Reduction of multiple reaction monitoring protein target list using correlation analysisa.

High mass resolution mass spectrometry provides hundreds to thousands of protein identifications per sample, and quantification is typically performed using label-free quantification. However, the gold standard of quantitative proteomics is multiple reaction monitoring (MRM) using triple quadrupole mass spectrometers and stable isotope reference peptides. This raises the question how to reduce a large data set to a small one without losing essential information. Here we present the reduction of such a data set using correlation analysis of bovine dairy ingredients and derived products. We were able to explain the variance in the proteomics data set using only 9 proteins across all major dairy protein classes: caseins, whey, and milk fat globule membrane proteins. We term this method Trinity-MRM. The reproducibility of the protein extraction and Trinity-MRM methods was shown to be below 5% in independent experiments (multi-day single-user and single-day multi-user) using double cream. Further application of this reductionist approach might include screening of large sample cohorts for biologically interesting samples before analysis by high-resolution mass spectrometry or other omics methodologies.

Determination of Total Amino Acids in Infant Formulas, Adult Nutritionals, Dairy, and Cereal Matrixes by UHPLC-UV: Interlaboratory Validation Study, Final Action 2018.06.

BACKGROUND: A method for the quantification of total amino acids (including taurine and excluding tryptophan) using ultra- HPLC separation coupled to UV detection (UHPLC-UV) was granted First Action status (AOAC 2018.06) by the AOAC INTERNATIONAL Stakeholder Program for Infant Formula and Adult Nutritionals (SPIFAN) in 2018. OBJECTIVE: An interlaboratory study was conducted to further assess method performance against the AOAC Standard Method Performance Requirements (AOAC SMPR® 2014.013). Dairy and cereal matrixes were added to expand the scope of the method in collaboration with IDF (International Dairy Federation), ISO (International Organization for Standardization), and AACCI (American Association of Cereal Chemists International, now Cereals & Grains Association). METHODS: Sixteen different matrixes were chosen to cover the requirements of AOAC, IDF/ISO, and AACCI. Blind duplicate samples were organized into specific series to ensure that each pair was analyzed on the same day. Fifteen laboratories returned results. Data from four laboratories were considered invalid and removed from the dataset. Remaining data were assessed according to the Appendix D of the AOAC Official Methods of AnalysisSM (guidelines for collaborative study procedures). RESULTS: This method generally met the requirements listed in the SMPR for infant formulas and adult nutritionals, except for taurine. Method performance was comparable in dairy and cereal matrixes. Five different UHPLC instruments were used with either commercial or in-house reagents, demonstrating that the method is not limited to a single supplier. CONCLUSION: This method was recommended for Final Action in infant and adult/pediatric nutritional formulas by the AOAC SPIFAN Nutrients Expert Review Panel in April 2021, with the exception of taurine. The corresponding IDF/ISO Draft International Standard (DIS) was approved by national bodies in May 2022, and comments collected during the ballot were incorporated into this manuscript. HIGHLIGHTS: AOAC Official Method 2018.06 for the determination of total amino acids in infant formulas, adult nutritionals, dairy, and cereal matrixes was successfully validated in an interlaboratory study.

Total Amino Acids by UHPLC-UV in Infant Formulas and Adult Nutritionals, First Action 2018.06.

Background: An acid hydrolysis ultrahigh-performance LC-UV method was evaluated for the determination of total amino acids in infant formula and adult/pediatric nutritional formula. Objective: It was assessed for compliance against AOAC INTERNATIONAL Standard Method Performance Requirements (SMPR®) established by the Stakeholder Panel for Infant Formula and Adult Nutritionals (SPIFAN). Methods: A single-laboratory validation (SLV) study was conducted as a first step in the process to validate the method. In this SLV, 17 SPIFAN matrices representing a range of infant formula and adult nutritional products were evaluated for their amino acid content. Results: The analytical range was found to be within the needs for all products; some may require a dilution. Evaluation of trueness performed on Standard Reference Material 1849a (Infant/Adult Nutritional Formula) showed all compounds met the SMPR theoretical value, with exceptions for threonine and tyrosine. These may have a bias for the National Institute of Standards and Technology (NIST) data, depending on hydrolysis used in the determination of the NIST certificate of analysis. Conclusions: Based on the results of this SLV, this method met the SMPR and was approved as a First Action method by the AOAC Expert Review Panel on Nutrient Methods on August 28, 2018.

Quantification of Whey Protein Content in Milk-Based Infant Formula Powders by Sodium Dodecyl Sulfate-Capillary Gel Electrophoresis (SDS-CGE): Multilaboratory Testing Study, Final Action 2016.15.

A multilaboratory testing study was conducted on AOAC First Action Official Method SM 2016.15: Quantification of Whey Protein Content in Infant Formula Powders by Sodium Dodecyl Sulfate-Capillary Gel Electrophoresis (SDS-CGE). Nineteen laboratories participated in the analysis of duplicate blind-coded samples of 15 formula powder products for infants and young children. Electrophoregrams were recorded at UV220 nm and integrated. The normalized peak areas of whey and casein proteins were summed separately to calculate total whey protein content. Apart from one sample [NIST Standard Reference Material (SRM) 1849a], relative standard deviation of repeatability (RSDr) and reproducibility (RSDR) ranged from 0.83 to 2.11% and from 2.18 to 4.22%, respectively, and Horwitz ratios ranged from 1.02 to 1.85, meeting the precision limits specified in the whey protein Standard Method Performance Requirements and in the guidelines recommended for the Horwitz ratio. In these samples, the measured whey protein content was between 98 and 108% of the declared value. NIST SRM 1849a showed atypical results, with elevated RSDr (3.51%), RSDR (5.94%), Horwitz ratio (2.62), and recovery (134%). There is no clear reason for this. The percent whey protein value for NIST is calculated from the formulation and is not a reference or certified value. Multiple instrument models and makes, as well as capillary sources, were used in this collaborative study, demonstrating the robustness of the method. The method is fit-for-purpose for the quantification of whey protein content in milk-based formula powder products for infants and young children. It is not applicable to the analysis of hydrolyzed or plant protein-based infant formulas.

Quantification of Whey Protein Content in Infant Formulas by Sodium Dodecyl Sulfate-Capillary Gel Electrophoresis (SDS-CGE): Single-Laboratory Validation, First Action 2016.15.

Protein separation by sodium dodecyl sulfate-capillary gel electrophoresis, followed by UV absorption at 220 nm, allows for the quantification of major proteins in raw milk. In processed dairy samples such as skim milk powder (SMP) and infant formulas, signals from individual proteins are less resolved, but caseins still migrate as one family between two groups of whey proteins. In the first group, α-lactalbumin and ß-lactoglobulin migrate as two distinct peaks. Lactosylated adducts show delayed migration times and interfere with peak separation, but both native and modified forms as well as other low-MW whey proteins still elute before the caseins. The second group contains high-MW whey proteins (including bovine serum albumin, lactoferrin, and immunoglobulins) and elutes after the caseins. Caseins and whey proteins can thus be considered two distinct nonoverlapping families whose ratio can be established based on integrated areas without the need for a calibration curve. Because mass-to-area response factors for whey proteins and caseins are different, an area correction factor was determined from experimental measurement using SMP. Method performance assessed on five infant formulas showed RSDs of 0.2-1.2% (within day) and 0.5-1.1% (multiple days), with average recoveries between 97.4 and 106.4% of added whey protein. Forty-three different infant formulas and milk powders were analyzed. Of the 41 samples with manufacturer claims, the measured whey protein content was in close agreement with declared values, falling within 5% of the declared value in 76% of samples and within 10% in 95% of samples.

Amyloid-beta oligomerization is associated with the generation of a typical peptide fragment fingerprint.

Amyloid-beta (Aß) peptide oligomerization plays a central role in the pathogenesis of Alzheimer's disease (AD), and Aß oligomers are collectively considered an appealing therapeutic target for the treatment of AD. However, the molecular mechanisms leading to the pathologic accumulation of oligomers are unclear, and the exact structural composition of oligomers is being debated. Using targeted and quantitative mass spectrometry, we reveal site-specific Aß autocleavage during the early phase of aggregation, producing a typical Aß fragment signature and that truncated Aß peptides can form stable oligomeric complexes with full-length Aß peptide. We show that the use of novel anti-Aß antibodies raised against these truncated Aß isoforms allows for monitoring and targeting the accumulation of truncated Aß fragments. Antibody-enabled screening of transgenic models of AD as well as human postmortem brain tissue and cerebrospinal fluid revealed that aggregation-associated Aß cleavage is a highly relevant clinical feature of AD.

Nodal·Gdf1 heterodimers with bound prodomains enable serum-independent nodal signaling and endoderm differentiation.

The TGFß family member Nodal is central to control pluripotent stem cell fate, but its use as a stem cell differentiation factor is limited by low specific activity. During development, Nodal depends on growth and differentiation factor (Gdf)-1 and on the shared co-receptor Cryptic to specify visceral left-right axis asymmetry. We therefore asked whether the functionality of Nodal can be augmented by Gdf1. Because Nodal and Gdf1 coimmunoprecipitate each other, they were predicted to form heterodimers, possibly to facilitate diffusion or to increase the affinity for signaling receptors. Here, we report that Gdf1 suppresses an unexpected dependence of Nodal on serum proteins and that it is critically required for non-autonomous signaling in cells expressing Cryptic. Nodal, Gdf1, and their cleaved propeptides copurified as a heterodimeric low molecular weight complex that stimulated Activin receptor (Acvr) signaling far more potently than Nodal alone. Although heterodimerization with Gdf1 did not increase binding of Nodal to Fc fusions of co-receptors or Acvr extracellular domains, it was essential for soluble Acvr2 to inhibit Nodal signaling. This implies that Gdf1 potentiates Nodal activity by stabilizing a low molecular weight fraction that is susceptible to neutralization by soluble Acvr2. Finally, in differentiating human ES cells, endodermal markers were more efficiently induced by Nodal·Gdf1 than by Nodal, suggesting that Nodal·Gdf1 is an attractive new reagent to direct stem cell differentiation.

Wnt5a can both activate and repress Wnt/ß-catenin signaling during mouse embryonic development.

Embryonic development is controlled by a small set of signal transduction pathways, with vastly different phenotypic outcomes depending on the time and place of their recruitment. How the same molecular machinery can elicit such specific and distinct responses, remains one of the outstanding questions in developmental biology. Part of the answer may lie in the high inherent genetic complexity of these signaling cascades, as observed for the Wnt-pathway. The mammalian genome encodes multiple Wnt proteins and receptors, each of which show dynamic and tightly controlled expression patterns in the embryo. Yet how these components interact in the context of the whole organism remains unknown. Here we report the generation of a novel, inducible transgenic mouse model that allows spatiotemporal control over the expression of Wnt5a, a protein implicated in many developmental processes and multiple Wnt-signaling responses. We show that ectopic Wnt5a expression from E10.5 onwards results in a variety of developmental defects, including loss of hair follicles and reduced bone formation in the skull. Moreover, we find that Wnt5a can have dual signaling activities during mouse embryonic development. Specifically, Wnt5a is capable of both inducing and repressing ß-catenin/TCF signaling in vivo, depending on the time and site of expression and the receptors expressed by receiving cells. These experiments show for the first time that a single mammalian Wnt protein can have multiple signaling activities in vivo, thereby furthering our understanding of how signaling specificity is achieved in a complex developmental context.

Secreted Wingless-interacting molecule (Swim) promotes long-range signaling by maintaining Wingless solubility.

Lipid-modified Wnt/Wingless (Wg) proteins can signal to their target cells in a short- or long-range manner. How these hydrophobic proteins travel through the extracellular environment remains an outstanding question. Here, we report on a Wg binding protein, Secreted Wg-interacting molecule (Swim), that facilitates Wg diffusion through the extracellular matrix. Swim, a putative member of the Lipocalin family of extracellular transport proteins, binds to Wg with nanomolar affinity in a lipid-dependent manner. In quantitative signaling assays, Swim is sufficient to maintain the solubility and activity of purified Wg. In Drosophila, swim RNAi phenotypes resemble wg loss-of-function phenotypes in long-range signaling. We propose that Swim is a cofactor that promotes long-range Wg signaling in vivo by maintaining the solubility of Wg.

The microenvironment patterns the pluripotent mouse epiblast through paracrine Furin and Pace4 proteolytic activities.

The fate of pluripotent cells in early mouse embryos is controlled by graded Nodal signals that are activated by the endoproteases Furin and Pace4. Soluble forms of Furin and Pace4 cleave proNodal in vitro and after secretion in transfected cells, but direct evidence for paracrine activity in vivo is elusive. Here, we show that Furin and Pace4 are released by the extraembryonic microenvironment, and that they cleave a membrane-bound reporter substrate in adjacent epiblast cells and activate Nodal to maintain pluripotency. Secreted Pace4 and Furin also stimulated mesoderm formation, whereas endoderm was only induced by Pace4, correlating with a difference in the spatiotemporal distribution of these proteolytic activities. Our analysis of paracrine Furin and Pace4 activities and their in vivo functions significantly advances our understanding of how the epiblast is patterned by its microenvironment. Adding cell-cell communication to the pleiotropic portfolio of these proteases provides a new framework to study proprotein processing also in other relevant contexts.

Wnt proteins promote bone regeneration.

The Wnt signaling pathway plays a central role in bone development and homeostasis. In most cases, Wnt ligands promote bone growth, which has led to speculation that Wnt factors could be used to stimulate bone healing. We gained insights into the mechanism by which Wnt signaling regulates adult bone repair through the use of the mouse strain Axin2(LacZ/LacZ) in which the cellular response to Wnt is increased. We found that bone healing after injury is accelerated in Axin2(LacZ/LacZ) mice, a consequence of more robust proliferation and earlier differentiation of skeletal stem and progenitor cells. In parallel, we devised a biochemical strategy to increase the duration and strength of Wnt signaling at the sites of skeletal injury. Purified Wnt3a was packaged in liposomal vesicles and delivered to skeletal defects, where it stimulated the proliferation of skeletal progenitor cells and accelerated their differentiation into osteoblasts, cells responsible for bone growth. The end result was faster bone regeneration. Because Wnt signaling is conserved in mammalian tissue repair, this protein-based approach may have widespread applications in regenerative medicine.

Lentiviral vectors to probe and manipulate the Wnt signaling pathway.

BACKGROUND: The Wnt signaling pathway plays key roles in development, adult tissue homeostasis and stem cell maintenance. Further understanding of the function of Wnt signaling in specific cell types could benefit from lentiviral vectors expressing reporters for the Wnt pathway or vectors interfering with signaling. METHODOLOGY/PRINCIPAL FINDINGS: We have developed a set of fluorescent and luminescent lentiviral vectors that report Wnt signaling activity and discriminate between negative and uninfected cells. These vectors possess a 7xTcf-eGFP or 7xTcf-FFluc (Firefly Luciferase) reporter cassette followed by either an SV40-mCherry or SV40-Puro(R) (puromycin N-acetyltransferase) selection cassette. We have also constructed a vector that allows drug-based selection of cells with activated Wnt signaling by placing Puro(R) under the control of the 7xTcf promoter. Lastly, we have expressed dominant-negative Tcf4 (dnTcf4) or constitutively active beta-catenin (beta-catenin(4A)) from the hEF1alpha promoter in a SV40-Puro(R) or SV40-mCherry backbone to create vectors that inhibit or activate the Wnt signaling pathway. These vectors will be made available to the scientific community through Addgene. CONCLUSIONS: These novel lentiviruses are efficient tools to probe and manipulate Wnt signaling. The use of a selection cassette in Wnt-reporter viruses enables discriminating between uninfected and non-responsive cells, an important requirement for experiments where selection of clones is not possible. The use of a chemiluminescent readout enables quantification of signaling. Finally, selectable vectors can be used to either inhibit or activate the Wnt signaling pathway. Altogether, these vectors can probe and modulate the Wnt signaling pathway in experimental settings where persistence of the transgene or gene transfer cannot be accomplished by non-viral techniques.

A study on the interactions between heparan sulfate proteoglycans and Wnt proteins.

The Wnt signaling pathway plays key roles in development and adult homeostasis. Wnt proteins are secreted, lipid-modified glycoproteins. They can form morphogen gradients that are regulated at the level of protein secretion, diffusion, and internalization. These gradients can only exist if the hydrophobic Wnt proteins are prevented from aggregating in the extracellular environment. Heparan sulfate proteoglycans (HSPGs) are necessary for proper activity of Wnt proteins and influence their distribution along the morphogenetic gradient. In this study, we show that HSPGs are able to maintain the solubility of Wnt proteins, thus stabilizing their signaling activity. Our results suggest that the role of HSPGs is not only to concentrate Wnt molecules at the cell surface but also to prevent them from aggregating in the extracellular environment.

Wnt signaling mediates self-organization and axis formation in embryoid bodies.

Embryonic stem cells (ESCs) form descendants of all three germ layers when differentiated as aggregates, termed embryoid bodies. In vivo, differentiation of cells depends on signals and morphogen gradients that provide instructive and positional cues, but do such gradients exist in embryoid bodies? We report here the establishment of anteroposterior polarity and the formation of a primitive streak-like region in the embryoid body, dependent on local activation of the Wnt pathway. In this region, cells undergo an epithelial-to-mesenchymal transition and differentiate into mesendodermal progenitors. Exogenous Wnt3a protein posteriorizes the embryoid body, resulting in predominantly mesendodermal differentiation. Conversely, inhibiting Wnt signaling promotes anterior character and results in neurectodermal differentiation. The activation of Wnt signaling and primitive streak formation requires external signals but is self-reinforcing after initiation. Our findings show that the Wnt pathway mediates the local execution of a gastrulation-like process in the embryoid body, which displays an unexpected degree of self-organization.

Wnt/beta-catenin signaling in murine hepatic transit amplifying progenitor cells.

BACKGROUND & AIMS: Oval cells are postnatal hepatic progenitors with high proliferative potential and bipotent differentiation ability to become hepatocytes and cholangiocytes. Because Wnt/beta-catenin signaling is a known regulatory pathway for liver development and regeneration, we studied the role of Wnt signaling in oval cells using a mouse model of chronic liver injury. METHODS: A 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-enriched diet was used to stimulate oval cell proliferation. Livers were harvested for histologic analysis and determination of Wnt family gene expression by quantitative reverse transcription-polymerase chain reaction and in situ hybridization. The transgenic beta-catenin reporter mouse (TOPGAL) was use to confirm canonical Wnt/beta-catenin signal transduction in proliferating oval cells within atypical ductal proliferations (ADPs). Confocal fluorescence microscopy and immunohistochemistry was used to confirm colocalization of beta-catenin with the oval cell antigen A-6. RESULTS: Several Wnt ligands were significantly induced in the liver of DDC-fed mice and localized to proliferating cells in and adjacent to the ADPs. Oval cells isolated from DDC-fed mouse livers showed the presence of active beta-catenin in the nucleus along with cell-cycle entry in response to purified Wnt3a in vitro. Moreover, Wnt3a-induced beta-catenin/T-cell factor/lymphoid enhancer factor (TCF/LEF) transcriptional activation was quantified by TCF/LEF luciferase reporter assays. CONCLUSIONS: From these data, we conclude that oval cells respond to Wnt ligands (Wnt3a) in vitro with an increase in amino-terminus dephosphorylated beta-catenin and cell-cycle entry and that canonical Wnt/beta-catenin/TCF signaling is active in proliferating facultative hepatic progenitor cells in vivo. These findings may lend insight to the consequences of increased canonical Wnt signaling during periods of chronic liver injury.

Wnt signaling mediates regional specification in the vertebrate face.

At early stages of development, the faces of vertebrate embryos look remarkably similar, yet within a very short timeframe they adopt species-specific facial characteristics. What are the mechanisms underlying this regional specification of the vertebrate face? Using transgenic Wnt reporter embryos we found a highly conserved pattern of Wnt responsiveness in the developing mouse face that later corresponded to derivatives of the frontonasal and maxillary prominences. We explored the consequences of disrupting Wnt signaling, first using a genetic approach. Mice carrying compound null mutations in the nuclear mediators Lef1 and Tcf4 exhibited radically altered facial features that culminated in a hyperteloric appearance and a foreshortened midface. We also used a biochemical approach to perturb Wnt signaling and found that in utero delivery of a Wnt antagonist, Dkk1, produced similar midfacial malformations. We tested the hypothesis that Wnt signaling is an evolutionarily conserved mechanism controlling facial morphogenesis by determining the pattern of Wnt responsiveness in avian faces, and then by evaluating the consequences of Wnt inhibition in the chick face. Collectively, these data elucidate a new role for Wnt signaling in regional specification of the vertebrate face, and suggest possible mechanisms whereby species-specific facial features are generated.

Fusion of the BCL9 HD2 domain to E1A increases the cytopathic effect of an oncolytic adenovirus that targets colon cancer cells.

BACKGROUND: The Wnt signaling pathway is activated by mutations in the APC and beta-catenin genes in many types of human cancer. beta-catenin is stabilized by these mutations and activates transcription in part by acting as a bridge between Tcf/LEF proteins and the HD2 domain of the BCL9 coactivator. We have previously described oncolytic adenoviruses with binding sites for Tcf/LEF transcription factors inserted into the early viral promoters. These viruses replicate selectively in cells with activation of the Wnt pathway. To increase the activity of these viruses we have fused the viral transactivator E1A to the BCL9 HD2 domain. METHODS: Luciferase assays, co-immunoprecipitation and Western blotting, immunofluorescent cell staining and cytopathic effect assays were used to characterize the E1A-HD2 fusion protein and virus in vitro. Growth curves of subcutaneous SW620 colon cancer xenografts were used to characterize the virus in vivo. RESULTS: The E1A-HD2 fusion protein binds to beta-catenin in vivo and activates a Tcf-regulated luciferase reporter better than wild-type E1A in cells with activated Wnt signaling. Expression of the E1A-HD2 protein promotes nuclear import of beta-catenin, mediated by the strong nuclear localization signal in E1A. Tcf-regulated viruses expressing the fusion protein show increased expression of viral proteins and a five-fold increase in cytopathic effect (CPE) in colorectal cancer cell lines. There was no change in viral protein expression or CPE in HeLa cells, indicating that E1A-HD2 viruses retain selectivity for cells with activation of the Wnt signaling pathway. Despite increasing the cytopathic effect of the virus in vitro, fusion of the HD2 domain to E1A did not increase the burst size of the virus in vitro or the anti-tumor effect of the virus in an SW620 xenograft model in vivo. CONCLUSION: Despite an increase in the nuclear pool of beta-catenin, the effects on viral activity in colon cancer cells were small, suggesting that factors acting downstream of beta-catenin are limiting for viral replication and toxicity in these cells. The approach of fusing E1A to a protein domain implicated in oncogenic signaling could be used to selectively increase the activity of oncolytic viruses targeting several other pathways defective in cancer.