Extracellular vesicles from mouse trophoblast cells: Effects on neural progenitor cells and potential participants in the placenta-brain axis†.

The fetal brain of the mouse is thought to be dependent upon the placenta as a source of serotonin (5-hydroxytryptamine; 5-HT) and other factors. How factors reach the developing brain remains uncertain but are postulated here to be part of the cargo carried by placental extracellular vesicles (EV). We have analyzed the protein, catecholamine, and small RNA content of EV from mouse trophoblast stem cells (TSC) and TSC differentiated into parietal trophoblast giant cells (pTGC), potential primary purveyors of 5-HT. Current studies examined how exposure of mouse neural progenitor cells (NPC) to EV from either TSC or pTGC affect their transcriptome profiles. The EV from trophoblast cells contained relatively high amounts of 5-HT, as well as dopamine and norepinephrine, but there were no significant differences between EV derived from pTGC and from TSC. Content of miRNA and small nucleolar (sno)RNA, however, did differ according to EV source, and snoRNA were upregulated in EV from pTGC. The primary inferred targets of the microRNA (miRNA) from both pTGC and TSC were mRNA enriched in the fetal brain. NPC readily internalized EV, leading to changes in their transcriptome profiles. Transcripts regulated were mainly ones enriched in neural tissues. The transcripts in EV-treated NPC that demonstrated a likely complementarity with miRNA in EV were mainly up- rather than downregulated, with functions linked to neuronal processes. Our results are consistent with placenta-derived EV providing direct support for fetal brain development and being an integral part of the placenta-brain axis.

Human Keratinocyte Responses to Woodsmoke Chemicals.

Air pollution consists of complex mixtures of chemicals with serious deleterious health effects from acute and chronic exposure. To help understand the mechanisms by which adverse effects occur, the present work examines the responses of cultured human epidermal keratinocytes to specific chemicals commonly found in woodsmoke. Our earlier findings with liquid smoke flavoring (aqueous extract of charred wood) revealed that such extracts stimulated the expression of genes associated with oxidative stress and proinflammatory response, activated the aryl hydrocarbon receptor, thereby inducing cytochrome P4501A1 activity, and induced cross-linked envelope formation, a lethal event ordinarily occurring during terminal differentiation. The present results showed that furfural produced transcriptional responses resembling those of liquid smoke, cyclohexanedione activated the aryl hydrocarbon receptor, and several chemicals induced envelope formation. Of these, syringol permeabilized the cells to the egress of lactate dehydrogenase at a concentration close to that yielding envelope formation, while furfural induced envelope formation without permeabilization detectable in this way. Furfural (but not syringol) stimulated the incorporation of amines into cell proteins in extracts in the absence of transglutaminase activity. Nevertheless, both chemicals substantially increased the amount of cellular protein incorporated into envelopes and greatly altered the envelope protein profile. Moreover, the proportion of keratin in the envelopes was dramatically increased. These findings are consistent with the chemically induced protein cross-linking in the cells. Elucidating mechanisms by which this phenomenon occurs may help understand how smoke chemicals interact with proteins to elicit cellular responses, interpret bioassays of complex pollutant mixtures, and suggest additional sensitive ways to monitor exposures.

Metabolic Enzyme Alterations and Astrocyte Dysfunction in a Murine Model of Alexander Disease With Severe Reactive Gliosis.

Alexander disease (AxD) is a rare and fatal neurodegenerative disorder caused by mutations in the gene encoding glial fibrillary acidic protein (GFAP). In this report, a mouse model of AxD (GFAPTg;Gfap+/R236H) was analyzed that contains a heterozygous R236H point mutation in murine Gfap as well as a transgene with a GFAP promoter to overexpress human GFAP. Using label-free quantitative proteomic comparisons of brain tissue from GFAPTg;Gfap+/R236H versus wild-type mice confirmed upregulation of the glutathione metabolism pathway and indicated proteins were elevated in the peroxisome proliferator-activated receptor (PPAR) signaling pathway, which had not been reported previously in AxD. Relative protein-level differences were confirmed by a targeted proteomics assay, including proteins related to astrocytes and oligodendrocytes. Of particular interest was the decreased level of the oligodendrocyte protein, 2-hydroxyacylsphingosine 1-beta-galactosyltransferase (Ugt8), since Ugt8-deficient mice exhibit a phenotype similar to GFAPTg;Gfap+/R236H mice (e.g., tremors, ataxia, hind-limb paralysis). In addition, decreased levels of myelin-associated proteins were found in the GFAPTg;Gfap+/R236H mice, consistent with the role of Ugt8 in myelin synthesis. Fabp7 upregulation in GFAPTg;Gfap+/R236H mice was also selected for further investigation due to its uncharacterized association to AxD, critical function in astrocyte proliferation, and functional ability to inhibit the anti-inflammatory PPAR signaling pathway in models of amyotrophic lateral sclerosis (ALS). Within Gfap+ astrocytes, Fabp7 was markedly increased in the hippocampus, a brain region subjected to extensive pathology and chronic reactive gliosis in GFAPTg;Gfap+/R236H mice. Last, to determine whether the findings in GFAPTg;Gfap+/R236H mice are present in the human condition, AxD patient and control samples were analyzed by Western blot, which indicated that Type I AxD patients have a significant fourfold upregulation of FABP7. However, immunohistochemistry analysis showed that UGT8 accumulates in AxD patient subpial brain regions where abundant amounts of Rosenthal fibers are located, which was not observed in the GFAPTg;Gfap+/R236H mice.

Mitochondrial and Proteasome Dysfunction Occurs in the Hearts of Mice Treated with Triazine Herbicide Prometryn.

Prometryn is a methylthio-s-triazine herbicide used to control the growth of annual broadleaf and grass weeds in many cultivated plants. Significant traces of prometryn are documented in the environment, mainly in waters, soil, and plants used for human and domestic consumption. Previous studies have shown that triazine herbicides have carcinogenic potential in humans. However, there is limited information about the effects of prometryn on the cardiac system in the literature, or the mechanisms and signaling pathways underlying any potential cytotoxic effects are not known. It is important to understand the possible effects of exogenous compounds such as prometryn on the heart. To determine the mechanisms and signaling pathways affected by prometryn (185 mg/kg every 48 h for seven days), we performed proteomic profiling of male mice heart with quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) using ten-plex tandem mass tag (TMT) labeling. The data suggest that several major pathways, including energy metabolism, protein degradation, fatty acid metabolism, calcium signaling, and antioxidant defense system were altered in the hearts of prometryn-treated mice. Proteasome and immunoproteasome activity assays and expression levels showed proteasome dysfunction in the hearts of prometryn-treated mice. The results suggest that prometryn induced changes in mitochondrial function and various signaling pathways within the heart, particularly affecting stress-related responses.

Blood Proteome Profiling Reveals Biomarkers and Pathway Alterations in Fragile X PM at Risk for Developing FXTAS.

Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) is a neurodegenerative disorder associated with the FMR1 premutation. Currently, it is not possible to determine when and if individual premutation carriers will develop FXTAS. Thus, with the aim to identify biomarkers for early diagnosis, development, and progression of FXTAS, along with associated dysregulated pathways, we performed blood proteomic profiling of premutation carriers (PM) who, as part of an ongoing longitudinal study, emerged into two distinct groups: those who developed symptoms of FXTAS (converters, CON) over time (at subsequent visits) and those who did not (non-converters, NCON). We compared these groups to age-matched healthy controls (HC). We assessed CGG repeat allele size by Southern blot and PCR analysis. The proteomic profile was obtained by liquid chromatography mass spectrometry (LC-MS/MS). We identified several significantly differentiated proteins between HC and the PM groups at Visit 1 (V1), Visit 2 (V2), and between the visits. We further reported the dysregulated protein pathways, including sphingolipid and amino acid metabolism. Our findings are in agreement with previous studies showing that pathways involved in mitochondrial bioenergetics, as observed in other neurodegenerative disorders, are significantly altered and appear to contribute to the development of FXTAS. Lastly, we compared the blood proteome of the PM who developed FXTAS over time with the CSF proteome of the FXTAS patients recently reported and found eight significantly differentially expressed proteins in common. To our knowledge, this is the first report of longitudinal proteomic profiling and the identification of unique biomarkers and dysregulated protein pathways in FXTAS.

AtTRAPPC11/ROG2: A Role for TRAPPs in Maintenance of the Plant Trans-Golgi Network/Early Endosome Organization and Function.

The dynamic trans-Golgi network/early endosome (TGN/EE) facilitates cargo sorting and trafficking and plays a vital role in plant development and environmental response. Transport protein particles (TRAPPs) are multi-protein complexes acting as guanine nucleotide exchange factors and possibly as tethers, regulating intracellular trafficking. TRAPPs are essential in all eukaryotic cells and are implicated in a number of human diseases. It has been proposed that they also play crucial roles in plants; however, our current knowledge about the structure and function of plant TRAPPs is very limited. Here, we identified and characterized AtTRAPPC11/RESPONSE TO OLIGOGALACTURONIDE2 (AtTRAPPC11/ROG2), a TGN/EE-associated, evolutionarily conserved TRAPP protein in Arabidopsis (Arabidopsis thaliana). AtTRAPPC11/ROG2 regulates TGN integrity, as evidenced by altered TGN/EE association of several residents, including SYNTAXIN OF PLANTS61, and altered vesicle morphology in attrappc11/rog2 mutants. Furthermore, endocytic traffic and brefeldin A body formation are perturbed in attrappc11/rog2, suggesting a role for AtTRAPPC11/ROG2 in regulation of endosomal function. Proteomic analysis showed that AtTRAPPC11/ROG2 defines a hitherto uncharacterized TRAPPIII complex in plants. In addition, attrappc11/rog2 mutants are hypersensitive to salinity, indicating an undescribed role of TRAPPs in stress responses. Overall, our study illustrates the plasticity of the endomembrane system through TRAPP protein functions and opens new avenues to explore this dynamic network.

De Novo Arginine Synthesis Is Required for Full Virulence of Xanthomonas arboricola pv. juglandis During Walnut Bacterial Blight Disease.

Walnut blight (WB) disease caused by Xanthomonas arboricola pv. juglandis (Xaj) threatens orchards worldwide. Nitrogen metabolism in this bacterial pathogen is dependent on arginine, a nitrogen-enriched amino acid that can either be synthesized or provided by the plant host. The arginine biosynthetic pathway uses argininosuccinate synthase (argG), associated with increased bacterial virulence. We examined the effects of bacterial arginine and nitrogen metabolism on the plant response during WB by proteomic analysis of the mutant strain Xaj argG-. Phenotypically, the mutant strain produced 42% fewer symptoms and survived in the plant tissue with 2.5-fold reduced growth compared with wild type, while showing itself to be auxotrophic for arginine in vitro. Proteomic analysis of infected tissue enabled the profiling of 676 Xaj proteins and 3,296 walnut proteins using isobaric labeling in a data-dependent acquisition approach. Comparative analysis of differentially expressed proteins revealed distinct plant responses. Xaj wild type (WT) triggered processes of catabolism and oxidative stress in the host under observed disease symptoms, while most of the host biosynthetic processes triggered by Xaj WT were inhibited during Xaj argG- infection. Overall, the Xaj proteins revealed a drastic shift in carbon and energy management induced by disruption of nitrogen metabolism while the top differentially expressed proteins included a Fis transcriptional regulator and a peptidyl-prolyl isomerase. Our results show the critical role of de novo arginine biosynthesis to sustain virulence and minimal growth during WB. This study is timely and critical as copper-based control methods are losing their effectiveness, and new sustainable methods are urgently needed in orchard environments.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Alternative LC-MS/MS Platforms and Data Acquisition Strategies for Proteomic Genotyping of Human Hair Shafts.

Protein is a major component of all biological evidence. Proteomic genotyping is the use of genetically variant peptides (GVPs) that contain single-amino-acid polymorphisms to infer the genotype of matching nonsynonymous single-nucleotide polymorphisms for the individual from whom the protein sample originated. This can be used to statistically associate an individual to evidence found at a crime scene. The utility of the inferred genotype increases as the detection of GVPs increases, which is the direct result of technology transfer to mass spectrometry platforms typically available. Digests of single (2 cm) human hair shafts from three European and two African subjects were analyzed using data-dependent acquisition on a Q-Exactive Plus Hybrid Quadrupole-Orbitrap system, data-independent acquisition and a variant of parallel reaction monitoring (PRM) on an Orbitrap Fusion Lumos Tribrid system, and multiple reaction monitoring (MRM) on an Agilent 6495 triple quadrupole system. In our hands, average GVP detection from a selected panel of 24 GVPs increased from 6.5 ± 1.1 and 3.1 ± 0.8 using data-dependent and -independent acquisition to 9.5 ± 0.7 and 11.7 ± 1.7 using PRM and MRM (p < 0.05), respectively. PRM resulted in a 1.3-fold increase in detection sensitivity, and MRM resulted in a 1.6-fold increase in detection sensitivity. This increase in biomarker detection has a functional impact on the statistical association of a protein sample and an individual. Increased biomarker sensitivity, using Markov Chain Monte Carlo modeling, produced a median-estimated random match probability of over 1 in 10 trillion from a single hair using targeted proteomics. For PRM and MRM, detected GVPs were validated by the inclusion of stable isotope-labeled peptides in each sample, which served also as a detection trigger. This research accomplishes two aims: the demonstration of utility for alternative analytical platforms in proteomic genotyping and the establishment of validation methods for the evaluation of inferred genotypes.

A Secreted Chorismate Mutase from Xanthomonas arboricola pv. juglandis Attenuates Virulence and Walnut Blight Symptoms.

Walnut blight is a significant above-ground disease of walnuts caused by Xanthomonas arboricola pv. juglandis (Xaj). The secreted form of chorismate mutase (CM), a key enzyme of the shikimate pathway regulating plant immunity, is highly conserved between plant-associated beta and gamma proteobacteria including phytopathogens belonging to the Xanthomonadaceae family. To define its role in walnut blight disease, a dysfunctional mutant of chorismate mutase was created in a copper resistant strain Xaj417 (XajCM). Infections of immature walnut Juglans regia (Jr) fruit with XajCM were hypervirulent compared with infections with the wildtype Xaj417 strain. The in vitro growth rate, size and cellular morphology were similar between the wild-type and XajCM mutant strains, however the quantification of bacterial cells by dPCR within walnut hull tissues showed a 27% increase in XajCM seven days post-infection. To define the mechanism of hypervirulence, proteome analysis was conducted to compare walnut hull tissues inoculated with the wild type to those inoculated with the XajCM mutant strain. Proteome analysis revealed 3296 Jr proteins (five decreased and ten increased with FDR ≤ 0.05) and 676 Xaj417 proteins (235 increased in XajCM with FDR ≤ 0.05). Interestingly, the most abundant protein in Xaj was a polygalacturonase, while in Jr it was a polygalacturonase inhibitor. These results suggest that this secreted chorismate mutase may be an important virulence suppressor gene that regulates Xaj417 virulence response, allowing for improved bacterial survival in the plant tissues.

Proteome Analysis of Walnut Bacterial Blight Disease.

The interaction between the plant host, walnut (Juglans regia; Jr), and a deadly pathogen (Xanthomonas arboricola pv. juglandis 417; Xaj) can lead to walnut bacterial blight (WB), which depletes walnut productivity by degrading the nut quality. Here, we dissect this pathosystem using tandem mass tag quantitative proteomics. Walnut hull tissues inoculated with Xaj were compared to mock-inoculated tissues, and 3972 proteins were identified, of which 3296 are from Jr and 676 from Xaj. Proteins with differential abundance include oxidoreductases, proteases, and enzymes involved in energy metabolism and amino acid interconversion pathways. Defense responses and plant hormone biosynthesis were also increased. Xaj proteins detected in infected tissues demonstrate its ability to adapt to the host microenvironment, limiting iron availability, coping with copper toxicity, and maintaining energy and intermediary metabolism. Secreted proteases and extracellular secretion apparatus such as type IV pilus for twitching motility and type III secretion effectors indicate putative factors recognized by the host. Taken together, these results suggest intense degradation processes, oxidative stress, and general arrest of the biosynthetic metabolism in infected nuts. Our results provide insights into molecular mechanisms and highlight potential molecular tools for early detection and disease control strategies.

A functional link between NAD+ homeostasis and N-terminal protein acetylation in Saccharomyces cerevisiae.

Nicotinamide adenine dinucleotide (NAD+) is an essential metabolite participating in cellular redox chemistry and signaling, and the complex regulation of NAD+ metabolism is not yet fully understood. To investigate this, we established a NAD+-intermediate specific reporter system to identify factors required for salvage of metabolically linked nicotinamide (NAM) and nicotinic acid (NA). Mutants lacking components of the NatB complex, NAT3 and MDM20, appeared as hits in this screen. NatB is an Nα-terminal acetyltransferase responsible for acetylation of the N terminus of specific Met-retained peptides. In NatB mutants, increased NA/NAM levels were concomitant with decreased NAD+ We identified the vacuolar pool of nicotinamide riboside (NR) as the source of this increased NA/NAM. This NR pool is increased by nitrogen starvation, suggesting NAD+ and related metabolites may be trafficked to the vacuole for recycling. Supporting this, increased NA/NAM release in NatB mutants was abolished by deleting the autophagy protein ATG14 We next examined Tpm1 (tropomyosin), whose function is regulated by NatB-mediated acetylation, and Tpm1 overexpression (TPM1-oe) was shown to restore some NatB mutant defects. Interestingly, although TPM1-oe largely suppressed NA/NAM release in NatB mutants, it did not restore NAD+ levels. We showed that decreased nicotinamide mononucleotide adenylyltransferase (Nma1/Nma2) levels probably caused the NAD+ defects, and NMA1-oe was sufficient to restore NAD+ NatB-mediated N-terminal acetylation of Nma1 and Nma2 appears essential for maintaining NAD+ levels. In summary, our results support a connection between NatB-mediated protein acetylation and NAD+ homeostasis. Our findings may contribute to understanding the molecular basis and regulation of NAD+ metabolism.

Cornification of nail keratinocytes requires autophagy for bulk degradation of intracellular proteins while sparing components of the cytoskeleton.

Epidermal keratinocytes undergo cornification to form the cellular building blocks of hard skin appendages such as nails and the protective layer on the surface of the skin. Cornification requires the cross-linking of structural proteins and the removal of other cellular components to form mechanically rigid and inert corneocytes. Autophagy has been proposed to contribute to this intracellular remodelling process, but its molecular targets in keratinocytes, if any, have remained elusive. Here, we deleted the essential autophagy factor Atg7 in K14-positive epithelia of mice and determined by proteomics the impact of this deletion on the abundance of individual proteins in cornified nails. The genetic suppression of autophagy in keratinocytes resulted in a significant increase in the number of proteins that survived cornification and in alterations of their abundance in the nail proteome. A broad range of enzymes and other non-structural proteins were elevated whereas the amounts of cytoskeletal proteins of the keratin and keratin-associated protein families, cytolinker proteins and desmosomal proteins were either unaltered or decreased in nails of mice lacking epithelial autophagy. Among the various types of non-cytoskeletal proteins, the subunits of the proteasome and of the TRiC/CCT chaperonin were most strongly elevated in mutant nails, indicating a particularly important role of autophagy in removing these large protein complexes during normal cornification. Taken together, the results of this study suggest that autophagy is active during nail keratinocyte cornification and its substrate specificity depends on the accessibility of proteins outside of the cytoskeleton and their presence in large complexes.

Human stratum corneum proteomics reveals cross-linking of a broad spectrum of proteins in cornified envelopes.

Defects in keratinocyte transglutaminase (TGM1), resulting in an improper protein scaffold for deposition of the lipid barrier, comprise a major source of autosomal recessive congenital ichthyosis. For that reason, the composition and formation of the cornified (cross-linked) protein envelope of the epidermis have been of considerable interest. Since the isopeptide cross-linked protein components are not individually isolable once incorporated, purified envelopes were analysed by mass spectrometry after trypsin digestion. Quantitative estimates of the identified components revealed some 170 proteins, each comprising at least 0.001% of the total, of which keratins were major constituents accounting for ≈74% of the total. Some prevalent non-keratin constituents such as keratinocyte proline-rich protein, loricrin and late envelope protein-7 were preferentially incorporated into envelopes. The results suggest a model where, as previously observed in hair shaft and nail plate, a diversity of cellular proteins are incorporated. They also help rationalize the minimal effect on epidermis of ablating genes for specific single envelope structural components. The quantitative profile of constituent proteins provides a foundation for future exploration of envelope perturbations that may occur in pathological conditions.

Corneocyte proteomics: Applications to skin biology and dermatology.

Advances in mass spectrometry-based proteomics now permit analysis of complex cellular structures. Application to epidermis and its appendages (nail plate, hair shaft) has revealed a wealth of information about their protein profiles. The results confirm known site-specific differences in levels of certain keratins and add great depth to our knowledge of site specificity of scores of other proteins, thereby connecting anatomy and pathology. An example is the evident overlap in protein profiles of hair shaft and nail plate, helping rationalize their sharing of certain dystrophic syndromes distinct from epidermis. In addition, interindividual differences in protein level are manifest as would be expected. This approach permits characterization of altered profiles as a result of disease, where the magnitude of perturbation can be quantified and monitored during treatment. Proteomic analysis has also clarified the nature of the isopeptide cross-linked residual insoluble material after vigorous extraction with protein denaturants, nearly intractable to analysis without fragmentation. These structures, including the cross-linked envelope of epidermal corneocytes, are comprised of hundreds of protein constituents, evidence for strengthening the terminal structure complementary to disulphide bonding. Along with other developing technologies, proteomic analysis is anticipated to find use in disease risk stratification, detection, diagnosis and prognosis after the discovery phase and clinical validation.

Proteomic analysis of hair shafts from monozygotic twins: Expression profiles and genetically variant peptides.

Forensic association of hair shaft evidence with individuals is currently assessed by comparing mitochondrial DNA haplotypes of reference and casework samples, primarily for exclusionary purposes. Present work tests and validates more recent proteomic approaches to extract quantitative transcriptional and genetic information from hair samples of monozygotic twin pairs, which would be predicted to partition away from unrelated individuals if the datasets contain identifying information. Protein expression profiles and polymorphic, genetically variant hair peptides were generated from ten pairs of monozygotic twins. Profiling using the protein tryptic digests revealed that samples from identical twins had typically an order of magnitude fewer protein expression differences than unrelated individuals. The data did not indicate that the degree of difference within twin pairs increased with age. In parallel, data from the digests were used to detect genetically variant peptides that result from common nonsynonymous single nucleotide polymorphisms in genes expressed in the hair follicle. Compilation of the variants permitted sorting of the samples by hierarchical clustering, permitting accurate matching of twin pairs. The results demonstrate that genetic differences are detectable by proteomic methods and provide a framework for developing quantitative statistical estimates of personal identification that increase the value of hair shaft evidence.

Absolute Quantification of Human Milk Caseins and the Whey/Casein Ratio during the First Year of Lactation.

Whey proteins and caseins in breast milk provide bioactivities and also have different amino acid composition. Accurate determination of these two major protein classes provides a better understanding of human milk composition and function, and further aids in developing improved infant formulas based on bovine whey proteins and caseins. In this study, we implemented a LC-MS/MS quantitative analysis based on iBAQ label-free quantitation, to estimate absolute concentrations of α-casein, ß-casein, and κ-casein in human milk samples (n = 88) collected between day 1 and day 360 postpartum. Total protein concentration ranged from 2.03 to 17.52 with a mean of 9.37 ± 3.65 g/L. Casein subunits ranged from 0.04 to 1.68 g/L (α-), 0.04 to 4.42 g/L (ß-), and 0.10 to 1.72 g/L (α-), with ß-casein having the highest average concentration among the three subunits. Calculated whey/casein ratio ranged from 45:55 to 97:3. Linear regression analyses show significant decreases in total protein, ß-casein, κ-casein, total casein, and a significant increase of whey/casein ratio during the course of lactation. Our study presents a novel and accurate quantitative analysis of human milk casein content, demonstrating a lower casein content than earlier believed, which has implications for improved infants formulas.

ABRF Proteome Informatics Research Group (iPRG) 2015 Study: Detection of Differentially Abundant Proteins in Label-Free Quantitative LC-MS/MS Experiments.

Detection of differentially abundant proteins in label-free quantitative shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS) experiments requires a series of computational steps that identify and quantify LC-MS features. It also requires statistical analyses that distinguish systematic changes in abundance between conditions from artifacts of biological and technical variation. The 2015 study of the Proteome Informatics Research Group (iPRG) of the Association of Biomolecular Resource Facilities (ABRF) aimed to evaluate the effects of the statistical analysis on the accuracy of the results. The study used LC-tandem mass spectra acquired from a controlled mixture, and made the data available to anonymous volunteer participants. The participants used methods of their choice to detect differentially abundant proteins, estimate the associated fold changes, and characterize the uncertainty of the results. The study found that multiple strategies (including the use of spectral counts versus peak intensities, and various software tools) could lead to accurate results, and that the performance was primarily determined by the analysts' expertise. This manuscript summarizes the outcome of the study, and provides representative examples of good computational and statistical practice. The data set generated as part of this study is publicly available.

Integrated Metabolomics and Proteomics Highlight Altered Nicotinamide- and Polyamine Pathways in Lung Adenocarcinoma.

Lung cancer is the leading cause of cancer mortality in the United States with non-small cell lung cancer (NSCLC) adenocarcinoma being the most common histological type. Early perturbations in cellular metabolism are a hallmark of cancer, but the extent of these changes in early stage lung adenocarcinoma remains largely unknown. In the current study, an integrated metabolomics and proteomics approach was utilized to characterize the biochemical and molecular alterations between malignant and matched control tissue from 27 subjects diagnosed with early stage lung adenocarcinoma. Differential analysis identified 71 metabolites and 1102 proteins that delineated tumor from control tissue. Integrated results indicated four major metabolic changes in early stage adenocarcinoma: (1) increased glycosylation and glutaminolysis; (2) elevated Nrf2 activation; (3) increase in nicotinic and nicotinamide salvaging pathways; and (4) elevated polyamine biosynthesis linked to differential regulation of the SAM/nicotinamide methyl-donor pathway. Genomic data from publicly available databases were included to strengthen proteomic findings. Our findings provide insight into the biochemical and molecular biological reprogramming that may accompanies early stage lung tumorigenesis and highlight potential therapeutic targets.

The 2012/2013 ABRF Proteomic Research Group Study: Assessing Longitudinal Intralaboratory Variability in Routine Peptide Liquid Chromatography Tandem Mass Spectrometry Analyses.

Questions concerning longitudinal data quality and reproducibility of proteomic laboratories spurred the Protein Research Group of the Association of Biomolecular Resource Facilities (ABRF-PRG) to design a study to systematically assess the reproducibility of proteomic laboratories over an extended period of time. Developed as an open study, initially 64 participants were recruited from the broader mass spectrometry community to analyze provided aliquots of a six bovine protein tryptic digest mixture every month for a period of nine months. Data were uploaded to a central repository, and the operators answered an accompanying survey. Ultimately, 45 laboratories submitted a minimum of eight LC-MSMS raw data files collected in data-dependent acquisition (DDA) mode. No standard operating procedures were enforced; rather the participants were encouraged to analyze the samples according to usual practices in the laboratory. Unlike previous studies, this investigation was not designed to compare laboratories or instrument configuration, but rather to assess the temporal intralaboratory reproducibility. The outcome of the study was reassuring with 80% of the participating laboratories performing analyses at a medium to high level of reproducibility and quality over the 9-month period. For the groups that had one or more outlying experiments, the major contributing factor that correlated to the survey data was the performance of preventative maintenance prior to the LC-MSMS analyses. Thus, the Protein Research Group of the Association of Biomolecular Resource Facilities recommends that laboratories closely scrutinize the quality control data following such events. Additionally, improved quality control recording is imperative. This longitudinal study provides evidence that mass spectrometry-based proteomics is reproducible. When quality control measures are strictly adhered to, such reproducibility is comparable among many disparate groups. Data from the study are available via ProteomeXchange under the accession code PXD002114.

Proteomic Analysis of Loricrin Knockout Mouse Epidermis.

The crosslinked envelope of the mammalian epidermal corneocyte serves as a scaffold for assembly of the lipid barrier of the epidermis. Thus, deficient envelope crosslinking by keratinocyte transglutaminase (TGM1) is a major cause of the human autosomal recessive congenital ichthyoses characterized by barrier defects. Expectations that loss of some envelope protein components would also confer an ichthyosis phenotype have been difficult to demonstrate. To help rationalize this observation, the protein profile of epidermis from loricrin knockout mice has been compared to that of wild type. Despite the mild phenotype of the knockout, some 40 proteins were incorporated into envelope material to significantly different extents compared to those of wild type. Nearly half were also incorporated to similarly altered extents into the disulfide bonded keratin network of the corneocyte. The results suggest that loss of loricrin alters their incorporation into envelopes as a consequence of protein-protein interactions during cell maturation. Mass spectrometric protein profiling revealed that keratin 1, keratin 10, and loricrin are prominent envelope components and that dozens of other proteins are also components. This finding helps rationalize the potential formation of functional envelopes, despite loss of a single component, due to the availability of many alternative transglutaminase substrates.