Development, validation, and prognostic evaluation of LiverPRO for the prediction of significant liver fibrosis in primary care: a prospective cohort study.

Background: Clinically significant liver fibrosis is associated with future adverse events in patients with steatotic liver disease. We designed a software tool for detection of clinically significant liver fibrosis in primary care.

Methods: In this prospective cohort study, we developed and validated LiverPRO using six independent cohorts from Denmark, Germany, and England that included patients from primary and secondary care with steatotic liver disease related to alcohol or metabolic dysfunction.

Using the ELF test, FIB-4 and NAFLD fibrosis score to screen the population for liver disease.

Background & Aims: There is a need for accurate biomarkers of fibrosis for population screening of alcohol-related and non-alcoholic fatty liver disease (ALD, NAFLD). We compared the performance of the enhanced liver fibrosis (ELF) test to the fibrosis-4 index (FIB-4) and NAFLD fibrosis score (NFS), using transient elastography as the reference standard.

Methods: We prospectively included participants from the general population, and people at risk of ALD or NAFLD.

Quantification of the anti-neoplastic polyacetylene falcarinol from carrots in human serum by LC-MS/MS.

Falcarinol is a polyacetylene which is found in carrots and known to have anti-neoplastic properties in rodents. Research in the bioactivity of falcarinol in humans require methods for quantification of falcarinol in human serum. Here we report the development of an LC-MS/MS method and its use to measure serum falcarinol concentrations in humans following intake of a carrot product.

Ultraviolet Photodissociation of Protonated Peptides and Proteins Can Proceed with H/D Scrambling.

Ultraviolet photodissociation (UVPD) has recently been introduced as an ion activation method for the determination of single-residue deuterium levels in H/D exchange tandem mass spectrometry experiments. In this regard, it is crucial to know which fragment ion types can be utilized for this purpose. UVPD yields rich product ion spectra where all possible backbone fragment ion types (a/x, b/y, and c/z) are typically observed.

Deglycosylation by the Acidic Glycosidase PNGase H Enables Analysis of N-Linked Glycoproteins by Hydrogen/Deuterium Exchange Mass Spectrometry.

Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) has become an important method to study the structural dynamics of proteins. However, glycoproteins represent a challenge to the traditional HDX-MS workflow for determining the deuterium uptake of the protein segments that contain the glycan. We have recently demonstrated the utility of the glycosidase PNGase A to enable HDX-MS analysis of N-glycosylated protein regions.

Long-Lived Folding Intermediates Predominate the Targeting-Competent Secretome.

Secretory preproteins carry signal peptides fused amino-terminally to mature domains. They are post-translationally targeted to cross the plasma membrane in non-folded states with the help of translocases, and fold only at their final destinations. The mechanism of this process of postponed folding is unknown, but is generally attributed to signal peptides and chaperones.

Conformational preludes to the latency transition in PAI-1 as determined by atomistic computer simulations and hydrogen/deuterium-exchange mass spectrometry.

Both function and dysfunction of serine protease inhibitors (serpins) involve massive conformational change in their tertiary structure but the dynamics facilitating these events remain poorly understood. We have studied the dynamic preludes to conformational change in the serpin plasminogen activator inhibitor 1 (PAI-1). We report the first multi-microsecond atomistic molecular dynamics simulations of PAI-1 and compare the data with experimental hydrogen/deuterium-exchange data (HDXMS).

Removal of N-Linked Glycosylations at Acidic pH by PNGase A Facilitates Hydrogen/Deuterium Exchange Mass Spectrometry Analysis of N-Linked Glycoproteins.

Protein glycosylation is the most frequent post-translational modification and is present on more than 50% of eukaryotic proteins. Glycosylation covers a wide subset of modifications involving many types of complex oligosaccharide structures, making structural analysis of glycoproteins and their glycans challenging for most analytical techniques. Hydrogen/deuterium exchange monitored by mass spectrometry is a sensitive technique for investigation of protein conformational dynamics of complex heterogeneous proteins in solution.

Analysis of Translocation-Competent Secretory Proteins by HDX-MS.

Protein folding is an intricate and precise process in living cells. Most exported proteins evade cytoplasmic folding, become targeted to the membrane, and then trafficked into/across membranes. Their targeting and translocation-competent states are nonnatively folded.

An Asymmetric Runaway Domain Swap Antithrombin Dimer as a Key Intermediate for Polymerization Revealed by Hydrogen/Deuterium-Exchange Mass Spectrometry.

Antithrombin deficiency is associated with increased risk of venous thrombosis. In certain families, this condition is caused by pathogenic polymerization of mutated antithrombin in the blood. To facilitate future development of pharmaceuticals against antithrombin polymerization, an improved understanding of the polymerogenic intermediates is crucial.

Copper(II) Ions Increase Plasminogen Activator Inhibitor Type 1 Dynamics in Key Structural Regions That Govern Stability.

Plasminogen activator inhibitor type 1 (PAI-1) regulates the fibrinolysis pathway by inhibiting the protease activity of plasminogen activators. PAI-1 works in concert with vitronectin (VN), an extracellular protein that aids in localization of active PAI-1 to tissues. The Peterson laboratory demonstrated that Cu(II) and other transition metals modulate the stability of PAI-1, exhibiting effects that are dependent on the presence or absence of the somatomedin B (SMB) domain of VN.

An RNA Aptamer Inhibits a Mutation-Induced Inactivating Misfolding of a Serpin.

Most serpins are fast and specific inhibitors of extracellular serine proteases controlling biological processes such as blood coagulation, fibrinolysis, tissue remodeling, and inflammation. The inhibitory activity of serpins is based on a conserved metastable structure and their conversion to a more stable state during reaction with the target protease. However, the metastable state also makes serpins vulnerable to mutations, resulting in disease caused by inactive and misfolded monomeric or polymeric forms ("serpinopathy").

Binding of NFκB Appears to Twist the Ankyrin Repeat Domain of IκBα.

Total internal reflection fluorescence-based single-molecule Förster resonance energy transfer (FRET) measurements were previously carried out on the ankyrin repeat domain (ARD) of IκBα, the temporally regulated inhibitor of canonical NFκB signaling. Under native conditions, most of the IκBα molecules showed stable, high FRET signals consistent with distances between the fluorophores estimated from the crystal structures of the NFκB(RelA/p50)-IκBα complex. Similar high FRET efficiencies were found when the IκBα molecules were either free or in complex with NFκB(RelA/p50), and were interpreted as being consistent with the crystallographically observed ARD structure.

Local transient unfolding of native state PAI-1 associated with serpin metastability.

The metastability of the native fold makes serpin (serine protease inhibitor) proteins prone to pathological conformational change, often by insertion of an extra β-strand into the central β-sheet A. How this insertion is made possible is a hitherto unresolved question. By the use of advanced hydrogen/deuterium-exchange mass spectrometry (HDX-MS) it is shown that the serpin plasminogen activator inhibitor 1 (PAI-1) transiently unfolds under native condition, on a second-to-minute time scale.

Dissecting the effect of RNA aptamer binding on the dynamics of plasminogen activator inhibitor 1 using hydrogen/deuterium exchange mass spectrometry.

RNA aptamers, selected from large synthetic libraries, are attracting increasing interest as protein ligands, with potential uses as prototype pharmaceuticals, conformational probes, and reagents for specific quantification of protein levels in biological samples. Very little is known, however, about their effects on protein conformation and dynamics. We have employed hydrogen/deuterium exchange (HDX) mass spectrometry to study the effect of RNA aptamers on the structural flexibility of the serpin plasminogen activator inhibitor-1 (PAI-1).

Hydrogen/deuterium exchange mass spectrometry reveals specific changes in the local flexibility of plasminogen activator inhibitor 1 upon binding to the somatomedin B domain of vitronectin.

The native fold of plasminogen activator inhibitor 1 (PAI-1) represents an active metastable conformation that spontaneously converts to an inactive latent form. Binding of the somatomedin B domain (SMB) of the endogenous cofactor vitronectin to PAI-1 delays the transition to the latent state and increases the thermal stability of the protein dramatically. We have used hydrogen/deuterium exchange mass spectrometry to assess the inherent structural flexibility of PAI-1 and to monitor the changes induced by SMB binding.

Spatially resolved protein hydrogen exchange measured by subzero-cooled chip-based nanoelectrospray ionization tandem mass spectrometry.

Mass spectrometry has become a valuable method for studying structural dynamics of proteins in solution by measuring their backbone amide hydrogen/deuterium exchange (HDX) kinetics. In a typical exchange experiment one or more proteins are incubated in deuterated buffer at physiological conditions. After a given period of deuteration, the exchange reaction is quenched by acidification (pH 2.

SET8 is degraded via PCNA-coupled CRL4(CDT2) ubiquitylation in S phase and after UV irradiation.

The eukaryotic cell cycle is regulated by multiple ubiquitin-mediated events, such as the timely destruction of cyclins and replication licensing factors. The histone H4 methyltransferase SET8 (Pr-Set7) is required for chromosome compaction in mitosis and for maintenance of genome integrity. In this study, we show that SET8 is targeted for degradation during S phase by the CRL4(CDT2) ubiquitin ligase in a proliferating cell nuclear antigen (PCNA)-dependent manner.

Dynamic histone H3 epigenome marking during the intraerythrocytic cycle of Plasmodium falciparum.

Epigenome profiling has led to the paradigm that promoters of active genes are decorated with H3K4me3 and H3K9ac marks. To explore the epigenome of Plasmodium falciparum asexual stages, we performed MS analysis of histone modifications and found a general preponderance of H3/H4 acetylation and H3K4me3. ChIP-on-chip profiling of H3, H3K4me3, H3K9me3, and H3K9ac from asynchronous parasites revealed an extensively euchromatic epigenome with heterochromatin restricted to variant surface antigen gene families (VSA) and a number of genes hitherto unlinked to VSA.

Global histone analysis by mass spectrometry reveals a high content of acetylated lysine residues in the malaria parasite Plasmodium falciparum.

Post-translational modifications (PTMs) of histone tails play a key role in epigenetic regulation of gene expression in a range of organisms from yeast to human; however, little is known about histone proteins from the parasite that causes malaria in humans, Plasmodium falciparum. We characterized P. falciparum histone PTMs using advanced mass spectrometry driven proteomics.

Automated 2D peptide separation on a 1D nano-LC-MS system.

Given the complexity of the mammalian proteome, high-resolution separation technologies are required to achieve comprehensive proteome coverage and to enhance the detection of low-abundance proteins. Among several technologies, Multidimensional Protein Identification Technology (MudPIT) enables the on-line separation of highly complex peptide mixtures directly coupled with mass spectrometry-based identification. Here, we present a variation of the traditional MudPIT protocol, combining highly sensitive chromatography using a nanoflow liquid chromatography system (nano-LC) with a two-dimensional precolumn in a vented column setup.

Utility of immonium ions for assignment of epsilon-N-acetyllysine-containing peptides by tandem mass spectrometry.

Tandem mass spectrometry (MS/MS) is a powerful tool for characterization of post-translationally modified proteins, including epsilon-N-acetyllysine-containing species. Previous reports indicate that epsilon-N-acetyllysine immonium ions are useful marker ions for peptides containing epsilon-N-acetyllysine, but the specificity and sensitivity of these ions for assignment of lysine acetylation by MS/MS have not been studied in detail. We investigated MS/MS data sets of 172 epsilon-N-acetyllysine tryptic peptides and 268 nonacetylated tryptic peptides to establish the utility and reliability of epsilon-N-acetyllysine immonium ions for identification and validation of acetylated peptides.

The histone methyltransferase SET8 is required for S-phase progression.

Chromatin structure and function is influenced by histone posttranslational modifications. SET8 (also known as PR-Set7 and SETD8) is a histone methyltransferase that monomethylates histonfe H4-K20. However, a function for SET8 in mammalian cell proliferation has not been determined.

Functional proteomics in histone research and epigenetics.

Post-translational modifications of histones comprise an important part of epigenetic gene regulation. Mass spectrometry and immunochemical techniques are currently the methods of choice for identification and quantitation of known and novel histone modifications. While peptide-centric mass spectrometry is a well-established tool for identification and quantification of histone modifications, recent technological advances have allowed discrete modification patterns to be assessed on intact histones.