Clint M. Alfaro

Ambient ionisation mass spectrometry for lipid profiling and structural analysis of mammalian oocytes, preimplantation embryos and stem cells.

Lipids play fundamental roles in mammalian embryo preimplantation development and cell fate. Triacylglycerol accumulates in oocytes and blastomeres as lipid droplets, phospholipids influence membrane functional properties, and essential fatty acid metabolism is important for maintaining the stemness of cells cultured in vitro. The growing impact that lipids have in the field of developmental biology makes analytical approaches to analyse structural information of great interest. This paper describes the concept and presents the results of lipid profiling by mass spectrometry (MS) of oocytes and preimplantation embryos, with special focus on ambient ionisation. Based on our previous experience with oocytes and embryos, we aim to convey that ambient MS is also valuable for stem cell differentiation analysis. Ambient ionisation MS allows the detection of a wide range of lipid classes (e.g. free fatty acids, cholesterol esters, phospholipids) in single oocytes, embryos and cell pellets, which are informative of in vitro culture impact, developmental and differentiation stages. Background on MS principles, the importance of underused MS scan modes for structural analysis of lipids, and statistical approaches used for data analysis are covered. We envisage that MS alone or in combination with other techniques will have a profound impact on the understanding of lipid metabolism, particularly in early embryo development and cell differentiation research.

Differential Lipid Profiles of Normal Human Brain Matter and Gliomas by Positive and Negative Mode Desorption Electrospray Ionization - Mass Spectrometry Imaging.

Desorption electrospray ionization—mass spectrometry (DESI-MS) imaging was used to analyze unmodified human brain tissue sections from 39 subjects sequentially in the positive and negative ionization modes. Acquisition of both MS polarities allowed more complete analysis of the human brain tumor lipidome as some phospholipids ionize preferentially in the positive and others in the negative ion mode. Normal brain parenchyma, comprised of grey matter and white matter, was differentiated from glioma using positive and negative ion mode DESI-MS lipid profiles with the aid of principal component analysis along with linear discriminant analysis. Principal component–linear discriminant analyses of the positive mode lipid profiles was able to distinguish grey matter, white matter, and glioma with an average sensitivity of 93.2% and specificity of 96.6%, while the negative mode lipid profiles had an average sensitivity of 94.1% and specificity of 97.4%. The positive and negative mode lipid profiles provided complementary information. Principal component–linear discriminant analysis of the combined positive and negative mode lipid profiles, via data fusion, resulted in approximately the same average sensitivity (94.7%) and specificity (97.6%) of the positive and negative modes when used individually. However, they complemented each other by improving the sensitivity and specificity of all classes (grey matter, white matter, and glioma) beyond 90% when used in combination. Further principal component analysis using the fused data resulted in the subgrouping of glioma into two groups associated with grey and white matter, respectively, a separation not apparent in the principal component analysis scores plots of the separate positive and negative mode data. The interrelationship of tumor cell percentage and the lipid profiles is discussed, and how such a measure could be used to measure residual tumor at surgical margins.

Feasibility of desorption electrospray ionization mass spectrometry for diagnosis of oral tongue squamous cell carcinoma

RATIONALE Desorption electrospray ionization mass spectrometry (DESI-MS) has demonstrated utility in differentiating tumor from adjacent normal tissue in both urologic and neurosurgical specimens. We sought to evaluate if this technique had similar accuracy in differentiating oral tongue squamous cell carcinoma (SCC) from adjacent normal epithelium due to current issues with late diagnosis of SCC in advanced stages. METHODS Fresh frozen samples of SCC and adjacent normal tissue were obtained by surgical resection. Resections were analyzed using DESI-MS sometimes by a blinded technologist. Normative spectra were obtained for separate regions containing SCC or adjacent normal epithelium. Principal Component Analysis and Linear Discriminant Analysis (PCA-LDA) of spectra were used to predict SCC versus normal tongue epithelium. Predictions were compared with pathology to assess accuracy in differentiating oral SCC from adjacent normal tissue. RESULTS Initial PCA score and loading plots showed clear separation of SCC and normal epithelial tissue using DESI-MS. PCA-LDA resulted in accuracy rates of 95% for SCC versus normal and 93% for SCC, adjacent normal and normal. Additional samples were blindly analyzed with PCA-LDA pixel-by-pixel predicted classifications as SCC or normal tongue epithelial tissue and compared against histopathology. The m/z 700-900 prediction model showed a 91% accuracy rate. CONCLUSIONS DESI-MS accurately differentiated oral SCC from adjacent normal epithelium. Classification of all typical tissue types and pixel predictions with additional classifications should increase confidence in the validation model.

N-Acetylaspartate and 2-Hydroxyglutarate Assessed in Human Brain Tissue by Mass Spectrometry as Neuronal Markers of Oncogenesis

To the Editor: Mass spectrometry (MS)1 assays are becoming crucial inputs into clinical decisions in personalized medicine (1). Several assays are finding application for the diagnosis of cancers because they can rapidly reveal the molecular content of minute biopsy tissue samples and provide diagnostic information that complements histopathological examination. For gliomas, which represent 75% of all malignant brain tumors, N-acetylaspartate (NAA) and 2-hydroxyglutarate (2HG) are markers of oncogenesis, and their MS measurement in brain tissue has the potential to improve glioma diagnosis. Here, we describe new results of a direct MS method applied to NAA and 2HG and discuss diagnostic and prognostic implications. We performed electrospray ionization (ESI) of brain tissue extracts with a triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode. Quantitative measurements were made on 28 human banked neurological specimens obtained from the Biorepository of the Methodist Hospital in Indianapolis (IRB # 1410015344). Adjacent tissue was examined by histopathology to identify the presence of the tumor, estimate degree of infiltration, which was measured as tumor cell percentage, and assess isocitrate dehydrogenase (IDH) mutation status. The tissue was weighed wet and extracted using methanol:water (3:2 v/v, 2 g/L) spiked with NAA-d3 and 2HG-d3. Extracts were homogenized and centrifuged. The supernatant was infused into the instrument and MRM transitions …