Iveta Klouckova

Analysis of Volatile Organic Compounds in Human Saliva by a Static Sorptive Extraction Method and Gas Chromatography-Mass Spectrometry

Human saliva not only helps control oral health (with anti-microbial proteins), but it may also play a role in chemical communication. As is the case with other mammalian species, human saliva contains peptides, proteins, and numerous volatile organic compounds (VOCs). A high-throughput analytical method is described for profiling a large number of saliva samples to screen the profiles of VOCs. Saliva samples were collected in a non-stimulated fashion. The method utilized static stir bar extraction followed by gas chromatography-mass spectrometry (GC-MS). The method provided excellent reproducibility for a wide range of salivary compounds, including alcohols, aldehydes, ketones, carboxylic acids, esters, amines, amides, lactones, and hydrocarbons. Furthermore, substantial overlap of salivary VOCs and the previously reported skin VOCs in the same subject group was found in this study by using pattern recognition analyses. Sensitivity, precision, and reproducibility of the method suggest that this technique has potential in physiological, metabolomic, pharmacokinetic, forensic, and toxicological studies of small organic compounds where a large number of human saliva samples are involved.

Analysis of site-specific glycosylation of renal and hepatic γ-glutamyl transpeptidase from normal human tissue

The cell surface glycoprotein γ-glutamyl transpeptidase (GGT) was isolated from healthy human kidney and liver to characterize its glycosylation in normal human tissue in vivo. GGT is expressed by a single cell type in the kidney. The spectrum of N-glycans released from kidney GGT constituted a subset of the N-glycans identified from renal membrane glycoproteins. Recent advances in mass spectrometry enabled us to identify the microheterogeneity and relative abundance of glycans on specific glycopeptides and revealed a broader spectrum of glycans than was observed among glycans enzymatically released from isolated GGT. A total of 36 glycan compositions, with 40 unique structures, were identified by site-specific glycan analysis. Up to 15 different glycans were observed at a single site, with site-specific variation in glycan composition. N-Glycans released from liver membrane glycoproteins included many glycans also identified in the kidney. However, analysis of hepatic GGT glycopeptides revealed 11 glycan compositions, with 12 unique structures, none of which were observed on kidney GGT. No variation in glycosylation was observed among multiple kidney and liver donors. Two glycosylation sites on renal GGT were modified exclusively by neutral glycans. In silico modeling of GGT predicts that these two glycans are located in clefts on the surface of the protein facing the cell membrane, and their synthesis may be subject to steric constraints. This is the first analysis at the level of individual glycopeptides of a human glycoprotein produced by two different tissues in vivo and provides novel insights into tissue-specific and site-specific glycosylation in normal human tissues.

Mapping site-specific protein N-glycosylations through liquid chromatography/mass spectrometry and targeted tandem mass spectrometry

Glycosylation is one of the most common posttranslational modifications (PTMs) of proteins, the characterization of which is commonly achieved through proteomic protocol, involving trypsin digestion followed by liquid chromatography/tandem mass spectrometry (LC/MS/MS). However, it is often not possible to characterize all glycopeptides in a complex sample because of the high complexity of glycoproteomic samples, and the relative lower abundances of glycopeptides in comparison to the unmodified peptides. We present here a targeted MS/MS analysis approach, which utilizes a previously developed computational tool, GlyPID, to guide multiple experiments, thus permitting a complete characterization of all N-glycosylation sites of glycoproteins present in a complex sample. We have tested our approach using model glycoproteins analyzed by high-resolution LTQ-FT MS. The results demonstrate a potential use of our method for a high-throughput characterization of complex mixtures of glycosylated proteins.

Elevated levels of hydroxylated phosphocholine lipids in the blood serum of breast cancer patients.

The difference in serum phospholipid content between stage-IV breast cancer patients and disease-free individuals was studied by employing a combination of chemometric statistical analysis tools and mass spectrometry. Chloroform-extracted serum samples were profiled for their lipid class composition and structure using precursor ion, neutral loss, and product ion tandem mass spectrometric (MS/MS) scanning experiments. Changes in the relative abundance of phospholipids in serum as a consequence of cancer progression, measured through electrospray ionization (ESI) mass spectrometry of flow-injected serum samples collected from 25 disease-free individuals and 50 patients diagnosed with stage-IV breast cancer, were statistically evaluated using principal component analysis (PCA), analysis of variance (ANOVA) and receiver operating characteristic (ROC) analysis. Lipids whose abundance changed significantly as a consequence of cancer progression were structurally characterized using product ion spectra, and independently quantified using precursor ion scan experiments against an internal standard of known concentration. Phosphocholine lipids that displayed a statistically significant change as a consequence of cancer progression were found to contain an oxidized fatty acid moiety as determined by MS3 experiments.

Increased protein nitration in mitochondrial diseases: evidence for vessel wall involvement.

Mitochondrial diseases (MD) are heterogeneous disorders because of impairment of respiratory chain function leading to oxidative stress. We hypothesized that in MD the vascular endothelium may be affected by increased oxidative/nitrative stress causing a reduction of nitric oxide availability. We therefore, investigated the pathobiology of vasculature in MD patients by assaying the presence of 3-nitrotyrosine in muscle biopsies followed by the proteomic identification of proteins which undergo tyrosine nitration. We then measured the flow-mediated vasodilatation as a proof of altered nitric oxide generation/bioactivity. Here, we show that 3-nitrotyrosine staining is specifically located in the small vessels of muscle tissue and that the reaction is stronger and more evident in a significant percentage of vessels from MD patients as compared with controls. Eleven specific proteins which are nitrated under pathological conditions were identified; most of them are involved in energy metabolism and are located mainly in mitochondria. In MD patients the flow-mediated vasodilatation was reduced whereas baseline arterial diameters, blood flow velocity and endothelium-independent vasodilatation were similar to controls. The present results provide evidence that in MD the vessel wall is a target of increased oxidative/nitrative stress.

A quantitative investigation of fucosylated serum glycoproteins with application to esophageal adenocarcinoma

Although glycoproteomic studies provide unique opportunities for cancer research, it has been necessary to develop specific methods for analysis of oncologically interesting glycoproteins. We describe a general, multimethodological approach for quantitative glycoproteomic analysis of fucosylated glycoproteins in human blood serum. A total of 136 putative fucosylated glycoproteins were identified with very high confidence in three clinically relevant sample pools (N=5 for each), with a mean CV of 3.1% observed for replicate analyses. Two samples were collected from subjects diagnosed with esophagus disease states, high‐grade dysplasia plus esophageal adenocarcinoma, while the third sample was representative of a disease‐free condition. Some glycoproteins, observed to be significantly upregulated in esophageal adenocarcinoma, i.e. more than twofold higher than in the disease‐free condition, are briefly discussed. Further investigation will be necessary to validate these findings; however, the method itself is demonstrated to be an effective tool for quantitative glycoproteomics of clinical samples.

Gel-free shotgun proteomic analysis of human milk.

The composition of milk has adapted during the evolution of the species to fulfill the specific nutritional needs of the offspring. Currently, it is widely recognized that milk benefits go beyond mere nutrition and serve as a source of a number of functional components to the newborn, particularly host defense effectors. However, the human milk proteome description is still incomplete, primarily because the detection of low-abundance proteins remains challenging. To overcome the limitations of the classical electrophoresis-based approach, previously separated milk fat globule membrane (MFGM) and whey protein fractions were analyzed by nanoflow-high performance liquid chromatography (HPLC)/Fourier Transform-Ion Cyclotron Resonance (FT-ICR) mass spectrometry (MS). This shotgun strategy showed an as yet unmatched potential to profile low-abundance proteins in human milk. Proteins associated with 301 different gene products were identified, some of which could be clustered into subsets of protein isoforms, thus providing one of the largest protein inventories of human milk. The identified proteins, which were derived from multiple metabolic pathways, are involved in different physiological functions, such as membrane trafficking, cell signaling, fat metabolism and transport, metabolite delivery, protein synthesis/proteolysis or folding, and immunity-related actions. Nevertheless, it appears clear from this study that the overall picture of the human milk proteome is still incomplete, although several protein signatures of milk evolution are emerging.

A computational approach for the identification of site-specific protein glycosylations through ion-trap mass spectrometry

Glycosylation is one of the most common post-translational modifications (PTMs) of proteins, the characterization of which is commonly achieved utilizing mass spectrometry (MS). However, its applicability is currently limited by the lack of computational tools capable of autmoated interpretation of high throughput MS experiments which would allow the characterization of glycosylation sites and their microheterogeneities. We present here a computational approach which overcomes this problem and allows the identification and assignment of the microheterogeneities of glycosylation sites of glycoproteins from liquid chromatography ion-trap-based mass spectrometry (LC/MS) data. This method was implemented in a software tool and tested on several model glycoproteins. The results demonstrate the potential of our computational approach in automating the high throughput identification of glycoproteins.