Andreia M. Porcari

Intact triacylglycerol profiles of fats and meats via thermal imprinting easy ambient sonic-spray ionization mass spectrometry

Thermal imprinting (TI) on a paper surface, using minimal solvent amounts, followed by direct analysis of the triacylglycerols (TAG) content via easy ambient sonic-spray ionization mass spectrometry (EASI-MS) is shown to provide a fast protocol to analyze TAG in meats and fats. The technique is simple, fast and eco-friendly requiring no hydrolysis, derivatization or chromatographic separation. The entire TI-EASI-MS protocol is performed in a few minutes and with minimal sample handling and solvent consumption. The TAG profiles obtained via TI-EASI-MS are shown to be quite similar to those obtained using GC and MALDI-MS analyses, and the imprinting and mailing of the imprinted paper in a sealed plastic bag is proposed for remote TI-EASI-MS analysis of meat and fat samples.

Rapid fingerprinting of sterols and related compounds in vegetable and animal oils and phytosterol enriched- margarines by transmission mode direct analysis in real time mass spectrometry.

Plant-derived sterols, often referred to as phytosterols, are important constituents of plant membranes where they assist in maintaining phospholipid bilayer stability. Consumption of phytosterols has been suggested to positively affect human health by reducing cholesterol levels in blood via inhibition of its absorption in the small intestine, thus protecting against heart attack and stroke. Sterols are challenging analytes for mass spectrometry, since their low polarity makes them difficult to ionize by both electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI), typically requiring derivatization steps to overcome their low ionization efficiencies. We present a fast and reliable method to characterize the composition of phytosterols in vegetable oils and enriched margarines. The method requires no derivatization steps or sample extraction procedures thanks to the use of transmission mode direct analysis in real time mass spectrometry (TM-DART-MS).

High throughput MS techniques for caviar lipidomics

The lipid profile of Sturgeon roe (caviar) was monitored by matrix assisted laser desorption/ionization mass spectrometry (MALDI(+)-MS), thermal imprinting easy ambient sonic-spray ionization mass spectrometry (TI-EASI(+)-MS) and electrospray mass spectrometry (ESI(+)-MS). Freshly salted and commercially salted pasteurized caviar samples of Atlantic sturgeon (Acipenser sturio) were stored either at +4 °C or at room temperature for 4 weeks. The different types of chemical information achieved by these MS techniques were compared: MALDI(+)-MS detects mainly phospholipids (PL) whereas TI-EASI(+)-MS allows monitoring of both triacylglycerols (TAG) and PL. ESI(+) coupled to Fourier transform ion cyclotron resonance high resolution mass spectrometer (FT-ICR-MS) and MS/MS experiments were used to fully characterize the detected lipids, ensure the absence of oxidation products in the degradation process and confirm the high efficiency of the thermal imprinting extraction. TI-EASI(+)-MS, via a more comprehensive profiling and easier operation, has therefore been demonstrated to provide caviar lipidomic profiles and discriminate its changes as a function of storage time and temperature. The data have also confirmed that hydrolysis is the main process of lipid degradation in caviar.

Spatial distribution of theobromine – a low MW drug – in tissues via matrix‐free NALDI‐MS imaging

The ability of nano-assisted laser desorption-ionization mass spectrometry imaging (NALDI-IMS) to provide selective chemical monitoring with appropriate spatial distribution of a low molecular drug in a biological tissue was investigated. NALDI-IMS is a matrix-free laser desorption ionization (LDI) protocol based on imprinting of tissue constituents on a nanostructured surface. Using the accumulation of theobromine in rat kidney as a model, NALDI-IMS was found to provide well-resolved images of the special distribution of this low molecular weight (MW) drug in tissue.

Variations in the Abundance of Lipid Biomarker Ions in Mass Spectrometry Images Correlate to Tissue Density

While mass spectrometry (MS) imaging is widely used to investigate the molecular composition of ex vivo slices of cancerous tumors, little is known about how variations in the cellular properties of cancer tissue can influence cancer biomarker ion images. To better understand the basis for variations in the abundances of cancer biomarker ions seen in MS images of relatively homogeneous ex vivo tumor samples, sections of snap frozen human breast cancer murine xenografts were subjected to desorption electrospray ionization mass spectrometry (DESI-MS) imaging. Serial sections were then stained with hematoxylin and eosin (H&E) and subjected to detailed morphometric cellular analysis, using a commercial digital pathology platform augmented with custom-tailored image analysis algorithms developed in-house. Gross morphological heterogeneities due to stroma, vasculature, and noncancer cells were mapped in the tumor and found to not correlate with the areas of suppressed cancer biomarker abundance. Instead, the ion abundances of major breast cancer biomarkers were found to correlate with the cytoplasmic area of cancer cells that comprised the tumor tissue. Therefore, detailed cellular analyses can be used to rationalize subtle heterogeneities in ion abundance in MS images, not explained by the presence of gross morphological heterogeneities such as stroma.

Molecular Signatures of High-Grade Cervical Lesions

Cervical cancer is the fourth most common neoplasia in women and the infection with human papilloma virus (HPV) is its necessary cause. Screening methods, currently based on cytology and HPV DNA tests, display low specificity/sensitivity, reducing the efficacy of cervical cancer screening programs. Herein, molecular signatures of cervical cytologic specimens revealed by liquid chromatography-mass spectrometry (LC-MS), were tested in their ability to provide a metabolomic screening for cervical cancer. These molecules were tested whether they could clinically differentiate insignificant HPV infections from precancerous lesions. For that, high-grade squamous intraepithelial lesions (HSIL)-related metabolites were compared to those of no cervical lesions in women with and without HPV infection. Samples were collected from women diagnosed with normal cervix (N = 40) and from those detected with HSIL from cytology and colposcopy (N = 40). Liquid-based cytology diagnosis, DNA HPV-detection test, and LC-MS analysis were carried out for all the samples. The same sample, in a customized collection medium, could be used for all the diagnostic techniques employed here. The metabolomic profile of cervical cancer provided by LC-MS was found to indicate unique molecular signatures for HSIL, being two ceramides and a sphingosine metabolite. These molecules occurred independently of women’s HPV status and could be related to the pre-neoplastic phenotype. Statistical models based on such findings could correctly discriminate and classify HSIL and no cervical lesion women. The results showcase the potential of LC-MS as an emerging technology for clinical use in cervical cancer screening, although further validation with a larger sample set is still necessary.

Multicenter Study Using Desorption-Electrospray-Ionization-Mass-Spectrometry Imaging for Breast-Cancer Diagnosis

The histological and molecular subtypes of breast cancer demand distinct therapeutic approaches. Invasive ductal carcinoma (IDC) is subtyped according to estrogen-receptor (ER), progesterone-receptor (PR), and HER2 status, among other markers. Desorption-electrospray-ionization-mass-spectrometry imaging (DESI-MSI) is an ambient-ionization MS technique that has been previously used to diagnose IDC. Aiming to investigate the robustness of ambient-ionization MS for IDC diagnosis and subtyping over diverse patient populations and interlaboratory use, we report a multicenter study using DESI-MSI to analyze samples from 103 patients independently analyzed in the United States and Brazil. The lipid profiles of IDC and normal breast tissues were consistent across different patient races and were unrelated to country of sample collection. Similar experimental parameters used in both laboratories yielded consistent mass-spectral data in mass-to-charge ratios ( m/ z) above 700, where complex lipids are observed. Statistical classifiers built using data acquired in the United States yielded 97.6% sensitivity, 96.7% specificity, and 97.6% accuracy for cancer diagnosis. Equivalent performance was observed for the intralaboratory validation set (99.2% accuracy) and, most remarkably, for the interlaboratory validation set independently acquired in Brazil (95.3% accuracy). Separate classification models built for ER and PR statuses as well as the status of their combined hormone receptor (HR) provided predictive accuracies (>89.0%), although low classification accuracies were achieved for HER2 status. Altogether, our multicenter study demonstrates that DESI-MSI is a robust and reproducible technology for rapid breast-cancer-tissue diagnosis and therefore is of value for clinical use.