Analytical Chemistry 2018

Alzheimer’s disease is becoming a public health crisis as the anticipated number of sufferers is expected to rise to 15 million in the next 30 years, at least in the US. These numbers are exacerbated when considering global incidences of disease. One strategy to combat this rise is to use ‘omics based approaches to better understand the complexity of the disease and to find biomarkers that can be useful for early disease diagnosis. Ultimately, it is desirable for these ‘omics approaches to lead to therapeutic targets and strategies to slow, prevent, or cure disease.

The RASR laboratory develops high-throughput proteomics approaches to understand health disparities in Alzheimer’s disease and the role of the periphery. In this presentation, Dr. Robinson will introduce how her laboratory is approaching these problems through the development of enhanced sample multiplexing techniques. Specifically, an approach developed in her laboratory, combined precursor isotopic labeling and isobaric tagging (cPILOT), has been demonstrated on up to 24 samples that can be analyzed in a single experiment. This capability has allowed studies that evaluate proteomic changes across multiple tissue types, genotypes, and ethnic populations. While there are inherent tradeoffs with a dual labeling approach the benefits gained in cost and time savings and comprehensive quantitative information are extremely valuable for this platform. These tradeoffs will be highlighted as well as considerations for the utility of enhanced multiplexing in enabling studies of Alzheimer’s disease.

Learning Objectives: 

1. Participants will learn two approaches for chemically labeling peptides in complex mixtures that can ultimately be combined into a single enhanced multiplexing approach.
2. Participants will become familiar with the benefits and challenges of multiplexed proteomics analyses.
3. Participants will understand how Alzheimer’s disease research can be advanced using multiplexing strategies. 

Analytical chemistry can take you to strange places - for me it has been to the bottom of a quarry, being covered in dirt, excavating sesame-seed sized fossils that allow us to work out when prehistoric creatures such as mammoths and Neanderthals roamed Europe.  Being able to identify (and isolate) a fraction of the protein contained within particular fossil shells which degrades at a predictable rate has allowed the development of a robust dating technique. This 'intra-crystalline' protein behaves as a closed system, and if this is targeted in a tiny calcite fossil (snail opercula, the little lids that close the aperture of snail shells) we can date back to at least 2.8 Ma.  With LC and MS analyses of protein in fossil material ranging from Great Barrier Reef corals to South African ostrich eggshell and Antarctic ice cores, the chirality of amino acids has helped us unravel the complexities of our past climate as well as providing insights into biomineralisation. 

Learning Objectives:

1. How critical it is to understand exactly where your molecules of interest are, and how they can be affected by their environment.
2. The importance of using multiple analytical techniques to provide complementary data.

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. 

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.

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. 

Two-dimensional (2D) 1H-13C methyl correlated NMR is increasingly being recognized as a powerful tool to characterize the higher order structure (HOS) of monoclonal antibody (mAb) therapeutics. 2D methyl NMR is well suited to the task as spectra can be readily be acquired on intact mAbs at natural isotopic abundance, and even small changes to chemical environment and structure manifest observable changes in the spectra, which can be interpreted at atomic resolution. This makes it possible to apply 2D NMR approaches directly to drug products in order to systematically characterize structural effects. Traditionally, such analysis has involved identifying specific changes to measured resonance peak parameters.  Recently, we have demonstrated a complementary approach using principal component analysis (PCA) to directly analyze the matrix of spectral data, correlating spectra according to similarities and differences in their overall shapes. This approach is particularly well-suited for spectra of mAbs, where many individual peaks may not be well resolved and peak parameters difficult to measure. Here we demonstrate the performance of the PCA method for discriminating structural variation among systematic sets of 2D NMR spectra using the NISTmAb reference material and illustrate how spectral variability identified by PCA may be correlated to structure.

Learning Objectives: 

1. 2D 1H-13C Methyl NMR spectra can be readily acquired on monoclonal antibody therapeutics at natural isotopic abundance.
2. Such spectra are suitable to assess the major conformation(s) of mAb drugs in solution.
3. Using multivariate statistics, such as principal component analysis mAb spectra can be classified based on minor structural perturbations with low levels of detection.

Analytical chemistry can take you to strange places - for me it has been to the bottom of a quarry, being covered in dirt, excavating sesame-seed sized fossils that allow us to work out when prehistoric creatures such as mammoths and Neanderthals roamed Europe.  Being able to identify (and isolate) a fraction of the protein contained within particular fossil shells which degrades at a predictable rate has allowed the development of a robust dating technique. This 'intra-crystalline' protein behaves as a closed system, and if this is targeted in a tiny calcite fossil (snail opercula, the little lids that close the aperture of snail shells) we can date back to at least 2.8 Ma.  With LC and MS analyses of protein in fossil material ranging from Great Barrier Reef corals to South African ostrich eggshell and Antarctic ice cores, the chirality of amino acids has helped us unravel the complexities of our past climate as well as providing insights into biomineralisation. 

Learning Objectives:

1. How critical it is to understand exactly where your molecules of interest are, and how they can be affected by their environment.
2. The importance of using multiple analytical techniques to provide complementary data.

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

This intensive workshop will introduce infrared spectroscopy, outline the various sample handling methods and provide guidance on the numerous transmission and reflectance methods available for infrared analysis. 

Concepts of infrared absorption spectroscopy will be introduced with a discussion of sample analysis methods and the wide selection of accessories used to obtain representative infrared spectra of analytical samples. Methods for obtaining the 'ideal' spectrum will be presented as well as how to get the most out of the large variety of available FT-IR sampling accessories. The common pitfalls to avoid during sample preparation will also be examined. Sampling techniques for transmission measurements using KBr pellets, mulls, liquid cells and films will be outlined in addition to presentations on Multiple Internal Reflectance (MIR, HATR), Diffuse Reflectance (DRIFTS), Specular Reflectance, Reflection-Absorption Spectroscopy (RAIRS) and Grazing Angle measurements. The various transmission and reflectance measurement experiments will be examined to determine which method, or accessory, can provide the infrared data required of specific samples. This workshop is intended for those participants just beginning infrared analysis up to participants interested in more advanced methods of infrared sample analysis.  

Learning Objectives: 

1. To review the basic theory of infrared absorption spectroscopy.  
2. To understand the various methods and accessories available to collect spectra as well as the advantages and disadvantages of the various accessories and methods.

Alzheimer’s disease is becoming a public health crisis as the anticipated number of sufferers is expected to rise to 15 million in the next 30 years, at least in the US. These numbers are exacerbated when considering global incidences of disease. One strategy to combat this rise is to use ‘omics based approaches to better understand the complexity of the disease and to find biomarkers that can be useful for early disease diagnosis. Ultimately, it is desirable for these ‘omics approaches to lead to therapeutic targets and strategies to slow, prevent, or cure disease.

The RASR laboratory develops high-throughput proteomics approaches to understand health disparities in Alzheimer’s disease and the role of the periphery. In this presentation, Dr. Robinson will introduce how her laboratory is approaching these problems through the development of enhanced sample multiplexing techniques. Specifically, an approach developed in her laboratory, combined precursor isotopic labeling and isobaric tagging (cPILOT), has been demonstrated on up to 24 samples that can be analyzed in a single experiment. This capability has allowed studies that evaluate proteomic changes across multiple tissue types, genotypes, and ethnic populations. While there are inherent tradeoffs with a dual labeling approach the benefits gained in cost and time savings and comprehensive quantitative information are extremely valuable for this platform. These tradeoffs will be highlighted as well as considerations for the utility of enhanced multiplexing in enabling studies of Alzheimer’s disease.

Learning Objectives: 

1. Participants will learn two approaches for chemically labeling peptides in complex mixtures that can ultimately be combined into a single enhanced multiplexing approach.
2. Participants will become familiar with the benefits and challenges of multiplexed proteomics analyses.
3. Participants will understand how Alzheimer’s disease research can be advanced using multiplexing strategies.