Analytical Chemistry 2018

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) has developed into a powerful tool for investigating biopharmaceuticals, protein interactions, and membrane protein dynamics. Since 2010 HDX-MS data has helped substantiate and protect >110 US patents. Increasingly, biopharma companies provide HDX-MS data to support their biologics license applications (BLAs). However, for HDX-MS to emerge as a tool for quality control and similarity evaluations of biotherapeutics, an understanding of HDX-MS reproducibility is needed. NIST has determined the precision of bottom-up HDX-MS (Figure 1) by the analysis of 78,900 peptide measurements from 15 laboratories. We have also developed improvements to HDX-MS methodology, including automated measurements of membrane protein drug targets and chromatography instrumentation, operating at -30 oC, that improves HDX-MS measurements by arresting back-exchange.

Learning Objectives: 

1. An interlaboratory comparison involving 15 laboratory cohort has established reproducibility of HDX-MS, facilitating its use in commerce.
2. NIST has developed a method that allows automated HDX-MS studies of membrane proteins, which comprise 60% of all drug targets.
3. Chromatography  at -30 oC reduces H for D back-exchange to 5 %, which improves the  D-uptake measurement by 3x to 10x. 

Ion mobility-mass spectrometry (IM-MS) is a rapid, gas-phase separation technique that has become an integral part of the analytical repertoire of techniques for the -omics. This method couples well with existing protocols (i.e., LC-MS) by affording an additional dimension of separation, and provides a characteristic measure of an analyte’s size, shape, and charge in the form of a collision cross section (CCS). CCS can be used to increase confidence in quantitative measurement of known compounds, thus reducing interferences and improving S:N ratios, and also serves as a defining feature in identification of unknowns. Furthermore, this separation based on structure (as opposed to mass-to-charge alone) allows for distinction of structural and stereoisomers of potential biological importance.

This presentation will cover the basic operating principles of IMS and current instrumentation; applications to metabolomics, lipidomics, and proteomics; and future directions for IM-MS, such as implementation in routine clinical analysis.

Learning Objectives: 

1. Understand the basic operating principles of IMS and current instrumentation
2. Understand current applications to metabolomics, lipidomics, and proteomics

In the postgenomic era, one expects the suite of chemical players in a brain region to be known and their functions uncovered. Perhaps surprisingly, many neurochemicals remain poorly characterized and for those that are known, their localization, dynamics and function are oftentimes unknown. Several approaches for assaying the chemical content within targeted brain regions and from individual brain cells are highlighted, including mass spectrometry imaging (MSI) and MS-based single cell measurements. Using these approaches, we can measure lipids, fatty acids, neurotransmitters and neuropeptides, among others. For single cell measurements, the cells of interest are scattered across a microscope slide, the exact cell positions determined via optical microscopy, and mass spectra are acquired only at the cell positions. The single cell assays allow differences in the metabolome and peptidome from supposedly homogeneous populations of cells to be explored. By obtaining information from tens of thousands of individual cells, rare cells are found and unusual neurochemicals are discovered. For select cells, follow-up capillary electrophoresis-mass spectrometry and other information rich assays can be performed. Several applications of single cell mass spectrometry are highlighted from the discovery of unusual metabolites to characterizing the both known and previously unknown neuropeptides and hormones in single cells. Our overarching goal is to uncover the complex chemical mosaic of the brain and pinpoint key cellular players involved in a range of physiological and pathological processes.

Learning Objectives:

1. Participants will understand why single cell measurements important.
2. Participants will describe the importance and how cells are prepared and measured with mass spectrometry.
3. Participants will learn about the chemical information gained from single cell mass spectrometry measurements and how it relates to cell function, health and disease.