Showing papers in "Journal of Proteome Research in 2010"
Vanderbilt University1, Texas A&M University2, National Institute of Standards and Technology3, Memorial University of Newfoundland4, University of Arizona5, Broad Institute6, Merck & Co.7, New York University8, Purdue University9, Buck Institute for Research on Aging10, Fred Hutchinson Cancer Research Center11, University of California, San Francisco12, National Institutes of Health13, Memorial Sloan Kettering Cancer Center14
TL;DR: The most repeatable peptides were those corresponding to conventional tryptic cleavage sites, those that produced intense MS signals, and those that resulted from proteins generating many distinct peptides.
Abstract: The complexity of proteomic instrumentation for LC-MS/MS introduces many possible sources of variability. Data-dependent sampling of peptides constitutes a stochastic element at the heart of discovery proteomics. Although this variation impacts the identification of peptides, proteomic identifications are far from completely random. In this study, we analyzed interlaboratory data sets from the NCI Clinical Proteomic Technology Assessment for Cancer to examine repeatability and reproducibility in peptide and protein identifications. Included data spanned 144 LC-MS/MS experiments on four Thermo LTQ and four Orbitrap instruments. Samples included yeast lysate, the NCI-20 defined dynamic range protein mix, and the Sigma UPS 1 defined equimolar protein mix. Some of our findings reinforced conventional wisdom, such as repeatability and reproducibility being higher for proteins than for peptides. Most lessons from the data, however, were more subtle. Orbitraps proved capable of higher repeatability and reproduci...
504 citations
TL;DR: It is shown, via a global proteomic approach, that TDP-43 has extensive interaction with proteins that regulate RNA metabolism, strongly suggesting that it has multiple roles in RNA metabolism and functions in both the nucleus and the cytoplasm.
Abstract: TDP-43 is a highly conserved and ubiquitously expressed member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family of proteins. Recently, TDP-43 was shown to be a major disease protein in the ubiquitinated inclusions characteristic of most cases of amyotrophic lateral sclerosis (ALS), tau-negative frontotemporal lobar degeneration (FTLD), and inclusion body myopathy. In these diseases, TDP-43 is redistributed from its predominantly nuclear location to ubiquitin-positive, cytoplasmic foci. The extent to which TDP-43 drives pathophysiology is unknown, but the identification of mutations in TDP-43 in familial forms of ALS and FTLD-U suggests an important role for this protein in pathogenesis. Little is known about TDP-43 function and only a few TDP-43 interacting proteins have been previously identified, which makes further insight into both the normal and pathological functions of TDP-43 difficult. Here we show, via a global proteomic approach, that TDP-43 has extensive interaction with proteins t...
426 citations
TL;DR: It is demonstrated that large portions of the proteome are simply inaccessible following digestion with a single protease and that multiple proteases, rather than technical replicates, provide a direct route to increase both protein identifications and proteome sequence coverage.
Abstract: Large-scale protein sequencing methods rely on enzymatic digestion of complex protein mixtures to generate a collection of peptides for mass spectrometric analysis. Here we examine the use of multiple proteases (trypsin, LysC, ArgC, AspN, and GluC) to improve both protein identification and characterization in the model organism Saccharomyces cerevisiae. Using a data-dependent, decision tree-based algorithm to tailor MS(2) fragmentation method to peptide precursor, we identified 92 095 unique peptides (609 665 total) mapping to 3908 proteins at a 1% false discovery rate (FDR). These results were a significant improvement upon data from a single protease digest (trypsin) - 27 822 unique peptides corresponding to 3313 proteins. The additional 595 protein identifications were mainly from those at low abundances (i.e., < 1000 copies/cell); sequence coverage for these proteins was likewise improved nearly 3-fold. We demonstrate that large portions of the proteome are simply inaccessible following digestion with a single protease and that multiple proteases, rather than technical replicates, provide a direct route to increase both protein identifications and proteome sequence coverage.
418 citations
TL;DR: The effects of top 7/top 14 immunodepletion on the shotgun proteomic analysis of human plasma was evaluated and it was found that either top 7 or top 14 immunoaffinity depletion resulted in a 25% increase in identified proteins compared to unfractionated plasma.
Abstract: Immunoaffinity depletion with antibodies to the top 7 or top 14 high-abundance plasma proteins is used to enhance detection of lower abundance proteins in both shotgun and targeted proteomic analyses. We evaluated the effects of top 7/top 14 immunodepletion on the shotgun proteomic analysis of human plasma. Our goal was to evaluate the impact of immunodepletion on detection of proteins across detectable ranges of abundance. The depletion columns afforded highly repeatable and efficient plasma protein fractionation. Relatively few nontargeted proteins were captured by the depletion columns. Analyses of unfractionated and immunodepleted plasma by peptide isoelectric focusing (IEF), followed by liquid chromatography−tandem mass spectrometry (LC−MS/MS), demonstrated enrichment of nontargeted plasma proteins by an average of 4-fold, as assessed by MS/MS spectral counting. Either top 7 or top 14 immunodepletion resulted in a 25% increase in identified proteins compared to unfractionated plasma. Although 23 low-...
315 citations
TL;DR: It is found that both of the surfactants (SDS and DOC) produced an increase in the overall yield of tryptic peptides from these 45 proteins, when compared to the more commonly used urea protocol, however, SDS can be a serious interference for subsequent mass spectrometry.
Abstract: Plasma biomarkers studies are based on the differential expression of proteins between different treatment groups or between diseased and control populations. Most mass spectrometry-based methods of protein quantitation, however, are based on the detection and quantitation of peptides, not intact proteins. For peptide-based protein quantitation to be accurate, the digestion protocols used in proteomic analyses must be both efficient and reproducible. There have been very few studies, however, where plasma denaturation/digestion protocols have been compared using absolute quantitation methods. In this paper, 14 combinations of heat, solvent [acetonitrile, methanol, trifluoroethanol], chaotropic agents [guanidine hydrochloride, urea], and surfactants [sodium dodecyl sulfate (SDS) and sodium deoxycholate (DOC)] were compared with respect to their effectiveness in improving subsequent tryptic digestion. These digestion protocols were evaluated by quantitating the production of proteotypic tryptic peptides fro...
312 citations
TL;DR: In this article, the urinary metabolic phenotypes of individuals diagnosed with autism using the DSM-IV-TR criteria (n = 39, male = 35, female = 4), together with their non-autistic siblings and age-matched healthy volunteers were characterized using 1H NMR spectroscopy and pattern recognition methods.
Abstract: Autism is an early onset developmental disorder with a severe life-long impact on behavior and social functioning that has associated metabolic abnormalities. The urinary metabolic phenotypes of individuals (age range=3−9 years old) diagnosed with autism using the DSM-IV-TR criteria (n = 39; male = 35; female = 4), together with their nonautistic siblings (n = 28; male = 14; female = 14) and age-matched healthy volunteers (n = 34, male = 17; female = 17) have been characterized for the first time using 1H NMR spectroscopy and pattern recognition methods. Novel findings associated with alterations in nicotinic acid metabolism within autistic individuals showing increased urinary excretion of N-methyl-2-pyridone-5-carboxamide, N-methyl nicotinic acid, and N-methyl nicotinamide indicate a perturbation in the tryptophan−nicotinic acid metabolic pathway. Multivariate statistical analysis indicated urinary patterns of the free amino acids, glutamate and taurine were significantly different between groups with t...
295 citations
TL;DR: Annotation of the neutral free oligosaccharides in milk is performed to develop a database for the rapid identification of oligosACcharide structures, which incorporates high performance nanoflow liquid chromatography and mass spectrometry for characterizing HMO structures.
Abstract: Human milk oligosaccharides (HMOs) perform a number of functions including serving as prebiotics to stimulate the growth of beneficial intestinal bacteria, as receptor analogues to inhibit binding of pathogens, and as substances that promote postnatal brain development. There is further evidence that HMOs participate in modulating the human immune system. Because the absorption, catabolism, and biological function of oligosaccharides (OS) have strong correlations with their structures, structure elucidation is key to advancing this research. Oligosaccharides are produced by competing enzymes that provide the large structural diversity and heterogeneity that characterizes this class of compounds. Unlike the proteome, there is no template for oligosaccharides, making it difficult to rapidly identify oligosaccharide structures. In this research, annotation of the neutral free oligosaccharides in milk is performed to develop a database for the rapid identification of oligosaccharide structures. Our strategy incorporates high performance nanoflow liquid chromatography and mass spectrometry for characterizing HMO structures. HPLC-Chip/TOF MS provides a sensitive and quantitative method for sample profiling. The reproducible retention time and accurate mass can be used to rapidly identify the OS structures in HMO samples. A library with 45 neutral OS structures has been constructed. The structures include information regarding the epitopes such as Lewis type, as well as information regarding the secretor status.
279 citations
TL;DR: The amount of dietary saturated fat may influence the proportion of saturated fatty acids in serum phospholipids and the degree of saturation of the constituent acyl group of plasma lysoPC in overweight/obese men.
Abstract: Obesity is currently epidemic in many countries worldwide and is strongly related to diabetes and cardiovascular disease. This study investigated the differences in metabolomic profiling between overweight/obese and normal-weight men. Overweight/obese (n=30) and age-matched, normal-weight men (n=30) were included. Anthropometric parameters, conventional metabolites, and biomarkers were measured. Metabolomic profiling was analyzed with UPLC-Q-TOF MS. Overweight/obese men showed higher levels of HOMA-IR, triglycerides, total cholesterol, and LDL-cholesterol, and lower levels of HDL-cholesterol and adiponectin than lean men. Overweight/obese men showed higher proportion of stearic acid and lower proportion of oleic acid in serum phospholipids. Additionally, overweight/obese individuals showed higher fat intake and lower ratio of polyunsaturated fatty acids to saturated fatty acids. We identified three lyso-phosphatidylcholine (lysoPC) as potential plasma markers and confirmed eight known metabolites for overweight/obesity men. Especially, overweight/obese subjects showed higher levels of lysoPC C14:0 and lysoPC C18:0 and lower levels of lysoPC C18:1 than lean subjects. Results confirmed abnormal metabolism of two branched-chain amino acids, two aromatic amino acids, and fatty acid synthesis and oxidation in overweight/obese men. Additionally, the amount of dietary saturated fat may influence the proportion of saturated fatty acids in serum phospholipids and the degree of saturation of the constituent acyl group of plasma lysoPC.
272 citations
TL;DR: An open source software environment, called MSQuant, which allows visualization and validation of peptide identification results directly on the raw mass spectrometric data, and iteratively recalibrates MS data thereby significantly increasing mass accuracy leading to fewer false positive peptide identifications.
Abstract: Mass spectrometry-based proteomics critically depends on algorithms for data interpretation. A current bottleneck in the rapid advance of proteomics technology is the closed nature and slow develop...
268 citations
TL;DR: This work represents the most extensive proteomic description of salinity responses of Arabidopsis and Thellungiella and has improved the knowledge of salt tolerance in glycophytes and halophytes.
Abstract: Salinity is a major abiotic stress affecting plant cultivation and productivity. Thellungiella halophila is a halophyte and has been used as a model for studying plant salt tolerance. Understanding the molecular mechanisms of salinity tolerance will facilitate the generation of salt tolerant crops. Here we report comparative leaf proteomics of Arabidopsis, a glycophyte, and its close relative Thellungiella, a halophyte, under different salt stress conditions. Proteins from control and NaCl treated Arabidopsis and Thellungiella leaf samples were extracted and separated by two-dimensional gel electrophoresis. A total of 88 protein spots from Arabidopsis gels and 37 protein spots from Thellungiella gels showed significant changes. Out of these spots, a total of 79 and 32 proteins were identified by mass spectrometry in Arabidopsis and Thellungiella, respectively. Most of the identified proteins were involved in photosynthesis, energy metabolism, and stress response in Arabidopsis and Thellungiella. As a complementary approach, isobaric tag for relative and absolute quantification (iTRAQ) LC-MS was used to identify crude microsomal proteins. A total of 31 and 32 differentially expressed proteins were identified in Arabidopsis and Thellungiella under salt treatment, respectively. Overall, there were more proteins changed in abundance in Arabidopsis than in Thellungiella. Distinct patterns of protein changes in the two species were observed. Collectively, this work represents the most extensive proteomic description of salinity responses of Arabidopsis and Thellungiella and has improved our knowledge of salt tolerance in glycophytes and halophytes.
266 citations
TL;DR: An in-depth analysis of phosphorylation sites in mouse brain reveals that 23% of identified sites are located on plasma membrane proteins, including a large number of ion channels and transporters.
Abstract: Taking advantage of the recently developed Filter Assisted Sample Preparation (FASP) method for sample preparation, we performed an in-depth analysis of phosphorylation sites in mouse brain. To maximize the number of detected phosphorylation sites, we fractionated proteins by size exclusion chromatography (SEC) or separated tryptic peptides on an anion exchanger (SAX) prior or after the TiO2-based phosphopeptide enrichment, respectively. SEC allowed analysis of minute tissue samples (1 mg total protein), and resulted in identification of more than 4000 sites in a single experiment, comprising eight fractions. SAX in a pipet tip format offered a convenient and rapid way to fractionate phosphopeptides and mapped more than 5000 sites in a single six fraction experiment. To enrich peptides containing phosphotyrosine residues, we describe a filter aided antibody capturing and elution (FACE) method that requires only the uncoupled instead of resin-immobilized capture reagent. In total, we identified 12 035 phos...
TL;DR: In this paper, the authors used Matrix-Aided Laser Desorption/ionization (MALDI) imaging mass spectrometry (IMS) to determine HER2 status directly from breast cancer tissues.
Abstract: Clinical laboratory testing for HER2 status in breast cancer tissues is critically important for therapeutic decision making. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a powerful tool for investigating proteins through the direct and morphology-driven analysis of tissue sections. We hypothesized that MALDI-IMS may determine HER2 status directly from breast cancer tissues. Breast cancer tissues (n = 48) predefined for HER2 status were subjected to MALDI-IMS, and protein profiles were obtained through direct analysis of tissue sections. Protein identification was performed by tissue microextraction and fractionation followed by top-down tandem mass spectrometry. A discovery and an independent validation set were used to predict HER2 status by applying proteomic classification algorithms. We found that specific protein/peptide expression changes strongly correlated with the HER2 overexpression. Among these, we identified m/z 8404 as cysteine-rich intestinal protein 1. The proteomic signature was able to accurately define HER2-positive from HER2-negative tissues, achieving high values for sensitivity of 83%, for specificity of 92%, and an overall accuracy of 89%. Our results underscore the potential of MALDI-IMS proteomic algorithms for morphology-driven tissue diagnostics such as HER2 testing and show that MALDI-IMS can reveal biologically significant molecular details from tissues which are not limited to traditional high-abundance proteins.
TL;DR: The focus of this review is on the use of proteomics tools and methods to identify oxidized proteins along with specific sites of oxidative damage and the consequences of protein oxidation.
Abstract: Excessive oxidative stress leaves a protein carbonylation fingerprint in biological systems. Carbonylation is an irreversible post-translational modification (PTM) that often leads to the loss of protein function and can be a component of multiple diseases. Protein carbonyl groups can be generated directly (by amino acids oxidation and the α-amidation pathway) or indirectly by forming adducts with lipid peroxidation products or glycation and advanced glycation end-products. Studies of oxidative stress are complicated by the low concentration of oxidation products and a wide array of routes by which proteins are carbonylated. The development of new selection and enrichment techniques coupled with advances in mass spectrometry are allowing the identification of hundreds of new carbonylated protein products from a broad range of proteins located at many sites in biological systems. The focus of this review is on the use of proteomics tools and methods to identify oxidized proteins along with specific sites o...
TL;DR: A protein-protein binding affinity benchmark consisting of binding constants (K(d)'s) for 81 complexes was used to assess the performance of nine commonly used scoring algorithms along with a free-energy prediction algorithm in their ability to predicting binding affinities.
Abstract: The design of an ideal scoring function for protein-protein docking that would also predict the binding affinity of a complex is one of the challenges in structural proteomics. Such a scoring function would open the route to in silico, large-scale annotation and prediction of complete interactomes. Here we present a protein-protein binding affinity benchmark consisting of binding constants (K(d)'s) for 81 complexes. This benchmark was used to assess the performance of nine commonly used scoring algorithms along with a free-energy prediction algorithm in their ability to predicting binding affinities. Our results reveal a poor correlation between binding affinity and scores for all algorithms tested. However, the diversity and validity of the benchmark is highlighted when binding affinity data are categorized according to the methodology by which they were determined. By further classifying the complexes into low, medium and high affinity groups, significant correlations emerge, some of which are retained after dividing the data into more classes, showing the robustness of these correlations. Despite this, accurate prediction of binding affinity remains outside our reach due to the large associated standard deviations of the average score within each group. All the above-mentioned observations indicate that improvements of existing scoring functions or design of new consensus tools will be required for accurate prediction of the binding affinity of a given protein-protein complex. The benchmark developed in this work will serve as an indispensable source to reach this goal.
TL;DR: FFPE biobank material can be analyzed by quantitative proteomics at the level of proteins and post-translational modifications, as well as fresh against FFPE tissue using the SILAC mouse and found no significant qualitative or quantitative differences.
Abstract: Tissue samples in biobanks are typically formalin-fixed and paraffin-embedded (FFPE), in which form they are preserved for decades. It has only recently been shown that proteins in FFPE tissues can be identified by mass spectrometry-based proteomics but analysis of post-translational modifications is thought to be difficult or impossible. The filter aided sample preparation (FASP) method can analyze proteomic samples solubilized in high concentrations of SDS and we use this feature to develop a simple protocol for FFPE analysis. Combination with simple pipet-tip based peptide fractionation identified about 5000 mouse liver proteins in 24 h measurement time—the same as in fresh tissue. Results from the FFPE-FASP procedure do not indicate any discernible changes due to storage time, hematoxylin staining or laser capture microdissection. We compared fresh against FFPE tissue using the SILAC mouse and found no significant qualitative or quantitative differences between these samples either at the protein or t...
TL;DR: 14 new phospholipid-associated proteins that migrate with traditionally defined HDL are identified, several of which further support roles for HDL in complement regulation and protease inhibition.
Abstract: Plasma levels of high density lipoprotein cholesterol (HDL-C) are inversely proportional to the incidence of cardiovascular disease. Recent applications of modern proteomic technologies have identified upward of 50 distinct proteins associated with HDL particles with many of these newly discovered proteins implicating HDL in nonlipid transport processes including complement activation, acute phase response and innate immunity. However, almost all MS-based proteomic studies on HDL to date have utilized density gradient ultracentrifugation techniques for HDL isolation prior to analysis. These involve high shear forces and salt concentrations that can disrupt HDL protein interactions and alter particle function. Here, we used high-resolution size exclusion chromatography to fractionate normal human plasma to 17 phospholipid-containing subfractions. Then, using a phospholipid binding resin, we identified proteins that associate with lipoproteins of various sizes by electrospray ionization mass spectrometry. We identified 14 new phospholipid-associated proteins that migrate with traditionally defined HDL, several of which further support roles for HDL in complement regulation and protease inhibition. The increased fractionation inherent to this method allowed us to visualize HDL protein distribution across particle size with unprecedented resolution. The observed heterogeneity across subfractions suggests the presence of HDL particle subpopulations each with distinct protein components that may prove to impart distinct physiological functions.
TL;DR: The increased concentration of the neurotoxins crotoxin and crotamine in South American rattlesnake venoms strongly argues that the gain of neurotoxicity and lethal venom activities to mammals may have represented the key axis along which overall venom toxicity has evolved during Crotalus durissus invasion of South America.
Abstract: We report a comparative venomic and antivenomic characterization of the venoms of newborn and adult specimens of the Central American rattlesnake, Crotalus simus, and of the subspecies cumanensis, durissus, ruruima, and terrificus of South American Crotalus durissus. Neonate and adult C. simus share about 50% of their venom proteome. The venom proteome of 6-week-old C. simus is predominantly made of the neurotoxic heterodimeric phospholipase A(2) (PLA(2) crotoxin) (55.9%) and serine proteinases (36%), whereas snake venom Zn(2+)-metalloproteinases (SVMPs), exclusively of class PIII, represent only 2% of the total venom proteins. In marked contrast, venom from adult C. simus comprises toxins from 7 protein families. A large proportion (71.7%) of these toxins are SVMPs, two-thirds of which belong to the PIII class. These toxin profiles correlate well with the overall biochemical and pharmacological features of venoms from adult (hemorrhagic) and newborn (neurotoxic) C. simus specimens. The venoms of the South American Crotalus subspecies belong to one of two distinct phenotypes. C. d. cumanensis exhibits high levels of SVMPs and low lethal potency (LD(50)), whereas C. d. subspecies terrificus, ruruima, and durissus have low SVMP activity and high neurotoxicity to mice. Their overall toxin compositions explain the outcome of envenomation by these species. Further, in all C. simus and C. durissus venoms, the concentration of neurotoxins (crotoxin and crotamine) is directly related with lethal activity, whereas lethality and metalloproteinase activity show an inverse relationship. The similar venom toxin profiles of newborn C. simus and adult C. durissus terrificus, ruruima, and durissus subspecies strongly suggests that the South American taxa have retained juvenile venom characteristics in the adult form (paedomorphism) along their North-South stepping-stone dispersal. The driving force behind paedomorphism is often competition or predation pressure. The increased concentration of the neurotoxins crotoxin and crotamine in South American rattlesnake venoms strongly argues that the gain of neurotoxicity and lethal venom activities to mammals may have represented the key axis along which overall venom toxicity has evolved during Crotalus durissus invasion of South America. The paedomorphic trend is supported by a decreasing LNC (lethal neurotoxicity coefficient, defined as the ratio between the average LD(50) of the venom and the crotoxin + crotamine concentration) along the North-South axis, coincident with the evolutionary dispersal pattern of the Neotropical rattlesnakes. The indistinguisable immunoreactivity patterns of Costa Rican and Venezuelan polyvalent antivenoms toward C. simus and C. durissus venoms strongly suggest the possibility of using these antivenoms indistinctly for the management of snakebites by adult C. simus and by certain C. d. cumanensis populations exhibiting a hemorrhagic venom phenotype. The antivenomic results also explain why the antivenoms effectively neutralize the hemorrhagic activity of adult C. simus venoms but does not protect against adult C. durissus sp. and newborn C. simus envenomations. The identification of evolutionary trends among tropical Crotalus, as reported here, may have an impact in defining the mixture of venoms for immunization to produce an effective pan-American anti-Crotalus antivenom.
TL;DR: A fast and robust method for lipid profiling utilizing liquid chromatography coupled with mass spectrometry has been demonstrated and validated and suggested an altered lipid metabolism associated with osteoarthritis and the release of arachidonic acid from phospholipids.
Abstract: A fast and robust method for lipid profiling utilizing liquid chromatography coupled with mass spectrometry has been demonstrated and validated for the analysis of human plasma. This method allowed quantification and identification of lipids in human plasma using parallel alternating low energy and high energy collision spectral acquisition modes. A total of 284 lipids were identified and quantified (as relative concentrations) in both positive and negative ion electrospray ionization mode. The method was validated with five nonendogenous lipids, and the linearity (r2 better than 0.994) and the intraday and interday repeatability (relative standard deviation, 4−6% and 5−8%, respectively) were satisfactory. The developed lipid profiling method was successfully applied for the analysis of plasma from osteoarthritis (OA) patients. The multivariate statistical analysis by partial least-squares-discrimination analysis suggested an altered lipid metabolism associated with osteoarthritis and the release of arach...
TL;DR: This study assessed the important metabonomic variations in urine associated with CRC and provided baseline information complementary to serum/plasma and tissue metabonomics for the complete elucidation of the underlying metabolic mechanisms of CRC.
Abstract: After our serum metabonomic study of colorectal cancer (CRC) patients recently published in J. Proteome Res., we profiled urine metabolites from the same group of CRC patients (before and after surgical operation) and 63 age-matched healthy volunteers using gas chromatography−mass spectrometry (GC−MS) in conjunction with a multivariate statistics technique. A parallel metabonomic study on a 1,2-dimethylhydrazine (DMH)-treated Sprague−Dawley rat model was also performed to identify significantly altered metabolites associated with chemically induced precancerous colorectal lesion. The orthogonal partial least-squares-discriminant analysis (OPLS-DA) models of metabonomic results demonstrated good separations between CRC patients or DMH-induced model rats and their healthy counterparts. The significantly increased tryptophan metabolism, and disturbed tricarboxylic acid (TCA) cycle and the gut microflora metabolism were observed in both the CRC patients and the rat model. The urinary metabolite profile of pos...
TL;DR: The first comparative proteome analysis of wound exudates obtained from normal healing or nonhealing (venous leg ulcer) human skin wounds is presented and reveals interesting results regarding the identification of new proteins with yet unknown functions in skin repair.
Abstract: Chronic wounds associated with vascular disease, diabetes mellitus, or aging are leading causes of morbidity in western countries and represent an unresolved clinical problem. The development of innovative strategies to promote tissue repair is therefore an important task that requires a more thorough analysis of the underlying molecular pathophysiology. We propose that the understanding of the complex biological events that control tissue repair or its failure largely benefits from a broad analytical approach as provided by novel proteomic methodologies. Here we present the first comparative proteome analysis of wound exudates obtained from normal healing or nonhealing (venous leg ulcer) human skin wounds. A total of 149 proteins were identified with high confidence. A minority of proteins was exclusively present in exudate of the healing wound (23 proteins) or the nonhealing wound (26 proteins). Of particular interest was the differential distribution of specific proteins among the two different healing...
TL;DR: This work proposes a new procedure for spatial segmentation of MALDI-imaging data sets that reduces pixel-to-pixel variability and improves the segmentation map significantly and analyzes two data sets using the proposed pipeline.
Abstract: In recent years, matrix-assisted laser desorption/ionization (MALDI)-imaging mass spectrometry has become a mature technology, allowing for reproducible high-resolution measurements to localize proteins and smaller molecules. However, despite this impressive technological advance, only a few papers have been published concerned with computational methods for MALDI-imaging data. We address this issue proposing a new procedure for spatial segmentation of MALDI-imaging data sets. This procedure clusters all spectra into different groups based on their similarity. This partition is represented by a segmentation map, which helps to understand the spatial structure of the sample. The core of our segmentation procedure is the edge-preserving denoising of images corresponding to specific masses that reduces pixel-to-pixel variability and improves the segmentation map significantly. Moreover, before applying denoising, we reduce the data set selecting peaks appearing in at least 1% of spectra. High dimensional dis...
TL;DR: To the authors' knowledge, this SDS-TCA approach enables the deepest proteome characterizations of microbes in soil to date, without significant biases in protein size, localization, or functional category compared to pure cultures.
Abstract: We present a novel direct protocol for deep proteome characterization of microorganisms in soil. The method employs thermally assisted detergent-based cellular lysis (SDS) of soil samples, followed by TCA precipitation for proteome extraction/cleanup prior to liquid chromatography-mass spectrometric characterization. This approach was developed and optimized using different soils inoculated with genome-sequenced bacteria (Gram-negative Pseudomonas putida or Gram-positive Arthrobacter chlorophenolicus). Direct soil protein extraction was compared to protein extraction from cells isolated from the soil matrix prior to lysis (indirect method). Each approach resulted in identification of greater than 500 unique proteins, with a wide range in molecular mass and functional categories. To our knowledge, this SDS-TCA approach enables the deepest proteome characterizations of microbes in soil to date, without significant biases in protein size, localization, or functional category compared to pure cultures. This protocol should provide a powerful tool for ecological studies of soil microbial communities.
TL;DR: Results suggest that TNF-alpha-induced secretome may play a pivotal role in inflammatory responses and that shotgun proteomic analysis will be useful for elucidation of the paracrine functions of mesenchymal stem cells.
Abstract: Human adipose tissue-derived mesenchymal stem cells (hASCs) are useful for regeneration of inflamed or injured tissues. To identify secreted hASC proteins during inflammation, hASCs were exposed to...
TL;DR: A nonredundant list of 1989 proteins is compiled and it is intuitively confirmed that red blood cells likely suffer of exacerbated oxidative stress and continuously strive against protein and cytoskeletal damage.
Abstract: Although a preliminary portrait of the red blood cell proteome and interactome has already been provided, the recent identification of 1578 gene products from the erythrocyte cytosol asks for an updated and improved view. In this paper, we exploit data available from recent literature to compile a nonredundant list of 1989 proteins and elaborate it with pathway and network analyses. Upon network analysis, it is intuitively confirmed that red blood cells likely suffer of exacerbated oxidative stress and continuously strive against protein and cytoskeletal damage. It also emerges that erythrocyte interaction networks display a high degree of maturity. Indeed, a series of core proteins were individuated to play a central role. A catalytic ring of proteins counteracting oxidative stress was individuated as well. In parallel, pathway analysis confirmed the validity of observations about the SEC23B gene role in CDA II in a fast and unbiased way.
TL;DR: One-hundred percent sensitivity in detecting BC was observed using urinary metabonomics versus 33% sensitivity achieved by urinary cytology, and urinary metabolism exhibited potential in the staging and grading of bladder tumors.
Abstract: Cystoscopy is considered the gold standard for the clinical diagnosis of human bladder cancer (BC). As cystoscopy is expensive and invasive, it may compromise patients' compliance and account for the failure in detecting recurrent BC in some patients. In this paper, we investigated the role of urinary metabonomics in the diagnosis of human BC. Gas chromatography/time-of-flight mass spectrometry was applied for the urinary metabolic profiling of 24 BC patients and 51 non-BC controls. The acquired data were analyzed using multivariate principal component analysis followed by orthogonal partial least-squares discriminant analysis (OPLS-DA). Model validity was verified using permutation tests and receiver operating characteristic (ROC) analysis. BC patients were clearly distinguished from non-BC subjects based on their global urinary metabolic profiles (OPLS-DA, 4 latent variables, R(2)X = 0.420, R(2)Y = 0.912 and Q(2) (cumulative) = 0.245; ROC AUC of 0.90; 15 marker metabolites). One-hundred percent sensitivity in detecting BC was observed using urinary metabonomics versus 33% sensitivity achieved by urinary cytology. Additionally, urinary metabonomics exhibited potential in the staging and grading of bladder tumors. In summary, urinary metabonomics is amenable for the noninvasive diagnosis of human BC.
TL;DR: It is concluded that HCD in the new configuration is now a viable method for large-scale phosphoproteome analysis alongside collisional induced dissociation, (CID) and electron capture/transfer dissociation (ECD/ETD).
Abstract: Mass spectrometry (MS)-based proteomics now enables the analysis of thousands of phosphorylation sites in single projects Among a wide range of analytical approaches, the combination of high resolution MS scans in an Orbitrap analyzer with low resolution MS/MS scans in a linear ion trap has proven to be particularly successful (“high-low” strategy) Here we investigate if the improved sensitivity of higher energy collisional dissociation (HCD) on an LTQ-Orbitrap Velos instrument allows a “high−high” strategy A high resolution MS scan was followed by up to 10 HCD MS/MS scans, and we achieved cycle times of about 3 s making the method compatible with chromatographic time scales Fragment mass accuracy increased about 50-fold compared to the “high-low” strategy Unexpectedly, the HCD approach mapped up to 16 000 total phosphorylation sites in one day’s measuring time — the same or better than the standard high-low strategy Reducing the target values from a standard of 30 000 to 5000 ions did not severely
TL;DR: Immunohistochemical analysis showed that the overexpression of trefoil factor 3 or growth/differentiation factor 15 in colorectal cancer was associated with lymph node metastatic behavior, and an accurate, sensitive, and robust label-free quantitation approach for differential analysis of cancer secretome.
Abstract: Lymph node metastasis is the major concern that causes death in colorectal cancers. However, biomarkers for cancer metastasis are still lacking. In this study, we applied an LC-MS/MS-based label-free quantitative proteomics approach to compare the differential secretome of a primary cell line SW480 and its lymph node metastatic cell line SW620 from the same colorectal cancer patient. We identified a total of 910 proteins from the conditioned media and 145 differential proteins between SW480 and SW620 (>1.5-fold change). The differential expression pattern of 6 candidate proteins was validated by Western blot analysis. Among them, trefoil factor 3 and growth/differentiation factor 15, two up-regulated proteins in SW620, were further analyzed in a large cohort of clinical tissue and serum samples. Sandwich ELISA assay showed that the serum levels of both proteins were significantly higher in lymph node metastatic colorectal cancers. Receiver operating characteristic curve analysis confirmed that serum trefoil factor 3 and growth/differentiation factor 15 could provide a discriminatory diagnostic test for predicting colorectal cancer metastasis. Immunohistochemical analysis also showed that the overexpression of trefoil factor 3 or growth/differentiation factor 15 in colorectal cancer was associated with lymph node metastatic behavior. This study showed an accurate, sensitive, and robust label-free quantitation approach for differential analysis of cancer secretome. The comparison of the cancer secretome in vitro is a feasible strategy to obtain valuable biomarkers for potential clinical application. Both trefoil factor 3 and growth/differentiation factor 15 could serve as potential biomarkers for the prediction of colorectal cancer metastasis.
TL;DR: The utility of MALDI MS for the analysis of tumor tissue in the elucidation of aberrant molecular changes in the tumor microenvironment is demonstrated.
Abstract: The rate of tumor recurrence post resection suggests that there are underlying molecular changes in nearby histologically normal tissue that go undetected by conventional diagnostic methods that utilize contrast agents and immunohistochemistry. MALDI MS is a molecular technology that has the specificity and sensitivity to monitor and identify molecular species indicative of these changes. The current study utilizes this technology to assess molecular distributions within a tumor and adjacent normal tissue in clear cell renal cell carcinoma biopsies. Results indicate that the histologically normal tissue adjacent to the tumor expresses many of the molecular characteristics of the tumor. Proteins of the mitochondrial electron transport system are examples of such distributions. This work demonstrates the utility of MALDI MS for the analysis of tumor tissue in the elucidation of aberrant molecular changes in the tumor microenvironment.
TL;DR: A striking characteristic of S. pneumoniae phosphoproteome is the large number of multiple species-specific phosphorylated sites, indicating that high level of protein phosphorylation may play important roles in regulating many metabolic pathways and bacterial virulence.
Abstract: Recent phosphoproteomic characterizations of Bacillus subtilis, Escherichia coli, Lactococcus lactis, Pseudomonas putida, and Pseudomonas aeruginosa have suggested that protein phosphorylation on serine, threonine, and tyrosine residues is a major regulatory post-translational modification in bacteria. In this study, we carried out a global and site-specific phosphoproteomic analysis on the Gram-positive pathogenic bacterium Streptococcus pneumoniae. One hundred and two unique phosphopeptides and 163 phosphorylation sites with distributions of 47%/44%/9% for Ser/Thr/Tyr phosphorylations from 84 S. pneumoniae proteins were identified through the combined use of TiO(2) enrichment and LC-MS/MS determination. The identified phosphoproteins were found to be involved in various biological processes including carbon/protein/nucleotide metabolisms, cell cycle and division regulation. A striking characteristic of S. pneumoniae phosphoproteome is the large number of multiple species-specific phosphorylated sites, indicating that high level of protein phosphorylation may play important roles in regulating many metabolic pathways and bacterial virulence.
TL;DR: A completely novel neuropeptide gene in Nasonia is discovered, coding for peptides containing the C-terminal sequence RYamide, which has orthologs in nearly all arthropods with a sequenced genome and its expression in mosquitoes was confirmed by mass spectrometry.
Abstract: Neuropeptides and protein hormones constitute a very important group of signaling molecules, regulating central physiological processes such as reproduction, development, and behavior. Using a bioi...