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.

A rapid MALDI-TOF mass spectrometry workflow for Drosophila melanogaster differential neuropeptidomics.

BACKGROUND: Neuropeptides are a diverse category of signaling molecules in the nervous system regulating a variety of processes including food intake, social behavior, circadian rhythms, learning, and memory. Both the identification and functional characterization of specific neuropeptides are ongoing fields of research. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of nervous tissues from a variety of organisms allows direct detection and identification of neuropeptides. Here, we demonstrate an analysis workflow that allows for the detection of differences in specific neuropeptides amongst a variety of neuropeptides being simultaneously measured. For sample preparation, we describe a straight-forward and rapid (minutes) method where individual adult Drosophila melanogaster brains are analyzed. Using a MATLAB-based data analysis workflow, also compatible with MALDI-TOF mass spectra obtained from other sample preparations and instrumentation, we demonstrate how changes in neuropeptides levels can be detected with this method. RESULTS: Over fifty isotopically resolved ion signals in the peptide mass range are reproducibly observed across experiments. MALDI-TOF MS profile spectra were used to statistically identify distinct relative differences in organ-wide endogenous levels of detected neuropeptides between biological conditions. In particular, three distinct levels of a particular neuropeptide, pigment dispersing factor, were detected by comparing groups of preprocessed spectra obtained from individual brains across three different D. melanogaster strains, each of which express different amounts of this neuropeptide. Using the same sample preparation, MALDI-TOF/TOF tandem mass spectrometry confirmed that at least 14 ion signals observed across experiments are indeed neuropeptides. Among the identified neuropeptides were three products of the neuropeptide-like precursor 1 gene previously not identified in the literature. CONCLUSIONS: Using MALDI-TOF MS and preprocessing/statistical analysis, changes in relative levels of a particular neuropeptide in D. melanogaster tissue can be statistically detected amongst a variety of neuropeptides. While the data analysis methods should be compatible with other sample preparations, the presented sample preparation method was sufficient to identify previously unconfirmed D. melanogaster neuropeptides.

Preparation of primary amine derivatives of the magic-size nanocluster (CdSe)13.

Four [(CdSe)13(RNH2)13] derivatives (R = n-propyl, n-pentyl, n-octyl, and oleyl) are prepared by reaction of Cd(OAc)2·2H2O and selenourea in the corresponding primary-amine solvent. Nanoclusters grow in spontaneously formed amine-bilayer templates and are characterized by elemental analysis, IR spectroscopy, UV-vis spectroscopy, TEM, and low-angle XRD. Derivative [(CdSe)13(n-propylamine)13] is isolated as a yellowish-white solid (MP 98 °C) on the gram scale. These compounds are the first derivatives of magic-size CdSe nanoclusters to be isolated in purity.

Capillary-channeled polymer (C-CP) films as processing platforms for protein analysis by matrix-assisted laser/desorption ionization mass spectrometry (MALDI-MS).

Polypropylene (PP) capillary-channeled polymer (C-CP) films have parallel, µm-sized channels that induce solution wicking via capillary action. Efficient mass transport from the solution phase to the channel surface leads to adsorption of hydrophobic protein solutes. The basic premise by which C-CP films can be used as media to manipulate analyte solutions (e.g., proteins in buffer), for the purpose of desalting or chromatographic separation prior to MALDI-MS analysis is presented here. Cytochrome c and myoglobin prepared in a Tris-HCl buffer, and ribonuclease A, lysozyme, and transferrin prepared in phosphate buffered saline (PBS), are used as the test solutions to demonstrate the desalting concept. Protein analysis is performed after deposition on a C-CP film with and without a water washing step, followed by spray deposition of a typical sinapinic acid matrix. Extracted MALDI mass spectra exhibit much improved signal-to-noise characteristics after water washing. A mixture of cytochrome c and myoglobin (2 µL of 2.5 µM each in Tris-HCl buffer) was applied, washed with water and spatially separated via simple capillary action (wicking) using a reversed-phase solvent composition of 0.1% trifluoroacetic acid (TFA) in 50:50 acetonitrile (ACN):H(2)O. Subsequent application of sinapinic acid followed by imaging of the film using MALDI-MS reveals that as the protein solution is wicked down the film, separation occurs.

Malignant granular cell tumor: a look into the diagnostic criteria.

Fanburg-Smith et al. classified granular cell tumors (GCTs) using six criteria with high Ki-67 and p53 in malignant cases. We aim to refine their classification and reproduce their immunohistochemical findings. We, first, classified our 48 cases according to Fanburg-Smith criteria (37 benign, seven atypical, and four malignant), and performed Ki-67 and p53 on a sample of tumors. Then, we reclassified them into 44 benign and four with uncertain malignant potential (GCT-UMP) using only necrosis and/or mitoses. (1) According to Fanburg-Smith criteria: Malignant cases were significantly younger than benign and atypical ones; occurred predominantly in males; were significantly larger in size; and showed a higher Ki-67 expression but an insignificant difference in p53 staining. (2) Comparative findings: The four malignant cases according to Fanburg-Smith corresponded to our four cases with UMP. The seven atypical cases and our benign group shared similar means, except for age. None of these atypical cases recurred or metastasized. Despite its small number, our preliminary study showed similar selectivity of two more reproducible criteria (vs six) in the classification of cases of GCT with potential aggressive behavior, preserving a role for Ki-67 in difficult cases. However, metastases remain the sole definite criterion for malignancy.

Tissue preparation for the in situ MALDI MS imaging of proteins, lipids, and small molecules at cellular resolution.

The resolution of MALDI MS imaging is limited by the displacement of analytes during matrix deposition or by laser focal diameter. Here we present three methods that minimize the displacement of analytes during matrix deposition, including a method where image resolution is not limited by the laser focal diameter. The first method, matrix solution fixation, simultaneously fixes tissue while depositing matrix and is optimal for analyzing proteins and for applications requiring a fast preparation time. This method is characterized by compatibility with histology methods and laser focal diameter-limited resolution. The second method, a sensor controlled aerosol, is characterized by aerosol droplet size-limited resolution and is optimal for small molecules, including lipids, peptides, and drug-like molecules. The third method, microinjection with matrix, selectively deposits matrix upon cells of interest, offers cellular resolution and is compatible with most analytes. A flow chart summarizing methods is provided so that users may design a tissue preparation strategy based upon their resources and experimental goals.

E-cadherin expression in primary carcinomas of the breast and its distant metastases.

INTRODUCTION: Aberrant expression of E-cadherin has been associated with the development of metastases in patients with breast cancer. Even though the expression of E-cadherin has been studied in primary breast tumors, little is known about its expression at the distant metastatic sites. We investigate the relationship between E-cadherin expression in primary breast carcinoma and their distant, non-nodal metastases. METHODS: Immunohistochemical analysis of E-cadherin was performed in tissues from 30 patients with primary invasive breast carcinoma and their distant metastases. E-cadherin expression was evaluated as normal or aberrant (decreased when compared with normal internal positive controls, or absent). RESULTS: Twenty-two (73%) invasive carcinomas were ductal, and eight (27%) were lobular. Of the primary invasive ductal carcinomas, 55% (12/22) had normal E-cadherin expression and 45% (10/22) had aberrant expression. All of the metastases expressed E-cadherin with the same intensity as (12 tumors) or with stronger intensity than (10 tumors) the corresponding primaries. Of the invasive lobular carcinomas, one of eight (12%) primary carcinomas and none of the metastases expressed E-cadherin in the cell membranes, but they accumulated the protein in the cytoplasm. CONCLUSION: Aberrant E-cadherin expression is frequent in invasive ductal carcinomas that progress to develop distant metastases. Distant metastases consistently express E-cadherin, often more strongly than the primary tumor. Invasive lobular carcinomas have a different pattern of E-cadherin expression, suggesting a different role for E-cadherin in this form of breast carcinoma.

Perineural invasion in adenoid cystic carcinoma: Its causation/promotion by brain-derived neurotrophic factor.

Adenoid cystic carcinoma (ACC) is a common salivary gland neoplasm with a lengthy clinical course and often late local recurrences after surgical resection. This is accounted for histologically by its infiltrative capacity and distinct propensity for perineural invasion. The expression of biological markers may help explain the clinicopathologic course in ACCs. Brain-derived neurotrophic factor (BDNF) is a growth factor known to be involved in neurogenesis. The aim of this study is to elucidate the expression of BDNF in ACCs, which is currently unknown. Twenty-nine cases of primary ACCs of the head and neck were immunostained to analyze BDNF protein expression. Staining intensity was described as focal or diffuse and graded on a 3-tiered scale. The study group comprised 20 adult females (age 30 to 78) and 9 adult males (age 22 to 70). Sites of involvement included the parotid gland (6 cases), nasopharynx (5), maxilla (4), palate (3), trachea (3), submandibular gland (2), buccal mucosa (2), mandible (1), tongue (1), lacrimal gland (1), and temporal region (1). All tumors exhibited diffuse cytoplasmic staining; 11 cases were classified as 1+ intensity, 12 cases as 2+, and six cases as 3+. Based on the results presented here, BDNF is unformly expressed by ACC and may play a causative role in its predilection for perineural invasion. ACCs may display neurogenesis with high levels of BDNF expression in some tumors. Further studies are warranted to gain better understanding of this possible relationship.