Best practices and benchmarks for intact protein analysis for top-down mass spectrometry.

One gene can give rise to many functionally distinct proteoforms, each of which has a characteristic molecular mass. Top-down mass spectrometry enables the analysis of intact proteins and proteoforms. Here members of the Consortium for Top-Down Proteomics provide a decision tree that guides researchers to robust protocols for mass analysis of intact proteins (antibodies, membrane proteins and others) from mixtures of varying complexity. We also present cross-platform analytical benchmarks using a protein standard sample, to allow users to gauge their proficiency.

Genetically Encoded Fluorescent Proteins Enable High-Throughput Assignment of Cell Cohorts Directly from MALDI-MS Images.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) provides a unique in situ chemical profile that can include drugs, nucleic acids, metabolites, lipids, and proteins. MSI of individual cells (of a known cell type) affords a unique insight into normal and disease-related processes and is a prerequisite for combining the results of MSI and other single-cell modalities (e.g. mass cytometry and next-generation sequencing). Technological barriers have prevented the high-throughput assignment of MSI spectra from solid tissue preparations to their cell type. These barriers include obtaining a suitable cell-identifying image (e.g. immunohistochemistry) and obtaining sufficiently accurate registration of the cell-identifying and MALDI-MS images. This study introduces a technique that overcame these barriers by assigning cell type directly from mass spectra. We hypothesized that, in MSI from mice with a defined fluorescent protein expression pattern, the fluorescent protein's molecular ion could be used to identify cell cohorts. A method was developed for the purification of enhanced yellow fluorescent protein (EYFP) from mice. To determine EYFP's molecular mass for MSI studies, we performed intact mass analysis and characterized the protein's primary structure and post-translational modifications through various techniques. MALDI-MSI methods were developed to enhance the detection of EYFP in situ, and by extraction of EYFP's molecular ion from MALDI-MS images, automated, whole-image assignment of cell cohorts was achieved. This method was validated using a well-characterized mouse line that expresses EYFP in motor and sensory neurons and should be applicable to hundreds of commercially available mice (and other animal) strains comprising a multitude of cell-specific fluorescent labels.

Secretion, isotopic labeling and deglycosylation of N-acylethanolamine acid amidase for biophysical studies.

N-acylethanolamine acid amidase (NAAA) is an N-terminal nucleophile (Ntn) enzyme with a catalytic cysteine residue that has highest activity at acidic pH. The most prominent substrate hydrolyzed is palmitoylethanolamine (PEA), which regulates inflammation. Inhibitors of NAAA have been shown to increase endogenous levels of PEA, and are of interest as potential treatments for inflammatory disorders and other maladies. Currently, there are no X-ray or NMR structures of NAAA available to inform medicinal chemistry. Additionally, there are a limited number of enzyme structures available that are within the Ntn-hydrolase family, have a catalytic cysteine residue, and have a high sequence homology. For these reasons, we developed expression and purification methods for the production of enzyme samples amenable to structural characterization. Mammalian cells are necessary for post-translational processing, including signal sequence cleavage and glycosylation, that are required for a correctly folded zymogen before conversion to active, and mature enzyme. We have identified an expression construct, mammalian cell line, specific media and additives to express and secrete hNAAA zymogen and we further optimized propagation conditions and show this secretion method is suitable for isotopic labeling of the protein. We refined purification methods to achieve a high degree of protein purity potentially suited to crystallography. Glycosylated proteins can present challenges to biophysical methods. Therefore we deglycosylate the enzyme and show that the activity of the mature enzyme is not affected by deglycosylation.

Imaging and Mapping of Tissue Constituents at the Single-Cell Level Using MALDI MSI and Quantitative Laser Scanning Cytometry.

For nearly a century, histopathology involved the laborious morphological analyses of tissues stained with broad-spectrum dyes (i.e., eosin to label proteins). With the advent of antibody-labeling, immunostaining (fluorescein and rhodamine for fluorescent labeling) and immunohistochemistry (DAB and hematoxylin), it became possible to identify specific immunological targets in cells and tissue preparations. Technical advances, including the development of monoclonal antibody technology, led to an ever-increasing palate of dyes, both fluorescent and chromatic. This provides an incredibly rich menu of molecular entities that can be visualized and quantified in cells-giving rise to the new discipline of Molecular Pathology. We describe the evolution of two analytical techniques, cytometry and mass spectrometry, which complement histopathological visual analysis by providing automated, cellular-resolution constituent maps. For the first time, laser scanning cytometry (LSC) and matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) are combined for the analysis of tissue sections. The utility of the marriage of these techniques is demonstrated by analyzing mouse brains with neuron-specific, genetically encoded, fluorescent proteins. We present a workflow that: (1) can be used with or without expensive matrix deposition methods, (2) uses LSC images to reveal the diverse landscape of neural tissue as well as the matrix, and (3) uses a tissue fixation method compatible with a DNA stain. The proposed workflow can be adapted for a variety of sample preparation and matrix deposition methods.