One electron less or one proton more: how do they differ?

From the NIST website and the literature, we have collected the Ionisation Energies (IE) of 3,052 and the Proton Affinities (PA) of 1,670 compounds. For 614 of these, both the IE and PA are known; this enables a study of the relationships between these quantities for a wide variety of molecules. From th

Proteomic Analysis of Human Osteoblastic Cells: Relevant Proteins and Functional Categories for Differentiation

Abstract Osteoblasts are the bone forming cells, capable of secreting an extracellular matrix with mineralization potential. The exact mechanism by which osteoblasts differentiate and form a mineralized extracellular matrix is presently not fully understood. To increase our knowledge about this process, we conducted proteomics analysis in human immortalized preosteoblasts (SV-HFO) able to differentiate and mineralize. We identified 381 proteins expressed during the time course of osteoblast differentiation. Gene ontology analysis revealed an overrepresentation of protein categories established as important players for osteoblast differentiation, bone formation, and mineralization such as pyrophosphatases. Proteins involved in antigen presentation, energy metabolism and cytoskeleton rearrangement constitute other overrepresented processes, whose function, albeit interesting, is not fully understood in the context of osteoblast differentiation and bone formation. Correlation analysis, based on quantitative data, revealed a biphasic osteoblast differentiation, encompassing a premineralization and a mineralization period. Identified differentially expressed proteins between mineralized and nonmineralized cells include cytoskeleton (e.g., CCT2, PLEC1, and FLNA) and extracellular matrix constituents (FN1, ANXA2, and LGALS1) among others. FT-ICR-MS data obtained for FN1, ANXA2, and LMNA shows a specific regulation of these proteins during the different phases of osteoblast differentiation. Taken together, this study increases our understanding of the proteomics changes that accompany osteoblast differentiation and may permit the discovery of novel modulators of bone formation

Metabolic changes related to the IDH1 mutation in gliomas preserve TCA-cycle activity: An investigation at the protein level

The discovery of the IDH1 R132H (IDH1 mut) mutation in low-grade glioma and the associated change in function of the IDH1 enzyme has increased the interest in glioma metabolism. In an earlier study, we found that changes in expression of genes involved in the aerobic glycolysis and the TCA cycle are associated with IDH1 mut. Here, we apply proteomics to FFPE samples of diffuse gliomas with or without IDH1 mutations, to map changes in protein levels associated with this mutation. We observed significant changes in the enz

Down-Regulation of Collagen Hydroxylation in Colorectal Liver Metastasis

Collagen is significantly upregulated in colorectal liver metastasis (CRLM) compared to liver tissue. Expression levels of specific collagen types in CRLM resemble those in colorectal cancer (CRC) and colon tissue. We investigated whether the collagen hydroxylation pattern from the primary tumor also migrates with the metastatic tumor. The degree of collagen alpha-1(I) hydroxylation in colon, CRC, liver, and CRLM tissue of the same individuals (n = 14) was studied with mass spectrometry. The degree of hydroxylation was investigated in 36 collagen alpha-1(I) peptides, covering 54% of the triple helical region. The degree of hydroxylation in liver tissue was similar to that in colon tissue. The overall degree of hydroxylation was significantly lower (9 ± 14%) in CRC tissue and also significantly lower (12 ± 22%) in CRLM tissue compared to colon. Furthermore, eleven peptides with a specific number of hydroxylations are significantly different between CRLM and liver tissue; these peptides could be candidates for the detection of CRLM. For one of these eleven peptides, a matching naturally occurring peptide in urine has been identified as being significantly different between patients suffering from CRLM and healthy controls. The hydroxylation pattern in CRLM resembles partly the pattern in liver, primary colorectal cancer and colon

Small Hydrophobic Protein of Human Metapneumovirus Does Not Affect Virus Replication and Host Gene Expression In Vitro

Human metapneumovirus (HMPV) encodes a small hydrophobic (SH) protein of unknown function. HMPV from which the SH open reading frame was deleted (HMPVΔSH) was viable and displayed similar replication kinetics, cytopathic effect and plaque size compared with wild type HMPV in several cell-lines. In addition, no differences were observed in infection efficiency or cell-to-cell spreading in human primary bronchial epithelial cells (HPBEC) cultured at an air-liquid interphase. Host gene expression was analyzed in A549 cells infected with HMPV or HMPVΔSH using microarrays and mass spectrometry (MS) based techniques at multiple time points post infection. Only minor differences were observed in mRNA or protein expression levels. A possible function of HMPV SH as apoptosis blocker, as proposed for several members of the family Paramyxoviridae, was rejected based on this analysis. So far, a clear phenotype of HMPV SH deletion mutants in vitro at the virus and host levels is absent

Data Processing Pipeline for Lipid Profiling of Carotid Atherosclerotic Plaque with Mass Spectrometry Imaging

Atherosclerosis is a lipid and inflammation-driven disease of the arteries that is characterized by gradual buildup of plaques in the vascular wall. A so-called vulnerable plaque, consisting of a lipid-rich necrotic core contained by a thin fibrous cap, may rupture and trigger thrombus formation, which can lead to ischemia in the heart (heart attack) or in the brain (stroke). In this study, we present a protocol to investigate the lipid composition of advanced human carotid plaques using matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI), providing a framework that should enable the discrimination of vulnerable from stable plaques based on lipid composition. We optimized the tissue preparation and imaging method

Peptide profiling of cerebrospinal fluid by mass spectrometry

The search for biomarkers is driven by the increasing clinical importance of early diagnosis. Reliable biomarkers can also assist in directing therapy, monitoring disease activity and the efficacy of treatment. In addition, the discovery of novel biomarkers might provide clues to the pathogenesis of a disease. The dynamic range of protein concentrations in body fluids exceeds 10 orders of magnitude. These huge differences in concentrations complicate the detection of proteins with low expression levels. Since all classical biomarkers have low expression levels (e.g., prostate-specific antigen: 2-4 μg/l; and CA125: 20-35 U/ml), new developments with respect to identification and validation techniques of the low-abundance proteins are required. This review will discuss the current status of profiling cerebrospinal fluid using mass spectrometry-based techniques, and new developments in this area

A mono-acyl phospholipid (20:1 lyso-PS) activates Toll-Like Receptor 2/6 hetero-dimer

Toll-like receptor 2 (TLR2) is an important pattern recognition receptor on the surface of host immune cells that binds a variety of ligands that are released by microorganisms as well as by damaged or dying host cells. According to the current concept, TLR2/1 and TLR2/6 heterodimers are activated by tri- or di-acylated ligands, respectively. However, also mono-acyl phospholipid containing lipid fractions derived from parasites, were reported to be able to activate TLR2. In order to provide conclusive evidence for the TLR2 activating capacity of mono-acyl phospholipids derived from pathogens, we developed a biosynthetic method to enzymatically convert commercially available phospholipids into several mono-acyl-phospholipid variants that were examined for their TLR2 activating capacity. These investigations demonstrated that 1-(11Z-eicosenoyl)-glycero-3-phosphoserine 20:1 (20:1 lyso-PS) is a true agonist of the TLR2/6 heterodimer and that its polar headgroup as well as the length of the acyl chain are crucial for TLR2 activation. In silico modelling further confirmed 20:1 mono-acyl PS as a ligand for TLR2/6 heterodimer, as this predicted that multiple hydrogen bonds are formed between the polar headgroup of 20:1 mono-acyl PS and amino acid residues of both TLR2 and TLR6. Future studies can now be performed to further assess the functions of 20:1 lyso-PS as an immunological mediator, because this enzymatic method enables its preparation in larger quantities than is possible by isolation from the parasite that naturally produces this compound, Schistosoma mansoni, the source of the original discovery (Van der Kleij et al., 2002)

Identifying Ca2+-Binding sites in proteins by liquid chromatography-mass spectrometry using Ca2+-Directed dissociations

Here we describe a new method to identify calcium-binding sites in proteins using high-resolution liquid chromatography- mass spectrometry in concert with calcium-directed collision-induced dissociations. Our method does not require any modifications to the liquid chromatography- mass spectrometry apparatus, uses standard digestion protocols, and can be applied to existing high-resolution MS data files. In contrast to NMR, our method is applicable to very small amounts of complex protein mixtures (femtomole level). Calcium-bound peptides can be identified using three criteria: (1) the calculated exact mass of the calcium containing peptide; (2) specific dissociations of the calcium-containing peptide from threonine and serine residues; and (3) the very similar retention times of the calcium-containing peptide and the free peptide