Concentration Of Macromolecules Research Articles

Crowding modulates the glycation of plasma proteins: In vitro analysis of structural modifications to albumin and transferrin and identification of sites of modification.

Protein modification occurs in biological milieus that are characterized by high concentrations of (macro)molecules (i.e. heterogeneous and packed environments). Recent data indicate that crowding can modulate the extent and rate of protein oxidation, however its effect on other post-translational modifications remains to be explored. In this work we hypothesized that crowding would affect the glycation of plasma proteins. Physiologically-relevant concentrations of albumin (35mgmL-1) and transferrin (2mgmL-1) were incubated with methylglyoxal and glyoxal (5μM-5mM), two α-oxoaldehyde metabolites that are elevated in the plasma of people with diabetes. Crowding was induced by adding dextran or ficoll polymers. Electrophoresis, electron microscopy, fluorescence spectroscopy and mass spectrometry were employed to investigate the structural consequences of glycation under crowded conditions. Our data demonstrate that crowding modulates the extent of formation of transferrin cross-links, and also the modification pathways in both albumin and transferrin. Arginine was the most susceptible residue to modification, with lysine and cysteine also affected. Loss of 0.48 and 7.28 arginine residues per protein molecule were determined on incubation with 500μM methylglyoxal for albumin and transferrin, respectively. Crowding did not influence the extent of loss of arginine and lysine for either protein, but the sites of modification, detected by LC-MS, were different between dilute and crowded conditions. These data confirm the relevance of studying modification processes under conditions that closely mimic biological milieus. These data unveil additional factors that influence the pattern and extent of protein modification, and their structural consequences, in biological systems.

Geobacter sulfurreducens' Unique Metabolism Results in Cells with a High Iron and Lipid Content.

Geobacter sulfurreducens is a ubiquitous iron-reducing bacterium in soils, and in engineered systems, it can respire an electrode to produce measurable electric current. Its unique metabolism, heavily dependent on an extensive network of cytochromes, requires a unique cell composition. In this work, we used metallomics, cell fraction and elemental analyses, and transcriptomics to study and analyze the cell composition of G. sulfurreducens. Elemental composition studies (C, H, O, N, and ash content) showed high C:O and H:O ratios of approximately 1.7:1 and 0.25:1, indicative of more reduced cell composition that is consistent with high lipid content. Our study shows that G. sulfurreducens cells have a large amount of iron (2 ± 0.2 μg/g dry weight) and lipids (32 ± 0.5% dry weight/dry weight) and that this composition does not change whether the cells are grown with a soluble or an insoluble electron acceptor. The high iron concentration, higher than similar microorganisms, is attributed to the production of cytochromes that are abundant in transcriptomic analyses in both solid and soluble electron acceptor growth. The unique cell composition of G. sulfurreducens must be considered when growing this microorganism for lab studies and commercial applications. IMPORTANCE Geobacter sulfurreducens is an electroactive microorganism. In nature, it grows on metallic minerals by transferring electrons to them, effectively "breathing" metals. In a manmade system, it respires an electrode to produce an electric current. It has become a model organism for the study of electroactive organisms. There are potential biotechnological applications of an organism that can bridge the gap between biology and electrical signal and, as a ubiquitous iron reducer in soils around the world, G. sulfurreducens has an impact on the global iron cycle. We measured the concentrations of metals, macromolecules, and basic elements in G. sulfurreducens to define this organism's composition. We also used gene expression data to discuss which proteins those metals could be associated with. We found that G. sulfurreducens has a large amount of lipid and iron compared to other bacteria-these observations are important for future microbiologists and biotechnologists working with the organism.

Macromolecular Crowding as an Intracellular Stimulus for Responsive Nanomaterials.

Stimuli-responsive materials are exploited in biological, materials, and sensing applications. We introduce a new endogenous stimulus, biomacromolecule crowding, which we achieve by leveraging changes in thermoresponsive properties of polymers upon high concentrations of crowding agents. We prepare poly(2-oxazoline) amphiphiles that exhibit lower critical solution temperatures (LCST) in serum above physiological temperature. These amphiphiles stabilize oil-in-water nanoemulsions at temperatures below the LCST but are ineffective surfactants above the LCST, resulting in emulsion fusion. We find that the transformations observed upon heating nanoemulsions above their surfactant's LCST can instead be induced at physiological temperatures through the addition of polymers and protein, rendering thermoresponsive materials "crowding responsive." We demonstrate that the cytosol is a stimulus for nanoemulsions, with droplet fusion occurring upon injection into cells of living zebrafish embryos. This report sets the stage for classes of thermoresponsive materials to respond to macromolecule concentration rather than temperature changes.

Hydrodynamic model for renal microvascular filtration: Effects of physiological and hemodynamic changes on glomerular size-selectivity.

The first step in renal urine formation is ultrafiltration across the glomerular barrier. The change in its nanostructure has been associated with nephrotic syndromes. Effects of physiological and hemodynamic factor alterations associated with diabetic nephropathy (DN) on glomerular permselectivity are examined through a mathematical model employing low-Reynolds-number hydrodynamics and hindered transport theory. Glomerular capillaries are represented as networks of cylindrical tubes with multilayered walls. Glomerular basement membrane (GBM) is a fibrous medium with bimodal fiber sizes. Epithelial slit fiber spacing follows a lognormal distribution based on reported electron micrographs with the highest resolution. Endothelial fenestrae are filled with fibers the size of glycosaminoglycans (GAGs). Effects of fiber-macromolecule steric and hydrodynamic interactions are included. Focusing on diabetic nephropathy, the physiological and hemodynamic factors employed in the computation are those reported for healthy humans and patients with early-but-overt diabetic nephropathy. The macromolecule concentration is obtained as a finite element solution of the convection-diffusion equation. Computed sieving coefficients averaged along the capillary length agree well with ficoll sieving coefficients from studies in humans for most solute radii. GBM thickening and the loss of the slit diaphragm hardly affect glomerular permselectivity. GAG volume fraction reduction in the endothelial fenestrae, however, significantly increases macromolecule filtration. Increased renal plasma flow rate (RPF), glomerular hypertension, and reduction of lumen osmotic pressure cause a slight sieving coefficient decrease. These effects are amplified by an increased macromolecule size. Our results indicate that glomerular hypertension and the reduction in the oncotic pressure decreases glomerular macromolecule filtration. Reduction of RPF and changes in the glomerular barrier structure associated with DN, however, increase the solute sieving. Damage to GAGs caused by hyperglycemia is likely to be the most prominent factor affecting glomerular size-selectivity.

Insights from auditory cortex for GABA+ magnetic resonance spectroscopy studies of aging.

Changes in levels of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) may underlie aging-related changes in brain function. GABA and co-edited macromolecules (GABA+) can be measured with MEGA-PRESS magnetic resonance spectroscopy (MRS). The current study investigated how changes in the aging brain impact the interpretation of GABA+ measures in bilateral auditory cortices of healthy young and older adults. Structural changes during aging appeared as decreasing proportion of grey matter in the MRS volume of interest and corresponding increase in cerebrospinal fluid. GABA+ referenced to H2 O without tissue correction declined in aging. This decline persisted after correcting for tissue differences in MR-visible H2 O and relaxation times but vanished after considering the different abundance of GABA+ in grey and white matter. However, GABA+ referenced to creatine and N-acetyl aspartate (NAA), which showed no dependence on tissue composition, decreased in aging. All GABA+ measures showed hemispheric asymmetry in young but not older adults. The study also considered aging-related effects on tissue segmentation and the impact of co-edited macromolecules. Tissue segmentation differed significantly between commonly used algorithms, but aging-related effects on tissue-corrected GABA+ were consistent across methods. Auditory cortex macromolecule concentration did not change with age, indicating that a decline in GABA caused the decrease in the compound GABA+ measure. Most likely, the macromolecule contribution to GABA+ leads to underestimating an aging-related decrease in GABA. Overall, considering multiple GABA+ measures using different reference signals strengthened the support for an aging-related decline in auditory cortex GABA levels.

Impact of Inert Crowders on Host-Guest Recognition Process.

Biological environments typically contain high concentrations (300-400 mg/mL) of different macromolecules at volume fractions as large as 30%-40%. Biomolecular recognition processes, a ubiquitous biological phenomena, occurring in such crowded heterogeneous media would differ significantly compared to the dilute buffer solutions. Here we quantify the potential impact of inert crowders on prototypical host-guest recognition process by explicit-solvent molecular dynamics (MD) simulations in atomic resolution. We demonstrate that the crowders, when smaller in size, would facilitate the binding process of the guest molecule by decreasing the free energy barrier for binding via excluded volume effect and desolvation of the host receptor. However, the extent of crowder-induced stabilization of a host-guest complex is found to be significantly higher when the guest molecule is sterically constricted to approach the host along a centrosymmetric direction, compared to its unrestricted, freely diffusive movement. A kinetic analysis of the recognition process reveals that the origin of a relatively stronger crowder impact during constricted movement of guest molecule lies in the relatively enhanced residence time of the guest inside the host by crowders. Together, our results suggest that the extent of impact of crowding on recognition processes would be contingent upon the presence or absence of constriction on ligand movement.

Water Treatment Using High Performance Antifouling Ultrafiltration Polyether Sulfone Membranes Incorporated with Activated Carbon

Membrane fouling is a continued critical challenge for ultrafiltration membranes performance. In this work, polyether sulfone (PES) ultrafiltration (UF) membranes were fabricated via phase-inversion method by incorporating varying concentrations of APTMS modified activated carbon (mAC). The mAC was thoroughly characterized and the fabricated membranes were studied for their surface morphology, functional groups, contact angle, water retention, swelling (%) porosity, and water flux. The hydrophilicity of mAC membranes also resulted in lower contact angle and higher values of porosity, roughness, water retention as well as water flux. Also, the membranes incorporated with mAC exhibited antibacterial performance against model test strains of gram-negative Ecoil and gram-positive S. aureus. The antifouling studies based on bovine serum albumin protein (BSA) solution filtration showed that mAC membranes have better BSA flux. The higher flux and antifouling characteristics of the mAC membranes were attributed to the electrostatic repulsion of the BSA protein from the unique functional properties of AC and network structure of APTMS. The novel mAC ultrafiltration membranes developed and studied in present work can provide higher flux and less BSA rejection thus can find antifouling applications for the isolation and concentration of proteins and macromolecules.

Effect of pH Dependent Substrate Inhibition on Glutamate Dehydrogenase Kinetics

Glutamate dehydrogenase (GDH) serves as the main regulated enzyme linking protein and carbohydrate metabolism. This enzyme converts the amino acid glutamate to the TCA intermediate, α‐ketoglutarate. While dysregulation of GDH is linked to hyperinsulinism and other diseases, the kinetics this enzyme of have only been analyzed under dilution conditions. However, cells are more crowded than these buffer solutions frequently used to collect enzyme kinetics data because their cytoplasm contains high concentrations of proteins, carbohydrates, and other macromolecules. Evidence has shown that this macromolecular crowding slow diffusion rates and changes protein conformations, which alters the kinetics of enzymes. Scientists often mimic the crowded environment of a cell using large, synthetic polymers, such as dextran, a glucose polymer, which excludes volume to other biological molecules in the same solution. While several studies have characterized these excluded volume effects on enzyme kinetics, this is the first study to analyze the effects of crowding on enzyme inhibition. Investigating how inhibitors like GTP and activators like leucine effect the kinetics of GDH will provide a deeper understanding of the regulation of this enzyme inside out bodies. Furthermore, saturating amounts of glutamate results in substrate inhibition of GDH by forming a “dead end complex” comprised of glutamate, NADH, and GDH at low pH values. To understand the effects of crowding on substrate inhibition, GDH Michaelis Menten kinetics were analyzed in the presence and absence of dextran at four pH values. These assays were repeated with glucose, the monomer of dextran as a control to separate chemical effects from excluded volume effects caused by the large size of dextran. The presence of dextran resulted in more substrate inhibition than in the presence of glucose and this inhibition that was observed at higher pH values than in buffer alone. Leucine, a known activator of GDH, operates by destabilizing the dead‐end complex. While the presence of leucine decreases substrate inhibition in dilute conditions, this compound was unable to active GDH in the presence of dextran. Overall, these results suggest that crowding promotes substrate inhibition by stabilizing the dead‐end complex of GDH which is a stronger interaction than leucine dissociation of the dead‐end complex. Therefore, macromolecular crowding in a cell could provide an additional level of regulation on GDH inside cells.

A Difference between In Vitro and In-Cell Protein Dimer Formation.

The high concentration of macromolecules in cells affects the stability of proteins and protein complexes via hard repulsions and chemical interactions, yet few studies have focused on chemical interactions. We characterized the domain-swapped dimer of the B1 domain of protein G in buffer and Escherichia coli cells by using heteronuclear, multidimensional nuclear magnetic resonance spectroscopy. In buffer, the monomer is a partially folded molten globule, but that species is not observed in cells. Experiments using urea suggest that the monomer is unfolded in cells, but again, the molten-globule form of the monomer is absent. The data suggest that attractive chemical interactions in the cytoplasm unfold the molten globule. We conclude that the intracellular environment not only modulates the stability of protein complexes but also can change the species present, reinforcing the idea that chemical interactions are more important than hard repulsions in cells.

Rapid Temperature-Dependent Rheological Measurements of Non-Newtonian Solutions Using a Machine-Learning Aided Microfluidic Rheometer.

Biofluids such as synovial fluid, blood plasma, and saliva contain several proteins which impart non-Newtonian properties to the biofluids. The concentration of such protein macromolecules in biofluids is regarded as an important biomarker for the diagnosis of several health conditions, including cardiovascular disorders, joint quality, and Alzheimer's. Existing technologies for the measurements of macromolecules in biofluids are limited; they require a long turnaround time, or require complex protocols, thus calling for alternative, more suitable, methodologies aimed at such measurements. According to the well-established relations for polymer solutions, the concentration of macromolecules in solutions can also be derived via measurement of rheological properties such as shear-viscosity and the longest relaxation time. We here introduce a microfluidic rheometer for rapid simultaneous measurement of shear viscosity and longest relaxation time of non-Newtonian solutions at different temperatures. At variance with previous technologies, our microfluidic rheometer provides a very short turnaround time of around 2 min or less thanks to the implementation of a machine-learning algorithm. We validated our platform on several aqueous solutions of poly(ethylene oxide). We also performed measurements on hyaluronic acid solutions in the clinical range for joint grade assessment. We observed monotonic behavior with the concentration for both rheological properties, thus speculating on their use as potential rheo-markers, i.e., rheological biomarkers, across several disease states.

Physical properties of the cytoplasm modulate the rates of microtubule polymerization and depolymerization.

The cytoplasm is a crowded, visco-elastic environment whose physical properties change according to physiological or developmental states. How the physical properties of the cytoplasm impact cellular functions invivo remains poorly understood. Here, we probe the effects of cytoplasmic concentration on microtubules by applying osmotic shifts to fission yeast, moss, and mammalian cells. We show that the rates of both microtubule polymerization and depolymerization scale linearly and inversely with cytoplasmic concentration; an increase in cytoplasmic concentration decreases the rates of microtubule polymerization and depolymerization proportionally, whereas a decrease in cytoplasmic concentration leads to the opposite. Numerous lines of evidence indicate that these effects are due to changes in cytoplasmic viscosity rather than cellular stress responses or macromolecular crowding per se. We reconstituted these effects on microtubules invitro by tuning viscosity. Our findings indicate that, even in normal conditions, the viscosity of the cytoplasm modulates the reactions that underlie microtubule dynamic behaviors.

Diffusion NMR of alpha-synuclein in the presence of bacterial cell lysate crowders

The crowded and heterogeneous intracellular milieu has a concentration of macromolecules up to 400 mg/ml. The causes of macromolecular crowding effects are excluded volume and ‘soft' nonspecific interactions, such as hydrogen bonds, electrostatic, Van der Waals, and hydrophobic interactions. Some physiological consequences of macromolecular crowding on proteins are on protein structure, protein folding, phase separation, aggregation, and protein-protein interaction. Traditionally, macromolecular crowding effects has been studied in artificial crowders like Ficoll and Dextran.

Counteractive effects of electrostatics and macromolecular crowding on actin bundle mechanics, organization, and secondary structure

The structural and mechanical properties of actin bundles are essential to eukaryotic cells aiding in cellular motility and contributing to structural support of plasma membrane. Bundle formation occurs in crowded intracellular environments containing high concentrations of ions and various macromolecules. While the roles of electrostatic interactions and macromolecular crowding on actin bundles has been independently explored, how the combined effects modulate the mechanics, organization, and structural transitioning of bundles has yet to fully established.

Stability and Activity of the 10-23 DNAzyme Under Molecular Crowding Conditions.

DNAzymes are biocatalysts that have been selected in vitro and their function inside cells (in vivo) is extremely low. Thus, almost all studies have been carried out in diluted solutions (in vitro). The cellular presence of molecules such as amino acids, polypeptides, alcohols, and sugars introduces forces that modify the kinetics and thermodynamics of DNAzyme-mediated catalysis. The crowded intracellular environment referred to as molecular crowding can be mimicked by adding high concentrations of natural or synthetic macromolecules to the reaction conditions. Here, we investigate the activity of the 10-23 DNAzyme and the stability of the DNAzyme:RNA complex under molecular crowding conditions. Therefore, we use a Förster resonance energy transfer (FRET)-based activity assay in combination with denaturing urea polyacrylamide gel electrophoresis and circular dichroism (CD) spectroscopy.

Macromolecular Crowding Induces a Binding Competent Transient Structure in Intrinsically Disordered Gab1

Intrinsically disordered proteins (IDPs) are an important class of proteins which lack tertiary structure elements. Their dynamic properties can depend on reversible post-translational modifications and the complex cellular milieu, which provides a crowded environment. Both influences the thermodynamic stability and folding of globular proteins as well as the conformational plasticity of IDPs. Here we investigate the intrinsically disordered C-terminal region (amino acids 613–694) of human Grb2-associated binding protein 1 (Gab1), which binds to the disease-relevant Src homolog region 2 (SH2) domain-containing protein tyrosine phosphatase SHP2 (PTPN11). This binding is mediated by phosphorylation at Tyr 627 and Tyr 659 in Gab1. We characterize induced structure in Gab1613–694 and binding to SHP2 by NMR, CD and ITC under non-crowding and crowding conditions, employing chemical and biological crowding agents and compare the results of the non-phosphorylated and tyrosine phosphorylated C-terminal Gab1 fragment. Our results show that under crowding conditions pre-structured motifs in two distinct regions of Gab1 are formed whereas phosphorylation has no impact on the dynamics and IDP character. These structured regions are identical to the binding regions towards SHP2. Therefore, biological crowders could induce some SHP2 binding capacity. Our results therefore indicate that high concentrations of macromolecules stabilize the preformed or excited binding state in the C-terminal Gab1 region and foster the binding to the SH2 tandem motif of SHP2, even in the absence of tyrosine phosphorylation.

Fabrication and Performance of Low-Fouling UF Membranes for the Treatment of Isolated Soy Protein Solutions

Consumers are becoming more conscious about the need to include functional and nutritional foods in their diet. This has increased the demand for food extracts rich in proteins and peptides with physiological effects that are used within the food and pharmaceutical industries. Among these protein extracts, soy protein and its derivatives are highlighted. Isolated soy protein (ISP) presents a protein content of at least 90%. Wastewaters generated during the production process contain small proteins (8–50 kDa), and it would be desirable to find a recovery treatment for these compounds. Ultrafiltration membranes (UF) are used for the fractionation and concentration of protein solutions. By the appropriate selection of the membrane pore size, larger soy proteins are retained and concentrated while carbohydrates and minerals are mostly recovered in the permeate. The accumulation and concentration of macromolecules in the proximity of the membrane surface generates one of the most important limitations inherent to the membrane technologies. In this work, three UF membranes based on polyethersulfone (PES) were fabricated. In two of them, polyethylene glycol (PEG) was added in their formulation to be used as a fouling prevention. The membrane fouling was evaluated by the study of flux decline models based on Hermia’s mechanisms.

Seawater Desalination by Modified Membrane Distillation: Effect of Hydrophilic Surface Modifying Macromolecules Addition into PVDF Hollow Fiber Membrane.

Hollow fiber membranes of polyvinylidene fluoride (PVDF) were prepared by incorporating varying concentrations of hydrophilic surface-modifying macromolecules (LSMM) and a constant amount of polyethylene glycol (PEG) additives. The membranes were fabricated by the dry-wet spinning technique. The prepared hollow fiber membranes were dip-coated by hydrophobic surface-modifying macromolecules (BSMM) as the final step fabrication. The additives combination is aimed to produce hollow fiber membranes with high flux permeation and high salt rejection in the matter of seawater desalination application. This study prepares hollow fiber membranes from the formulation of 18 wt. % of PVDF mixed with 5 wt. % of PEG and 3, 4, and 5 wt. % of LSMM. The membranes are then dip-coated with 1 wt. % of BSMM. The effect of LSMM loading on hydrophobicity, morphology, average pore size, surface porosity, and membrane performance is investigated. Coating modification on LSMM membranes showed an increase in contact angle up to 57% of pure, unmodified PVDF/PEG membranes, which made the fabricated membranes at least passable when hydrophobicity was considered as one main characteristic. Furthermore, The PVDF/PEG/4LSMM-BSMM membrane exhibits 161 °C of melting point as characterized by the DSC. This value indicates an improvement of thermal behavior shows so as the fabricated membranes are desirable for membrane distillation operation conditions range. Based on the results, it can be concluded that PVDF/PEG membranes with the use of LSMM and BSMM combination could enhance the permeate flux up to 81.32 kg·m−2·h−1 at the maximum, with stable salt rejection around 99.9%, and these are found to be potential for seawater desalination application.

The impeded diffusion fraction quantitative imaging assay demonstrated in multi-exponential diffusion phantom and prostate cancer.

To demonstrate a method for quantification of impeded diffusion fraction (IDF) using conventional clinical DWI protocols. The IDF formalism is introduced to quantify contribution from water coordinated by macromolecules to DWI voxel signal based on fundamentally different diffusion constants in vascular capillary, bulk free, and coordinated water compartments. IDF accuracy was studied as a function of b-value set. The IDF scaling with restricted compartment size and polyvinylpirrolidone (PVP) macromolecule concentration was compared to conventional apparent diffusion coefficient (ADC) and isotropic kurtosis model parameters for a diffusion phantom. An in vivo application was demonstrated for six prostate cancer (PCa) cases with low and high grade lesions annotated from whole mount histopathology. IDF linearly scaled with known restricted (vesicular) compartment size and PVP concentration in phantoms and increased with histopathologic score in PCa (from median 9% for atrophy up to 60% for Gleason 7). IDF via non-linear fit was independent of b-value subset selected between b = 0.1 and 2 ms/µm2 , including standard-of-care (SOC) PCa protocol. With maximum sensitivity for high grade PCa, the IDF threshold below 51% reduced false positive rate (FPR = 0/6) for low-grade PCa compared to apparent diffusion coefficient (ADC > 0.81 µm2 /ms) of PIRADS PCa scoring (FPR = 3/6). The proposed method may provide quantitative imaging assays of cancer grading using common SOC DWI protocols.

Forces of RBC interaction with single endothelial cells in stationary conditions: Measurements with laser tweezers

Red blood cells (RBCs) are able to interact and communicate with endothelial cells (ECs). Under some pathological or even normal conditions, the adhesion of RBCs to the endothelium can be observed. Presently, the mechanisms and many aspects of the interaction between RBCs and ECs are not fully understood. In this work, we considered the interaction of single RBCs with single ECs in vitro aiming to quantitatively determine the force of this interaction using laser tweezers. Measurements were performed under different concentrations of proaggregant macromolecules and in the presence or absence of tumor necrosis factor (TNF-[Formula: see text]) activating the ECs. We have shown that the strength of interaction depends on the concentration of fibrinogen or dextran proaggregant macromolecules in the environment. A nonlinear increase in the force of cells interaction (from 0.4 pN to 21 pN) was observed along with an increase in the fibrinogen concentration (from 3[Formula: see text]mg/mL to 9[Formula: see text]mg/mL) in blood plasma, as well as with the addition of dextran macromolecules (from 10[Formula: see text]mg/mL to 60[Formula: see text]mg/mL). Dextran with a higher molecular mass (500[Formula: see text]kDa) enhances the adhesion of RBCs to ECs greater compared to the dextran with a lower molecular mass (70[Formula: see text]kDa). With the preliminary activation of ECs with TNF-[Formula: see text], the force of interaction increases. Also, the adhesion of echinocytes to EC compared to discocytes is significantly higher. These results may help to better understand the process of interaction between RBCs and ECs.

Results and interpretation of a fitting challenge for MR spectroscopy set up by the MRS study group of ISMRM.

Fitting of MRS data plays an important role in the quantification of metabolite concentrations. Many different spectral fitting packages are used by the MRS community. A fitting challenge was set up to allow comparison of fitting methods on the basis of performance and robustness. Synthetic data were generated for 28 datasets. Short-echo time PRESS spectra were simulated using ideal pulses for the common metabolites at mostly near-normal brain concentrations. Macromolecular contributions were also included. Modulations of signal-to-noise ratio (SNR); lineshape type and width; concentrations of γ-aminobutyric acid, glutathione, and macromolecules; and inclusion of artifacts and lipid signals to mimic tumor spectra were included as challenges to be coped with. Twenty-six submissions were evaluated. Visually, most fit packages performed well with mostly noise-like residuals. However, striking differences in fit performance were found with bias problems also evident for well-known packages. In addition, often error bounds were not appropriately estimated and deduced confidence limits misleading. Soft constraints as used in LCModel were found to substantially influence the fitting results and their dependence on SNR. Substantial differences were found for accuracy and precision of fit results obtained by the multiple fit packages.