Molecular Weight Cut-off Filters Research Articles

Naturally Occurring Bioactive Oligosaccharides in Goat’s Milk-Infant Formula (P11-078-19)

ObjectivesHuman milk is rich in oligosaccharides (OS) that impart significant health benefits to the infant. When breast feeding is not possible, infant formula (IF) is used as an alternative. However, little is known about the presence of OS in IF and their potential health effects. This study was aimed at quantifying naturally occurring OS in a commercial goat’s milk IF and examining their prebiotic and anti-infection properties. MethodsOS were isolated from IF using molecular weight cut-off filters and HILIC-LC/MS system. Prebiotic activity of OS was assessed by co-incubating Bifidobacterium (BB12 and BB526) and Lactobacillus (LA4461 and LC2607) strains in growth media with or without OS anaerobically at 37°C for 24 hr and measuring OD600. Anti-adhesion potential of OS was determined by co-incubating E. coli NCTC 10418 and S. typhimurium, pre-incubated with or without OS for 2 hr at 37°C, with Caco-2 cells for 90 min. After PBS washes and cell lysis, supernatants were plated onto agars for measuring bacterial counts and calculating the anti-adhesion %. ResultsFourteen OS were quantified in goat’s milk IF, of which 5 were structurally similar to those reported in human milk. At concentrations similar to those in reconstituted IF, OS fractions were significantly effective in enhancing the growth of bifidobacteria and lactobacilli strains and preventing the adhesion of E. coli NCTC10418 (52.4%) and S. typhimurium (35%) to the Caco-2 cells. ConclusionsGoat’s milk IF contains several naturally occurring human milk-like OS that appear to promote the growth of beneficial gut microbes and protect against gut pathogens. Funding SourcesNo conflicts of interest were involved. Nuchev Pty Ltd. provided goat’s milk IF. Research costs were covered by RMIT University.

In Vitro and In Silico Approaches to Generating and Identifying Angiotensin-Converting Enzyme I Inhibitory Peptides from Green Macroalga Ulva lactuca.

A protein extract was generated from the macroalga Ulva lactuca, which was subsequently hydrolysed using the food-grade enzyme papain and angiotensin-converting Enzyme I and renin inhibitory peptides identified using a combination of enrichment strategies employing molecular weight cutoff filtration and mass spectrometry analysis. The generated hydrolysates with the most promising in vitro activity were further purified using preparative RP-HPLC and characterised. The 1 kDa hydrolysate (1 kDa-UFH), purified and collected by preparative RP-HPLC at minutes 41‒44 (Fr41‒44), displayed statistically higher ACE-I inhibitory activities ranging from 96.91% to 98.06%. A total of 48 novel peptides were identified from these four fractions by LC-MS/MS. A simulated gastrointestinal digestion of the identified peptide sequences was carried out using in silico enzyme cleavage simulation tools, resulting in 86 peptide sequences that were further assessed for their potential activity, toxicity and allergenicity using multiple predictive approaches. All the peptides obtained in this study were predicted to be non-toxic. However, 28 out of the 86 novel peptides released after the in silico gastrointestinal digestion were identified as potential allergens. The potential allergenicity of these peptides should be further explored to comply with the current labelling regulations in formulated food products containing U. lactuca protein hydrolysates.

200 Developing exosomes as a mediator for CRISPR/Cas-9 delivery

CRISPR/Cas systems present a powerful gene-editing tool with the potential for widespread therapeutic use; however, current methods of in vivo delivery such as adeno-associated viruses (AAV) may stimulate an immune response, creating the need for an alternative for delivery of CRISPR/Cas9. Exosomes are small vesicles that are released by cells and serve as a delivery system for RNA, proteins, and various molecules to other cells. The focus of this project was to use exosomes as a delivery system for Cas9, exploiting their high uptake by target cells and their ability to avoid the immune system in vivo. Porcine fetal fibroblasts (PFF) were grown to 80% confluency; after 48h, exosomes were isolated and concentrated from conditioned media by filtration with a 0.22-μm filter followed by 100-kDa molecular weight cutoff filter. Transmission electron microscopy, Western blotting for presence of CD81, and an uptake assay for exosomes stained with the lipophilic dye DiI (Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA) were used to characterise isolated exosomes, and average particle size was evaluated by NanoSight (Salisbury, United Kingdom). After characterisation, exosomes were loaded with Cas9 (PNA Bio, Newbury Park, CA, USA) using sonication, incubation with saponin, or extrusion. For each method of loading, 1.0×1011 exosomes and 500ng of Cas9 were used. For sonication, exosomes and Cas9 were sonicated 4 times: 4s on/2s off, left on ice for 2min, and then repeated for 4 more cycles. Loaded exosomes were then incubated at 37°C for 20min. For incubation with saponin, 100μL of 0.6% saponin solution was made in PBS, mixed with exosomes and Cas9, and then incubated on a shaker at 800 rpm for 20min. For extrusion, exosomes and Cas9 were extruded (Avanti Polar Lipids, Alabaster, AL, USA) 10, 15, or 20 times through a 0.22-μm filter. To evaluate efficiency of Cas9 loading into exosomes, loaded exosome samples were split in half, with one-half receiving a proteinase K digest (100μg mL−1) to remove free Cas9 and the other receiving no treatment. Proteinase K-treated and untreated samples were then compared side by side on Western blot staining for Cas9. ImageJ software (National Institutes for Health, Bethesda, MD, USA) was used to quantify band intensity and loading efficiency. With optimal conditions, our preliminary results show loading efficiency for sonication and saponin to be 16.7 and 19.2%, respectively, whereas loading by extrusion was undetectable. For CRISPR/Cas targeting, transgenic PFF carrying one copy of H2B-GFP were used to test delivery of ribonucleotide protein complex (RNP). To verify efficiency of the guide (g)RNA targeting green fluorescent protein (GFP), cells were nucleofected with Cas9 and gRNA. The DNA was extracted, PCR amplified, and sequenced (Eton Bioscience, San Diego, CA, USA) and then evaluated for indels with TIDE, resulting in a 53.2% cleavage efficiency. Next, exosomes will be loaded with RNP to knockout GFP in H2B-GFP cells, and targeting efficiency will be evaluated by flow cytometry and TIDE. We hypothesise that based on loading efficiency and target cell uptake, exosomes will present a safe and efficient method for in vitro and in vivo delivery of Cas9. The financial support of the Comparative Medicine Institute is gratefully acknowledged.

Detection of Amyloid Beta (Aβ) Oligomeric Composition Using Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI MS).

The use of MALDI MS as a fast and direct method to detect the Aβ oligomers of different masses is examined in this paper. Experimental results suggest that Aβ oligomers are ionized and detected as singly charged ions, and thus, the resulting mass spectrum directly reports the oligomer size distribution. Validation experiments were performed to verify the MS data against artifacts. Mass spectra collected from modified Aβ peptides with different propensities for aggregation were compared. Generally, the relative intensities of multimers were higher from samples where oligomerization was expected to be more favorable, and vice versa. MALDI MS was also able to detect the differences in oligomeric composition before and after the incubation/oligomerization step. Such differences in sample composition were also independently confirmed with an in vitro Aβ toxicity study on primary rat cortical neurons. An additional validation was accomplished through removal of oligomers from the sample using molecular weight cutoff filters; the resulting MS data correctly reflected the removal at the expected cutoff points. The results collectively validated the ability of MALDI MS to assess the monomeric/multimeric composition of Aβ samples. Graphical Abstract ᅟ.

Peptidomic Analysis of Endometrial Tissue from Patients with Ovarian Endometriosis

Background/Aims: Ovarian endometriosis (OvE) is ovarian cyst that is lined with endometrial tissue. They are found in 17–44% of women with endometriosis. Their clinical manifestations include pelvic pain, dysmenorrhea, dyspareunia, and infertility. Although the incidence of OvE has increased yearly, the exact pathogenesis of OvE is still unclear. We used peptidomics, an emerging branch of proteomics, to identify differentially expressed peptides in order to determine the possible roles of these peptides in the pathogenesis of OvE. Methods: The ectopic and eutopic endometria of OvE were used to extract peptides with 10-kDa molecular weight cutoff filters, and the peptide precursor proteins were then identified with PEAKS software, followed by quantification with the TMT labeling method and subsequent analysis by liquid chromatography-tandem mass spectrometry. Gene ontology (GO) analysis, pathway analysis, SMART, and SABLE were used to study the possible functions of these peptide according to their precursor proteins’ function. The effects of peptides derived from VCAM-1 (PDFV) on endometrial stromal cell (ESC) migration and invasion were examined with wound healing assays and Transwell assays and the expression of E-cadherin was detected by western blotting. Results: A total of 491 peptides were identified with abundant differences between the two groups of samples (p < 0.05, and absolute fold change ≥ 2). SMART and SABLE database showed that 42 of the 491 peptides were located in the conserved structural domains of their protein precursors and contained secondary structure and, among them, 2 peptides’ precursor proteins were associated with the cell proliferation. Additionally, 5 peptides’ precursor proteins were associated with endometriosis. Our study confirmed that PDFV promoted ESC migration and invasion and reduced E-cadherin expression (p < 0.05). Conclusion: PDFV and its precursor protein VCAM-1 may be involved in the process of OvE formation by reducing the expression of E-cadherin. The peptidomics analysis provides new insight for future studies of the mechanisms of OvE development.

Construction of Viable Soil Defined Media Using Quantitative Metabolomics Analysis of Soil Metabolites.

Exometabolomics enables analysis of metabolite utilization of low molecular weight organic substances by soil bacteria. Environmentally-based defined media are needed to examine ecologically relevant patterns of substrate utilization. Here, we describe an approach for the construction of defined media using untargeted characterization of water soluble soil microbial metabolites from a saprolite soil collected from the Oak Ridge Field Research Center (ORFRC). To broadly characterize metabolites, both liquid chromatography mass spectrometry (LC/MS) and gas chromatography mass spectrometry (GC/MS) were used. With this approach, 96 metabolites were identified, including amino acids, amino acid derivatives, sugars, sugar alcohols, mono- and di-carboxylic acids, nucleobases, and nucleosides. From this pool of metabolites, 25 were quantified. Molecular weight cut-off filtration determined the fraction of carbon accounted for by the quantified metabolites and revealed that these soil metabolites have an uneven quantitative distribution (e.g., trehalose accounted for 9.9% of the <1 kDa fraction). This quantitative information was used to formulate two soil defined media (SDM), one containing 23 metabolites (SDM1) and one containing 46 (SDM2). To evaluate the viability of the SDM, we examined the growth of 30 phylogenetically diverse soil bacterial isolates from the ORFRC field site. The simpler SDM1 supported the growth of 13 isolates while the more complex SDM2 supported 15 isolates. To investigate SDM1 substrate preferences, one isolate, Pseudomonas corrugata strain FW300-N2E2 was selected for a time-series exometabolomics analysis. Interestingly, it was found that this organism preferred lower-abundance substrates such as guanine, glycine, proline and arginine and glucose and did not utilize the more abundant substrates maltose, mannitol, trehalose and uridine. These results demonstrate the viability and utility of using exometabolomics to construct a tractable environmentally relevant media. We anticipate that this approach can be expanded to other environments to enhance isolation and characterization of diverse microbial communities.

Isotype-specific agglutination-PCR (ISAP): A sensitive and multiplex method for measuring allergen-specific IgE

Isotype-specific agglutination-PCR (ISAP): A sensitive and multiplex method for measuring allergen-specific IgE

Sample Preparation for Endopeptidomic Analysis in Human Cerebrospinal Fluid.

This protocol describes a method developed to identify endogenous peptides in human cerebrospinal fluid (CSF). For this purpose, a previously developed method based on molecular weight cut-off (MWCO) filtration and mass spectrometric analysis was combined with an offline high-pH reverse phase HPLC pre-fractionation step. Secretion into CSF is the main pathway for removal of molecules shed by cells of the central nervous system. Thus, many processes in the central nervous system are reflected in the CSF, rendering it a valuable diagnostic fluid. CSF has a complex composition, containing proteins that span a concentration range of 8 - 9 orders of magnitude. Besides proteins, previous studies have also demonstrated the presence of a large number of endogenous peptides. While less extensively studied than proteins, these may also hold potential interest as biomarkers. Endogenous peptides were separated from the CSF protein content through MWCO filtration. By removing a majority of the protein content from the sample, it is possible to increase the sample volume studied and thereby also the total amount of the endogenous peptides. The complexity of the filtrated peptide mixture was addressed by including a reverse phase (RP) HPLC pre-fractionation step at alkaline pH prior to LC-MS analysis. The fractionation was combined with a simple concatenation scheme where 60 fractions were pooled into 12, analysis time consumption could thereby be reduced while still largely avoiding co-elution. Automated peptide identification was performed by using three different peptide/protein identification software programs and subsequently combining the results. The different programs were complementary rather than comparable with less than 15% of the identifications overlapped between the three.

Analysis of procainamide-derivatised heparan sulphate disaccharides in biological samples using hydrophilic interaction liquid chromatography mass spectrometry

Glycosaminoglycans (GAGs) are a family of linear heteropolysaccharides made up of repeating disaccharide units that are found on the surface and extracellular matrix of animal cells. They are known to play a critical role in a wide range of cellular processes including proliferation, differentiation and invasion. To elucidate the mechanism of action of these molecules, it is essential to quantify their disaccharide composition. Analytical methods that have been reported involve either chemical or enzymatic depolymerisation of GAGs followed by separation of non-derivatised (native) or derivatised disaccharide subunits and detection by either UV/fluorescence or MS. However, the measurement of these disaccharides is challenging due to their hydrophilic and labile nature. Here we report a pre-column LC-MS method for the quantification of GAG disaccharide subunits. Heparan sulphate (HS) was extracted from cell lines using a combination of molecular weight cutoff and anion exchange spin filters and digested using a mixture of heparinases I, II and III. The resulting subunits were derivatised with procainamide, separated using hydrophilic interaction liquid chromatography and detected using electrospray ionisation operated in positive ion mode. Eight HS disaccharides were separated and detected together with an internal standard. The limit of detection was found to be in the range 0.6-4.9ng/mL. Analysis of HS extracted from all cell lines tested in this study revealed a significant variation in their composition with the most abundant disaccharide being the non-sulphated ∆UA-GlcNAc. Some structural functional relationships are discussed demonstrating the viability of the pre-column method for studying GAG biology. Graphical abstract Extraction and HILIC UPLC-MS analysis of procainamide-labelled heparan sulphate disaccharides.

Label-free quantitative comparison of cerebrospinal fluid glycoproteins and endogenous peptides in subjects with Alzheimer's disease, mild cognitive impairment, and healthy individuals.

The goal of this study is to investigate putative molecular dynamic changes in cerebrospinal fluids (CSFs) collected from individuals with mild cognitive impairment (MCI) and Alzheimer's disease (AD) as compared to healthy controls. The CSF samples from 12 subjects comprised of four cognitively normal individuals and eight patients with MCI and AD, respectively. Two aliquots of each CSF samples (total 1 mL) of each participant are used for this study. Endogenous peptide separations are performed using 10000 molecular weight cut-off filters followed by LC-MS/MS identification and quantitation while lectin-enrichment chromatography is used to enrich glycoproteins in CSF followed by trypsin digestion and subsequent LC-MS/MS for shotgun identification and label-free quantitation. Using an optimized submicrogram peptide separation with molecular weight cut-off filtration and an in house-constructed database, 645 peptides are identified. Glycoproteins are enriched by lectin affinity chromatography, resulting in 795 identified proteins. The discovery and alterations of proSAAS-derived peptides and transthyretin are described and their roles in AD are discussed. Comprehensive identification of endogenous CSF peptidome is achieved. Fifteen proteins are found to be differentially expressed among the three groups. The dynamic changes of transthyretin are reported for the first time.

Paraprotein-Related Kidney Disease: Evaluation and Treatment of Myeloma Cast Nephropathy.

Nearly 50% of patients with multiple myeloma develop renal disease, most commonly from AKI caused by cast nephropathy. Development of AKI is associated with poor 1-year survival and reduces the therapeutic options available to patients. There is a great need for more effective therapies. Cast nephropathy is caused by the interaction and aggregation of filtered free light chains and Tamm-Horsfall protein causing intratubular obstruction and damage. The key to treating cast nephropathy is rapid lowering of free light chains, because this correlates with renal recovery. Newer chemotherapy agents rapidly lower free light chains and have been referred to as renoprotective. There is additional great interest in using extracorporeal therapies to remove serum free light chains. Small trials initially showed benefit of therapeutic plasma exchange to improve renal outcomes in cast nephropathy, but a large randomized trial of therapeutic plasma exchange failed to show benefit. A newer technique is extended high-cutoff hemodialysis. This modality uses a high molecular weight cutoff filter to remove free light chains. To date, trials of high-cutoff hemodialysis use in patients with cast nephropathy have been encouraging. However, there are no randomized trials showing the benefit of high-cutoff hemodialysis when used in addition to newer chemotherapeutic regimens. Until these studies are available, high-cutoff hemodialysis cannot be recommended as standard of care.

Components of yeast (Sacchromyces cervisiae) extract as defined media additives that support the growth and productivity of CHO cells

Yeast and plant hydrolysates are used as media supplements to support the growth and productivity of CHO cultures for biopharmaceutical production. Through fractionation of a yeast lysate and metabolic analysis of a fraction that had bioactivity equivalent to commercial yeast extract (YE), bioactive components were identified that promoted growth and productivity of two recombinant CHO cell lines (CHO-Luc and CHO-hFcEG2) equivalent to or greater than YE-supplemented media. Autolysis of the yeast lysate was not necessary for full activity, suggesting that the active components are present in untreated yeast cells. A bioactive fraction (3KF) of the yeast lysate was isolated from the permeate using a 3kDa molecular weight cut-off (MWCO) filter. Supplementation of this 3KF fraction into the base media supported growth of CHO-Luc cells over eight passages equivalent to YE-supplemented media. The 3KF fraction was fractionated further by a cation exchange spin column using a stepwise pH elution. Metabolomic analysis of a bioactive fraction isolated at high pH identified several arginine and lysine-containing peptides as well as two polyamines, spermine and spermidine, with 3.5× and 4.5× higher levels compared to a fraction showing no bioactivity. The addition of a mixture of polyamines and their precursors (putrescine, spermine, spermidine, ornithine and citrulline) as well as increasing the concentration of some of the components of the original base medium resulted in a chemically-defined (CD) formulation that produced an equivalent viable cell density (VCD) and productivity of the CHO-Luc cells as the YE-supplemented medium. The VCD of the CHO-hFcEG2 culture in the CD medium was 1.9× greater and with equivalent productivity to the YE-supplemented media.

Peptidome profiling of umbilical cord plasma associated with gestational diabetes-induced fetal macrosomia

Fetal macrosomia, defined as a birth weight ≥4000g, may affect 15–45% of newborns of women with gestational diabetes mellitus (GDM). The associations between endogenous peptides and gestational diabetes-induced macrosomia have not been investigated extensively by peptidome analysis. Here, we analyzed the umbilical cord plasma by combining ultrafiltration using molecular weight cut-off filters and liquid chromatography–tandem mass spectrometry (LC–MS/MS) to investigate potential associations of GDM with macrosomia. As macrosomic babies have increased susceptibility to obesity, diabetes and cardiovascular diseases in later life, we also aimed to identify specific biomarkers to detect these future diseases. Thirty pairs of GDM mothers and controls were randomly divided into three subgroups. We identified 235 peptides of around 1000–3000Da, originating from 115 proteins. Analyzing the cleavage sites revealed that these peptides were cleaved in regulation, which may reflect the protease activity and distribution in umbilical cord plasma. Four identified peptides, of 2471.7, 1077.2, 1446.5 and 2372.7Da, were significantly differentially expressed in the GDM macrosomia groups compared with controls, whose precursors may play a critical role in developing GDM macrosomia. We provide for the first time a validated GDM macrosomia peptidome profile and identify potential biomarkers linking the effects of macrosomia to later-life diseases. Biological significanceFetal macrosomia is the predominant adverse outcome of gestational diabetes mellitus (GDM), which is a frequent medical condition during pregnancy. Till now, the detailed molecular mechanisms underlying gestational diabetes-induced macrosomia are still not elucidated. With high detection sensitivity and high throughput of peptidome technology, it is now possible to systemically identify peptides possibly involved in the umbilical cord plasma of GDM induced macrosomia cases. With LC–MS/MS based quantification, totally, we identified 235 peptides originated from 115 precursor proteins. And four peptides of 2471.7, 1077.2, 1446.5 and 2372.7Da differentially expressed between GDM cases and compared controls. A precursor protein of 1077.2Da was fibrinogen alpha chain (FGA), which was also identified in the Ai et al. [29] study with a downregulated manner in the serum samples of GDM cases. And further analysis the cleavage pattern of the identified peptides revealed that the enzymes in tissues cleaved the protein according to their rules. Thus, this quantitative peptidome approach can identify related peptides that may play a role in the gestational diabetes-induced macrosomia, and give candidate biomarkers contributing to the development of later-life diseases in macrosomic babies.

Amicon-adapted enhanced FASP: an in-solution digestion-based alternative sample preparation method to FASP

Sample preparation is a crucial step for liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics. Sodium dodecyl sulfate (SDS) is a powerful denaturing detergent that allows for long-term preservation of protein integrity. However, as it inhibits trypsin and interferes with LC-MS/MS analyses, it must be removed from samples prior to these experiments. The Filter-Aided Sample Preparation (FASP) method is actually one of the preferred and simplest methods for such purpose. Nonetheless, there exist great disparities in the quality of outcomes when comparing FASP to other protocols depending on the authors, and recent reports have pointed to concerns regarding its depth of proteome coverage. To address these issues, we propose an Amicon-adapted in-solution-based enhanced FASP (eFASP) approach that relies on current best practices in comprehensive proteomics sample preparation. Human megakaryoblastic leukaemia cancer cells’ protein extracts were treated in parallel with both Amicon-adapted eFASP and FASP, quantified for remaining SDS and then analyzed with a 1-hr gradient LC-MS/MS run. The Amicon-adapted eFASP utilizes a passivated low molecular weight cut-off Amicon filter, and incorporates a cleaning step with a high-content deoxycholate buffer and a ‘one-step-two-enzymes’ trypsin/Lys-C in-solution digestion. Amicon-adapted eFASP was found more reproducible and deepened proteome coverage, especially for membrane proteins. As compared to FASP, Amicon-adapted eFASP removed much of SDS from high-protein samples and reached a notable depth of proteome coverage with nearly 1,700 proteins identified in a 1 hr LC-MS/MS single-run analysis without prior fractionation. Amicon-adapted eFASP can therefore be regarded as a simple and reliable sample preparation approach for comprehensive proteomics.

Revalorization of a peach (Prunus persica (L.) Batsch) byproduct: Extraction and characterization of ACE-inhibitory peptides from peach stones

The value-added use of peach stone for production of a high protein product was investigated. Different parameters were optimized in order to obtain the highest protein yield in the extraction of peach seed proteins. Optimal conditions enabled the extraction of 43 g of protein per 100 g of dried and defatted milled seeds. Different enzymes and parameters were also tried in order to obtain the highest hydrolysis degree in the digestion of this peach seed protein isolate. Thermolysin was the enzyme that yielded the extract with the highest ACE-inhibition capability. After ultrafiltration through different molecular weight cut-off filters, potential antihypertensive peptides remained in the fraction below 3 kDa. Three different peptides (LYSPH, LYTPH, and HLLP) resisting simulated gastrointestinal digestion and ACE activity and demonstrating antihypertensive in vitro capacity were identified by HPLC-MS/MS. This is the first time that potential antihypertensive peptides have been isolated from peach stones.

Amicon-adapted enhanced FASP: an in-solution digestion-based alternative sample preparation method to FASP

Sample preparation is a crucial step for liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics. Sodium dodecyl sulfate (SDS) is a powerful denaturing detergent that allows for long-term preservation of protein integrity. However, as it inhibits trypsin and interferes with LC-MS/MS analyses, it must be removed from samples prior to these experiments. The Filter-Aided Sample Preparation (FASP) method is actually one of the preferred and simplest methods for such purpose. Nonetheless, there exist great disparities in the quality of outcomes when comparing FASP to other protocols depending on the authors, and recent reports have pointed to concerns regarding its depth of proteome coverage. To address these issues, we propose an Amicon-adapted in-solution-based enhanced FASP (eFASP) approach that relies on current best practices in comprehensive proteomics sample preparation. Human megakaryoblastic leukaemia cancer cells’ protein extracts were treated in parallel with both Amicon-adapted eFASP and FASP, quantified for remaining SDS and then analyzed with a 1-hr gradient LC-MS/MS run. The Amicon-adapted eFASP utilizes a passivated low molecular weight cut-off Amicon filter, and incorporates a cleaning step with a high-content deoxycholate buffer and a ‘one-step-two-enzymes’ trypsin/Lys-C in-solution digestion. Amicon-adapted eFASP was found more reproducible and deepened proteome coverage, especially for membrane proteins. As compared to FASP, Amicon-adapted eFASP removed much of SDS from high-protein samples and reached a notable depth of proteome coverage with nearly 1,700 proteins identified in a 1 hr LC-MS/MS single-run analysis without prior fractionation. Amicon-adapted eFASP can therefore be regarded as a simple and reliable sample preparation approach for comprehensive proteomics.

Leaching test of gamma-emitting Cs, Ru, Zr, and U from neutron-irradiated UO2/ZrO2 solid solutions in non-filtered surface seawater

The leaching behaviors of gamma-ray radionuclides, Cs-137, Ru-103, and Zr-95, produced by neutron irradiation of UO2/ZrO2 solid solutions, in real surface seawater were investigated under atmospheric conditions. The fraction of radionuclide inventory leached in the seawater was in the order of Cs > Ru (∼U) ≫ Zr, indicating that the fraction was significantly affected by the chemical state of the radionuclides. However, the amount of soluble nuclides was proportional to that of uranium regardless of whether the solid solutions were prepared under an oxidative or reductive environment. A tiny fraction of Ru was filtered out by a 3 kDa nominal molecular weight cut-off filter after the 160 d leaching test, suggesting a different behavior from its ionic form, but Cs and U did not form a colloid-like species in seawater.

Quantitating metabolites in protein precipitated serum using NMR spectroscopy.

Quantitative NMR-based metabolite profiling is challenged by the deleterious effects of abundant proteins in the intact blood plasma/serum, which underscores the need for alternative approaches. Protein removal by ultrafiltration using low molecular weight cutoff filters thus represents an important step. However, protein precipitation, an alternative and simple approach for protein removal, lacks detailed quantitative assessment for use in NMR based metabolomics. In this study, we have comprehensively evaluated the performance of protein precipitation using methanol, acetonitrile, perchloric acid, and trichloroacetic acid and ultrafiltration approaches using 1D and 2D NMR, based on the identification and absolute quantitation of 44 human blood metabolites, including a few identified for the first time in the NMR spectra of human serum. We also investigated the use of a “smart isotope tag,” 15N-cholamine for further resolution enhancement, which resulted in the detection of a number of additional metabolites. 1H NMR of both protein precipitated and ultrafiltered serum detected all 44 metabolites with comparable reproducibility (average CV, 3.7% for precipitation; 3.6% for filtration). However, nearly half of the quantified metabolites in ultrafiltered serum exhibited 10–74% lower concentrations; specifically, tryptophan, benzoate, and 2-oxoisocaproate showed much lower concentrations compared to protein precipitated serum. These results indicate that protein precipitation using methanol offers a reliable approach for routine NMR-based metabolomics of human blood serum/plasma and should be considered as an alternative to ultrafiltration. Importantly, protein precipitation, which is commonly used by mass spectrometry (MS), promises avenues for direct comparison and correlation of metabolite data obtained from the two analytical platforms to exploit their combined strength in the metabolomics of blood.

Treatment of acute kidney injury with cast nephropathy.

Nearly 50% of patients with multiple myeloma develop renal disease; acute kidney injury (AKI) from cast nephropathy, or "myeloma kidney" is the most common type. Development of AKI is associated with worse 1-year survival and reduces the therapeutic options available to patients. Therefore, there is a great need to develop more effective therapies. Cast nephropathy is due to the interaction and aggregation of filtered free light chains (FLCs) and Tamm- Horsfall protein (THP) causing intratubular obstruction and damage. The key to treating cast nephropathy is rapid lowering of FLCs as this correlates with renal recovery. Newer chemotherapy agents lower FLCs and have been referred to as "renoprotective". However there remains great interest in using various extracorporeal therapies to remove serum FLCs. Initially, therapeutic plasma exchange (TPE) was thought to improve renal outcomes in cast nephropathy based on small trials. The largest randomized trial of TPE, however, failed to show any benefit. A newer technique is extended high cut-off hemodialysis (HCO-HD). This modality uses a high molecular weight cut-off filter to remove FLCs. To date, trials with HCO-HD in patients with cast nephropathy have been encouraging. However, there are no randomized trials demonstrating the benefit of HCOHD when used in addition to newer chemotherapeutic regimens. Until these studies are available, HCO-HD cannot be recommended as standard of care.

Addendum: Digestion and depletion of abundant proteins improves proteomic coverage.

Addendum: Digestion and depletion of abundant proteins improves proteomic coverage.