Discovery and identification of serum potential biomarkers for pulmonary tuberculosis using iTRAQ‐coupled two‐dimensional LC‐MS/MS

Pulmonary tuberculosis (TB) caused by Mycobacterium tuberculosis is a chronic disease. Currently, there are no sufficiently validated biomarkers for early diagnosis of TB infection. In this study, a panel of potential serum biomarkers was identified between patients with pulmonary TB and healthy controls by using iTRAQ‐coupled 2D LC‐MS/MS technique. Among 100 differentially expressed proteins screened, 45 proteins were upregulated (>1.25‐fold at p < 0.05) and 55 proteins were downregulated (<0.8‐fold at p < 0.05) in the TB serum. Bioinformatics analysis revealed that the differentially expressed proteins were related to the response to stimulus, the metabolic and immune system processes. The significantly differential expression of apolipoprotein CII (APOCII), CD5 antigen‐like (CD5L), hyaluronan‐binding protein 2 (HABP2), and retinol‐binding protein 4 (RBP4) was further confirmed using immunoblotting and ELISA analysis. By forward stepwise multivariate regression analysis, a panel of serum biomarkers including APOCII, CD5L, and RBP4 was obtained to form the disease diagnostic model. The receiver operation characteristic curve of the diagnostic model was 0.98 (sensitivity = 93.42%, specificity = 92.86%). In conclusion, APOCII, CD5L, HABP2, and RBP4 may be potential protein biomarkers of pulmonary TB. Our research provides useful data for early diagnosis of TB.     

Identification of β integrin‐like‐ and fibronectin‐like proteins in the bivalve mollusk Mytilus trossulus

Integrins play a key role in the intermediation and coordination between cells and extracellular matrix components. In this study, we first determined the presence of the β integrin‐like protein and its presumptive ligand, fibronectin‐like protein, during development and in some adult tissues of the bivalve mollusc Mytilus trossulus. We found that β integrin‐like protein expression correlated with the development and differentiation of the digestive system in larvae. Besides the presence of β integrin‐like protein in the digestive epithelial larval cells, this protein was detected in the hemocytes and some adult tissues of M. trossulus. The fibronectin‐like protein was detected firstly at the blastula stage and later, the FN‐LP‐immunoreactive cells were scattered in the trochophore larvae. The fibronectin‐like protein was not expressed in the β integrin‐positive cells of either the veliger stage larvae or the adult mussel tissues and the primary hemocyte cell culture. Despite the β integrin‐ and fibronectin‐like proteins being expressed in different cell types of mussel larvae, we do not exclude the possibility of direct interaction between these two proteins during M. trossulus development or in adult tissues.     

Identification of kaempferol‐regulated proteins in rat calvarial osteoblasts during mineralization by proteomics

Kaempferol, a flavonoid, promotes osteoblast mineralization in vitro and bone formation in vivo; however, its mechanism of action is yet unknown. We adopted proteomic approach to identify the differential effect of kaempferol on rat primary calvarial osteoblasts during mineralization. The primary rat calvarial osteoblasts were treated with kaempferol (5.0 μM) for 9 days under mineralizing condition that resulted in significant increase in alkaline phosphatase activity and mineralization of the cells. Further, 2‐D analysis of the kaempferol‐treated osteoblast lysates revealed 18 differentially expressed proteins (nine upregulated and nine downregulated) on the basis of >/<2.0‐fold as cut‐off (p<0.01) that were then identified by MALDI‐TOF MS. These included cytoskeletal proteins, intracellular signaling protein, chaperone, extracellular matrix protein, and proteins involved in glycolysis and cell–matrix interactions. Proteomics data were confirmed by Western blotting and quantitative real‐time PCR by randomly selecting two upregulated and two downregulated proteins. Western blot analysis confirmed upregulation of HSP‐70 and cytokeratin‐14 levels, and downregulation of aldose reductase and caldesmon expression. We further demonstrated that kaempferol treatment inhibits aldose reductase activity in osteoblasts indicating an altered cellular metabolism by decelerating polyol pathway that was associated with the kaempferol‐induced osteoblast mineralization. In conclusion, this is a first comprehensive study on the differential regulation of proteins by kaempferol in primary osteoblast, which would further help to elucidate the role of the identified proteins in the process of osteoblast mineralization.     

Developmental sex differences in calbindin‐D28K and calretinin immunoreactivity in the neonatal rat hypothalamus

The proteins calbindin‐D_28K and calretinin buffer intracellular calcium and are speculated to be involved in the integration of neuronal signaling. Using Western blot analysis, we compared the levels of calbindin‐D_28K and calretinin in the developing male and female rat hypothalamus on postnatal days (PN) 0, PN2, PN4, PN6, PN8, and PN10. Analysis of variance (ANOVA) of mean calbindin levels indicated a significant effect of sex (p ≤ .001) and age (p ≤ .0001) and a significant interaction (p ≤ .02). Post‐hoc Neuman‐Keuls analysis revealed that PN0 and PN2 males had significantly elevated calbindin levels over PN0 and PN2 females (p ≤ .05). ANOVA of mean calretinin levels from the same animals also indicated a significant effect of sex (p ≤ .002) and a significant interaction between sex and age (p ≤ .001). Post‐hoc analysis indicated males had significantly elevated calretinin levels over PN0, PN4 (p ≤ .05) and PN6 (p ≤ .01) females. Immunocytochemical analyses indicated calbindin‐immunopositive staining for cell bodies in the central subdivision of the medial preoptic nucleus, paraventricular nucleus, arcuate nucleus, and dorsomedial nucleus, and an area immediately surrounding the ventromedial nucleus (VMN). Calbindin immunoreactivity was absent from the ventrolateral VMN, but lightly stained cell bodies were observed in the dorsomedial VMN. The sex differences observed in calcium binding proteins parallel our previously observed sex differences in excitatory γ‐aminobutyric acid and glutamate early in development and may be related to mechanisms of sexual differentiation of the brain. © 2000 John Wiley & Sons, Inc. J Neurobiol 42: 315–322, 2000     

Proteome profiling of aging in mouse models: Differential expression of proteins involved in metabolism,transport, and stress response in kidney

Aging is a time‐dependent complex biological phenomenon observed in various organs and organelles of all living organisms. To understand the molecular mechanism of age‐associated functional loss in aging kidneys, we have analyzed the expression of proteins in the kidneys of young (19–22 wk) and old (24 months) C57/BL6 male mice using 2‐DE followed by LC‐MS/MS. We found that expression levels of 49 proteins were upregulated (p ≤ 0.05), while that of only ten proteins were downregulated (p ≤ 0.05) due to aging. The proteins identified belong to three broad functional categories: (i) metabolism (e.g., aldehyde dehydrogenase family, ATP synthase β‐subunit, malate dehydrogenase, NADH dehydrogenase (ubiquinone), hydroxy acid oxidase 2), (ii) transport (e.g., transferrin), and (iii) chaperone/stress response (e.g., Ig‐binding protein, low density lipoprotein receptor‐related protein associated protein 1, selenium‐binding proteins (SBPs)). Some proteins with unknown functions were also identified as being differentially expressed. ATP synthase β subunit, transferrin, fumarate hydratase, SBPs, and albumin are present in multiple forms, possibly arising due to proteolysis or PTMs. The above functional categories suggest specific mechanisms and pathways for age‐related kidney degeneration.     

Phosphatidylinositol 3‐kinase‐mediated regulation of neuronal apoptosis and necrosis by insulin and IGF‐I

We examined effects of two insulin‐like growth factors, insulin and insulin‐like growth factor‐I (IGF‐I), against apoptosis, excitotoxicity, and free radical neurotoxicity in cortical cell cultures. Like IGF‐I, insulin attenuated serum deprivation‐induced neuronal apoptosis in a dose‐dependent manner at 10–100 ng/mL. The anti‐apoptosis effect of insulin against serum deprivation disappeared by addition of a broad protein kinase inhibitor, staurosporine, but not by calphostin C, a selective protein kinase C inhibitor. Addition of PD98059, a mitogen‐activated protein kinase kinase (MAPKK) inhibitor, blocked insulin‐induced activation of extracellular signal‐regulated protein kinases (ERK1/2) without altering the neuroprotective effect of insulin. Cortical neurons underwent activation of phosphatidylinositol (PI) 3‐kinase as early as 1 min after exposure to insulin. Inclusion of wortmannin or LY294002, selective inhibitors of PI 3‐K, reversed the insulin effect against apoptosis. In contrast to the anti‐apoptosis effect, neither insulin nor IGF‐I protected excitotoxic neuronal necrosis following continuous exposure to 15 μM N‐methyl‐d ‐aspartate or 40 μM kainate for 24 h. Surprisingly, concurrent inclusion of 50 ng/mL insulin or IGF‐I aggravated free radical‐induced neuronal necrosis over 24 h following continuous exposure to 10 μM Fe²⁺ or 100 μM buthionine sulfoximine. Wortmannin or LY294002 also reversed this potentiation effect of insulin. These results suggest that insulin‐ like growth factors act as anti‐apoptosis factor and pro‐oxidant depending uon the activation of PI 3‐kinase. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 536–546, 1999     

Helicobacter pylori proteomics by 2‐DE/MS, 1‐DE‐LC/MS and functional data mining

With its predicted proteome of 1550 proteins (data set Etalon) Helicobacter pylori 26695 represents a perfect model system of medium complexity for investigating basic questions in proteomics. We analyzed urea‐solubilized proteins by 2‐DE/MS (data set 2‐DE) and by 1‐DE‐LC/MS (Supprot); proteins insoluble in 9 M urea but solubilized by SDS (Pellet); proteins precipitating in the Sephadex layer at the application side of IEF (Sephadex) by 1‐DE‐LC/MS; and proteins precipitating close to the application side within the IEF gel by LC/MS (Startline). The experimental proteomics data of H. pylori comprising 567 proteins (protein coverage: 36.6%) were stored in the Proteome Database System for Microbial Research ( http://www.mpiib‐berlin.mpg.de/2D‐PAGE/ ), which gives access to raw mass spectra (MALDI‐TOF/TOF) in T2D format, as well as to text files of peak lists. For data mining the protein mapping and comparison tool PROMPT ( http://webclu.bio.wzw.tum.de/prompt/ ) was used. The percentage of proteins with transmembrane regions, relative to all proteins detected, was 0, 0.2, 0, 0.5, 3.8 and 6.3% for 2‐DE, Supprot, Startline, Sephadex, Pellet, and Etalon, respectively. 2‐DE does not separate membrane proteins because they are insoluble in 9 M urea/70 mM DTT and 2% CHAPS. SDS solubilizes a considerable portion of the urea‐insoluble proteins and makes them accessible for separation by SDS‐PAGE and LC. The 2‐DE/MS analysis with urea‐solubilized proteins and the 1‐DE‐LC/MS analysis with the urea‐insoluble protein fraction (Pellet) are complementary procedures in the pursuit of a complete proteome analysis. Access to the PROMPT‐generated diagrams in the Proteome Database allows the mining of experimental data with respect to other functional aspects.     

Quantification of in vivo oxidative damage in Caenorhabditis elegans during aging by endogenous F3‐isoprostane measurement

Oxidative damage is thought to be a major cause in development of pathologies and aging. However, quantification of oxidative damage is methodologically difficult. Here, we present a robust liquid chromatography–tandem mass spectrometry (LC‐MS/MS) approach for accurate, sensitive, and linear in vivo quantification of endogenous oxidative damage in the nematode Caenorhabditis elegans, based on F3‐isoprostanes. F3‐isoprostanes are prostaglandin‐like markers of oxidative damage derived from lipid peroxidation by Reactive Oxygen Species (ROS). Oxidative damage was quantified in whole animals and in multiple cellular compartments, including mitochondria and peroxisomes. Mutants of the mitochondrial electron transport proteins mev‐1 and clk‐1 showed increased oxidative damage levels. Furthermore, analysis of Superoxide Dismutase (sod) and Catalase (ctl) mutants uncovered that oxidative damage levels cannot be inferred from the phenotype of resistance to pro‐oxidants alone and revealed high oxidative damage in a small group of chemosensory neurons. Longitudinal analysis of aging nematodes revealed that oxidative damage increased specifically with postreproductive age. Remarkably, aging of the stress‐resistant and long‐lived daf‐2 insulin/IGF‐1 receptor mutant involved distinct daf‐16‐dependent phases of oxidative damage including a temporal increase at young adulthood. These observations are consistent with a hormetic response to ROS.     

The N‐terminal tripeptide of insulin‐like growth factor‐I protects against β‐amyloid‐induced somatostatin depletion by calcium and glycogen synthase kinase 3β modulation

The protective effects of insulin‐like growth factor I on the somatostatin (SRIF) system in the temporal cortex after β‐amyloid (Aβ) injury may be mediated through its N‐terminal tripeptide glycine‐proline‐glutamate (GPE). GPE is cleaved to cyclo[Pro‐Gly] (cPG), a metabolite suggested to mediate in neuroprotective actions. We evaluated the effects of GPE and cPG in the temporal cortex of Aβ25–35‐treated rats on SRIF and SRIF receptor protein and mRNA levels, adenylyl cyclase activity, cell death, Aβ25–35 accumulation, cytosolic calcium levels ([Ca²⁺]_c) and the intracellular signaling mechanisms involved. GPE and cPG did not change Aβ25–35 levels, but GPE partially restored SRIF and SRIF receptor 2 protein content and mRNA levels and protected against cell death after Aβ25–35 insult, which was coincident with Akt activation and glycogen synthase kinase 3β inhibition. In addition, GPE displaced glutamate from NMDA receptors and blocked the glutamate induced rise in cytosolic calcium in isolated rat neurons and moderately increased Ca²⁺ influx per se. Our findings suggest that GPE, but not its metabolite, mimics insulin‐like growth factor I effects on the SRIF system through a mechanism independent of Aβ clearance that involves modulation of calcium and glycogen synthase kinase 3β signaling.     

A label‐free internal standard method for the differential analysis of bioactive lupin proteins using nano HPLC‐Chip coupled with Ion Trap mass spectrometry

Quantitative proteomics based on MS is useful for pointing out the differences in some food proteomes relevant to human nutrition. Stable isotope label‐free (SIF) techniques are suitable for comparing an unlimited number of samples by the use of relatively simple experimental workflows. We have developed an internal standard label‐free method based on the intensities of peptide precursor ions from MS/MS spectra, collected in data dependent runs, for the simultaneous qualitative characterization and relative quantification of storage proteins of Lupinus albus seeds in protein extracts of four lupin cultivars (cv Adam, Arés, Lucky, Multitalia). The use of an innovative microfluidic system, the HPLC‐Chip, coupled with a classical IT mass spectrometer, has allowed a complete qualitative characterization of all proteins. In particular, the homology search mode has permitted to identify single amino acid substitutions in the sequences of vicilins (β‐conglutin precursor and vicilin‐like protein). The MS/MS sequencing of substituted peptides confirms the high heterogeneity of vicilins according to the peculiar characteristics of the vicilin‐encoding gene family. Two suitable bioinformatics parameters were optimized for the differential analyses of the main bioactive proteins: the “normalized protein average of common reproducible peptides” (N‐ACRP) for γ‐conglutin, which is a homogeneous protein, and the “normalized protein mean peptide spectral intensity” (N‐MEAN) for the highly heterogenous class of the vicilins.     

Twoplex 12/13C6 aniline stable isotope and linkage‐specific sialic acid labeling 2D‐LC‐MS workflow for quantitative N‐glycomics

Quantitative glycomics represents an actively expanding research field ranging from the discovery of disease‐associated glycan alterations to the quantitative characterization of N‐glycans on therapeutic proteins. Commonly used analytical platforms for comparative relative quantitation of complex glycan samples include MALDI‐TOF‐MS or chromatographic glycan profiling with subsequent data alignment and statistical evaluation. Limitations of such approaches include run‐to‐run technical variation and the potential introduction of subjectivity during data processing. Here, we introduce an offline 2D LC‐MS^E workflow for the fractionation and relative quantitation of twoplex isotopically labeled N‐linked oligosaccharides using neutral ¹²C₆ and ¹³C₆ aniline (Δmass = 6 Da). Additional linkage‐specific derivatization of sialic acids using 4‐(4,6‐dimethoxy‐1,3,5‐trizain‐2‐yl)‐4‐methylmorpholinium chloride offered simultaneous and advanced in‐depth structural characterization. The potential of the method was demonstrated for the differential analysis of structurally defined N‐glycans released from serum proteins of patients diagnosed with various stages of colorectal cancer. The described twoplex ¹²C₆/¹³C₆ aniline 2D LC‐MS platform is ideally suited for differential glycomic analysis of structurally complex N‐glycan pools due to combination and analysis of samples in a single LC‐MS injection and the associated minimization in technical variation.     

Central insulin‐like growth factor‐1 (IGF‐1) restores whole‐body insulin action in a model of age‐related insulin resistance and IGF‐1 decline

Low insulin‐like growth factor‐1 (IGF‐1) signaling is associated with improved longevity, but is paradoxically linked with several age‐related diseases in humans. Insulin‐like growth factor‐1 has proven to be particularly beneficial to the brain, where it confers protection against features of neuronal and cognitive decline. While aging is characterized by central insulin resistance in the face of hyperinsulinemia, the somatotropic axis markedly declines in older humans. Thus, we hypothesized that increasing IGF‐1 in the brain may prove to be a novel therapeutic alternative to overcome central insulin resistance and restore whole‐body insulin action in aging. Utilizing hyperinsulinemic‐euglycemic clamps, we show that old insulin‐resistant rats with age‐related declines in IGF‐1 level demonstrate markedly improved whole‐body insulin action, when treated with central IGF‐1, as compared to central vehicle or insulin (P < 0.05). Furthermore, central IGF‐1, but not insulin, suppressed hepatic glucose production and increased glucose disposal rates in aging rats (P < 0.05). Taken together, IGF‐1 action in the brain and periphery provides a ‘balance’ between its beneficial and detrimental actions. Therefore, we propose that strategies aimed at ‘tipping the balance’ of IGF‐1 action centrally are the optimal approach to achieve healthy aging and longevity in humans.     

Life‐style changes of a halophilic archaeon analyzed by quantitative proteomics

Quantitative proteomics based on isotopic labeling has become the method of choice to accurately determine changes in protein abundance in highly complex mixtures. Isotope‐coded protein labeling (ICPL), which is based on the nicotinoylation of proteins at lysine residues and free N‐termini was used as a simple, reliable and fast method for the comparative analysis of three different cellular states of the halophilic archaeon Halobacterium salinarum through pairwise comparison. The labeled proteins were subjected to SDS‐PAGE, in‐gel digested and the proteolytic peptides were separated by LC and analyzed by MALDI‐TOF/TOF MS. Automated quantitation was performed by comparing the MS peptide signals of ¹²C and ¹³C nicotinoylated isotopic peptide pairs. The transitions between (i) aerobic growth in complex versus synthetic medium and (ii) aerobic versus anaerobic/phototrophic growth, both in complex medium, provide a wide span in nutrient and energy supply for the cell and thus allowed optimal studies of proteome changes. In these two studies, 559 and 643 proteins, respectively, could be quantified allowing a detailed analysis of the adaptation of H. salinarum to changes of its living conditions. The subtle cellular response to a wide variation of nutrient and energy supply demonstrates a fine tuning of the cellular protein inventory.     

Protein profile of Lupinus texensis phloem sap exudates: Searching for Fe‐ and Zn‐containing proteins

The aim of this study was to obtain a comprehensive overview of the phloem sap protein profile of Lupinus texensis, with a special focus on proteins binding Fe and Zn. L. texensis was chosen as model plant given the simplicity to obtain exudates from sieve elements. Protein profiling by 2DE revealed 249 spots, and 54 of them were unambiguously identified by MALDI‐MS and ESI‐MS/MS. The largest number of identified protein species belongs to protein modification/turnover and general metabolism (19–21%), followed by redox homeostasis (9%) and defense and cell structural components (7%). This protein profile is similar to that reported in other plant species, suggesting that the phloem sap proteome is quite conserved. Staining of 2DE gels for Fe‐containing proteins and affinity chromatography experiments revealed the presence of two low molecular weight Fe‐binding proteins in phloem sap: a metallothionein‐like protein type 2B identified in the Fe‐affinity chromatography, and a second protein identified with both Fe staining methods. This protein species had a molecular weight of 13.5 kDa, a pI of 5.6 and 51% homology to a phloem‐specific protein from Medicago truncatula. Zinc affinity chromatography revealed four Zn‐binding proteins in phloem sap, one belonging to the dehydrin family and three Zn finger proteins.     

Spectral Libraries for SWATH‐MS Assays for Drosophila melanogaster and Solanum lycopersicum

Quantitative proteomics methods have emerged as powerful tools for measuring protein expression changes at the proteome level. Using MS‐based approaches, it is now possible to routinely quantify thousands of proteins. However, prefractionation of the samples at the protein or peptide level is usually necessary to go deep into the proteome, increasing both MS analysis time and technical variability. Recently, a new MS acquisition method named SWATH is introduced with the potential to provide good coverage of the proteome as well as a good measurement precision without prior sample fractionation. In contrast to shotgun‐based MS however, a library containing experimental acquired spectra is necessary for the bioinformatics analysis of SWATH data. In this study, spectral libraries for two widely used models are built to study crop ripening or animal embryogenesis, Solanum lycopersicum (tomato) and Drosophila melanogaster, respectively. The spectral libraries comprise fragments for 5197 and 6040 proteins for S. lycopersicum and D. melanogaster, respectively, and allow reproducible quantification for thousands of peptides per MS analysis. The spectral libraries and all MS data are available in the MassIVE repository with the dataset identifiers MSV000081074 and MSV000081075 and the PRIDE repository with the dataset identifiers PXD006493 and PXD006495.     

Gene expression associated with indicated faster growth of crossbreeding in Sinocyclocheilus grahami

In this study, pure‐breeding and crossbreeding of Sinocyclocheilus grahami were established to test heterosis and expression of growth‐related genes which were examined in the first‐generation (F1). Genetic distances results showed that Muyang river (M) and Sanbaimu reservoir (S) populations had smaller interspecific distances (genetic distance: 0.000) while larger distances were shown to other two populations (0.024 of Jiuzhai vauclusian spring [J] and 0.044 of Chenglong vauclusian spring [C]). Full‐sib families were created by using the Muyang river population as females: MM, MS, MJ and MC. Growth performance had significant differences between each group of F1 hybrids; the fastest growth group was shown in MJ while the slowest was in MS. For growth‐related genes, the fast growth group of MJ hybrids showed significantly higher levels of ghrelin expression in strong expression tissues of the intestine and liver. Among the four combinations, mRNA levels of insulin‐like growth factor I (IGF‐I) were the highest expression in MJ hybrids than others in all tested tissues. Myostatin b and somatostatin1 (SST1) were strongly expressed in the brain and MJ hybrids showed relatively lower expression level. In the muscle, slow growth group of MS hybrids showed extremely higher expression levels of these two genes, especially SST1 gene. In conclusion, associating growth with growth‐related genes expression indicated faster growth of crossbreeding in S. grahami and can make a contribution to commercial fish production in the future.     

General method for selective labelling of double‐chain cysteine‐rich peptides with a lanthanide chelate via solid‐phase synthesis

The use of lanthanides in preference to radioisotopes as probes for various biological assays has gained enormous popularity. The introduction of lanthanide chelates to peptides/proteins can be carried out either in solution using a commercially available labelling kit or by solid‐phase peptide synthesis using an appropriate lanthanide chelate. Herein, a detailed protocol for the latter is provided for the labelling of peptides or small proteins with diethylenetriamine‐N, N, N″, N″‐tetra‐tert‐butyl acetate‐N′‐acetic acid (DTPA) chelate or other similar chelates on a solid support using a chimeric insulin‐like peptide composed of human insulin‐like peptide 5 (INSL5) A‐chain and relaxin‐3 B‐chain as a model peptide. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.