthetic rates of both fast and very slow turnover proteins simultaneously.
Speaker: Dwight E. Matthews, Professor Emeritus of Chemistry and Medicine, University of Vermont

12:30 - 1:30 p.m.

Lunch buffet

1:30 - 2 p.m.

Orbitrap mass spectrometry for Astrobiology
The search for extraterrestrial life requires confidence in what constitutes a biosignature. Life on other worlds may not mimic life on Earth, thus a non-Earthcentric (“agnostic”) understanding of what signifies life is essential. Molecular Assembly Theory, one of many complexity theories, is based on the idea that living organisms have the unique ability to produce complex molecular structures and abiotic systems do not. A Molecular Assembly index (MA) was developed based on the Assembly Theory of molecular complexity to differentiate between abiotic and biotic formation of molecules. This method works well to describe high molecular weight biological molecules. However, smaller, less complex molecules that can be synthesized by both living and non-living systems will have the same MA numbers; thus, their biogenicity cannot be distinguished by Assembly Theory alone. Further, diagenetic alteration of biological molecules can modify molecules, artificially depressing the MA value such that it no longer indicates biotic origin. We explore the use of Orbitrap mass spectrometry to approximate MA and combined with measurements of the isotopic composition of pure compounds as a tool for distinguishing life from non-living, chemical processes. We focus on amino acids as they are ubiquitous and produced by both biological and abiotic processes and test measurement requirements using both the Q Exactive and LTQ XL Orbitrap mass spectrometers.
Speaker: Gabriella M. Weiss, NASA Goddard Space Flight Center, Greenbelt, MD & University of Maryland Baltimore

2 - 2:45 p.m.

Share current and future projects

2:45 - 3:45 p.m.

Demo laboratory launch celebration and tours
Celebrate the launch of our new Isotope-Orbitrap demo laboratory and INSTAAR Stable Isotope demo laboratory with cake and drinks followed by tours of both laboratories

3:45 - 4:15 p.m.

Panel discussion: Shared resources

4:15 p.m.

User meeting wrap up

4:30 - 6 p.m.

Social Hour at the Sanitas Brewing Company
End the day with a social hour at the Sanitas Brewing Company for more networking, food and drinks, and fun!