Method development for proteome stabilization in human saliva

Human saliva is a biological fluid with emerging early detection and diagnostic potentials. However, the salivary proteome suffers from rapid degradation and thus compromises its translational and clinical utilities. Therefore, easy, reliable and practical methods are urgently required for the storage of human saliva samples. In this study, saliva samples from healthy subjects were collected and stored at room temperature (RT) and 4 °C for different lengths of time with and without specific protein stabilization treatments. SDS-PAGE was run to compare the protein profiling between samples. Reference proteins, β-actin and interleukin-1 β (IL1β), were chosen to evaluate salivary protein stability. Immunoassay was used for the detection of these target proteins. All data was compared with the positive control that had been kept at −80 °C. The results show that the salivary proteome that has been stored at 4 °C with added protease inhibitors was stable for approximately two weeks without significant degradation. By adding ethanol to the samples, the salivary proteome was stabilized at RT. After optimization, a simple, robust and convenient method is developed for the stabilization of proteins in human saliva that does not affect the downstream translational and clinical applications. The salivary proteome could be stabilized without significant degradation by adding ethanol at RT for about two weeks. This optimized method could greatly accelerate the clinical usage of saliva for future diagnosis.     

纳升液相色谱-高分辨串联质谱研究冻存条件对唾液多肽组的影响

唾液多肽组学为疾病相关的生物标记物研究提供了新方法,但冻存条件对分析结果的影响并不清楚。本研究采用氧化石墨烯-磷酸镧纳米复合材料分离富集唾液多肽,利用纳升液相色谱-高分辨串联质谱技术,考察唾液样品分别置于-80℃与-20℃冻存6个月后对唾液多肽组的影响。结果表明,在-80℃冻存条件下的唾液样品中,共鉴定出归属于33种蛋白的429条肽段;在-20℃冻存条件下的唾液样品中,鉴定出595条肽段,对应31种蛋白。实验结果表明,相比于-80℃,唾液置于-20℃条件下的新增加肽段主要来源于已有肽段的降解,并且唾液中的蛋白质也发生了一定降解。本研究在肽段序列水平上考察了冻存条件对多肽的影响,结果表明,-20℃冻存条件不适合长期保存用于多肽组分析的唾液样品。本研究结果可为相关医学研究提供借鉴。     

Salivary peptidome in type 1 diabetes mellitus

Diabetic patients show a high susceptibility to oral diseases of inflammatory, catabolic and chronic nature with potential impact on saliva composition. In this study, our purpose was to characterize type 1 diabetes‐induced alterations in the salivary peptidome aiming to find prospective biomarkers for type 1 diabetes oral health evaluation. Peptidomic analysis of saliva from controls (n = 5) and type 1 diabetic patients (n = 5) were performed by liquid chromatography followed by mass spectrometry. The proteolytic activity and metalloproteinases expression was accessed by zymography and slot blot analysis, respectively. Data evidenced a significant increase in the percentage of peptides in diabetic patients paralleled by a higher proteolytic activity, compared with healthy individuals. The nonsalivary gland protein fragments identified in saliva were mainly derived from collagen and extracellular matrix proteins, namely collagen type I. The cleavage site frequency analysis showed significant differences between healthy and type 1 diabetic individuals, highlighting the activity of proteases such as matrix metalloproteinase‐9 and cathepsin D. Our results highlight salivary collagen fragments as potential biomarkers to follow up diabetes‐related oral damage. Copyright © 2011 John Wiley & Sons, Ltd.     

Functional Analysis of Autoantibody Signatures in Rheumatoid Arthritis

For the identification of antigenic protein biomarkers for rheumatoid arthritis (RA), we conducted IgG profiling on high density protein microarrays. Plasma IgG of 96 human samples (healthy controls, osteoarthritis, seropositive and seronegative RA, n = 24 each) and time-series plasma of a pristane-induced arthritis (PIA) rat model (n = 24 total) were probed on AIT’s 16k protein microarray. To investigate the analogy of underlying disease pathways, differential reactivity analysis was conducted. A total of n = 602 differentially reactive antigens (DIRAGs) at a significance cutoff of p < 0.05 were identified between seropositive and seronegative RA for the human samples. Correlation with the clinical disease activity index revealed an inverse correlation of antibodies against self-proteins found in pathways relevant for antigen presentation and immune regulation. The PIA model showed n = 1291 significant DIRAGs within acute disease. Significant DIRAGs for (I) seropositive, (II) seronegative and (III) PIA were subjected to the Reactome pathway browser which also revealed pathways relevant for antigen presentation and immune regulation; of these, seven overlapping pathways had high significance. We therefore conclude that the PIA model reflects the biological similarities of the disease pathogenesis. Our data show that protein array analysis can elucidate biological differences and pathways relevant in disease as well be a useful additional layer of omics information.     

Double electrophoretic separation and lectin analyses of the component chains of secretory immunoglobulin A from human saliva

A new method is presented for the separation of secretory immunoglobulin A (SIgA) from salivary samples. Salivary proteins (from parotid or stimulated whole mouth saliva) were precipitated with methanol to concentrate SIgA from salivary samples whilst removing other salivary proteins. SIgA purified from breast milk and salivary proteins was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions. Following completion of electrophoresis the top strip of gel was removed and the proteins present reduced with dithiothreitol. The gel strip was then applied to the top of a second 10% SDS gel, and the proteins were electrophoresed and then stained by Coomassie Brilliant Blue R-250. Three major protein bands were stained in all samples corresponding in molecular mass to secretory component, alpha-heavy chain and light chains of SIgA. Separated proteins were also electroblotted onto nitrocellulose and stained by fluorescein isothiocyanate (FITC). Lectin analysis was then used to detect the O-glycans present on IgA1. Lectins from Helix aspersa and Arachis hypogaea were used to determine the amount of terminal N-acetyl galactosamine and nonsialylated O-glycans, respectively. Maclura pomifera lectin was used to determine the total amount of IgA1 present on the blots. The results indicate that SlgA in stimulated whole mouth saliva, stimulated parotid saliva and purified from breast milk contain similar O-glycans.     

Discovery of the serum biomarker proteins in severe preeclampsia by proteomic analysis

Preeclapsia (PE) is a severe disorder that occurs during pregnancy, leading to maternal and fetal morbidity and mortality. PE affects about 3-8% of all pregnancies. In this study, we conducted liquid chromatography- mass spectrometry/mass spectrometry (LC-MS/MS) to analyze serum samples depleted of the six most abundant proteins from normal and PE-affected pregnancies to profile serum proteins. A total of 237 proteins were confidently identified with <1% false discovery rate from the two groups of duplicate analysis. The expression levels of those identified proteins were compared semiquantitatively by spectral counting. To further validate the candidate proteins with a quantitative mass spectrometric method, selective reaction monitoring (SRM) and enzyme linked immune assay (ELISA) of serum samples collected from pregnant women with severe PE (n = 8) or normal pregnant women (n = 5) was conducted. α2- HS-glycoprotein (AHSG), retinol binding protein 4 (RBP4) and α-1-microglobulin/bikunin (AMBP) and Insulin like growth factor binding protein, acid labile subunit (IGFBP-ALS) were confirmed to be differentially expressed in PE using SRM (P<0.05). Among these proteins, AHSG was verified by ELISA and showed a statistically significant increase in PE samples when compared to controls.     

High Expression Level of α2-3-Linked Sialic Acids on Salivary Glycoproteins of Breastfeeding Women May Help to Protect Them from Avian Influenza Virus Infection

Terminal sialic acids (Sia) on soluble glycoprotein of saliva play an important role in the clearance of influenza virus. The aim of this study is to investigate the alteration of sialylation on the salivary proteins of women during the lactation period and its effect on the saliva binding ability to virus. In total, 210 saliva samples from postpartum women with and without breastfeeding were collected, and the expression level of α2-3/6-linked Sia on the whole salivary proteins and specific glycoproteins of IgA and MUC5B from different groups were tested and verified using lectin microarray, blotting analysis and ELISA based method. The H1N1 vaccine and three strains of Avian influenza virus (AIV) were used for the saliva binding assay. Results showed that the variation in salivary expression level of α2-3-linked Sia was much more obvious than the α2-6-linked Sia, which was up-regulated significantly in the breastfeeding groups compared to the non-breastfeeding groups at the same postpartum stage. Furthermore, the binding abilities of salivary glycoproteins to AIV strains and H1N1 vaccine were increased in breastfeeding groups accordingly. This finding adds new evidence for the maternal benefit of breastfeeding and provides new thinking to protect postpartum women from AIV infection.     

Synergistic Effect by Combining a gp120-Binding Protein and a gp41-Binding Antibody to Inactivate HIV-1 Virions and Inhibit HIV-1 Infection

Acquired immune deficiency syndrome (AIDS) has prevailed over the last 30 years. Although highly active antiretroviral therapy (HAART) has decreased mortality and efficiently controlled the progression of disease, no vaccine or curative drugs have been approved until now. A viral inactivator is expected to inactivate cell-free virions in the absence of target cells. Previously, we identified a gp120-binding protein, mD1.22, which can inactivate laboratory-adapted HIV-1. In this study, we have found that the gp41 N-terminal heptad repeat (NHR)-binding antibody D5 single-chain variable fragment (scFv) alone cannot inactivate HIV-1 at the high concentration tested. However, D5 scFv in the combination could enhance inactivation activity of mD1.22 against divergent HIV-1 strains, including HIV-1 laboratory-adapted strains, primary HIV-1 isolates, T20- and AZT-resistant strains, and LRA-reactivated virions. Combining mD1.22 and D5 scFv exhibited synergistic effect on inhibition of infection by divergent HIV-1 strains. These results suggest good potential to develop the strategy of combining a gp120-binding protein and a gp41-binding antibody for the treatment of HIV-1 infection.     

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of human parotid salivary proteins.

The proteins in human parotid saliva have been separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis into 20 or more well resolved species. The Coomassie Brilliant Blue (CBB) R-250 and silver staining procedures have been modified to overcome the problems encountered with staining of proline-rich proteins. By means of the CBB R-250 procedure which stains proline-rich proteins pink-violet, immunoblotting, concanavalin A binding, periodate-Schiff staining and zinc binding, all of the major proteins have been characterised. Substantial individual-to-individual differences were observed in the protein patterns formed. Comparison of parotid, submandibular, and whole saliva from a single individual indicated that fewer proline-rich proteins are expressed in submandibular saliva than in parotid, but whole saliva contains much lower levels than either duct secretion. The results will form a useful base for future research into the functions of salivary proteins.     

Label-free quantification of differentially expressed proteins in mouse liver cancer cells with high and low metastasis rates by a SWATH acquisition method

Label-free quantification is a valuable tool for the analysis of differentially expressed proteins identified by mass spectrometry methods.Herein,we used a new strategy:data-dependent acquisition mode identification combined with label-free quantification by SWATH acquisition mode,to study the differentially expressed proteins in mouse liver cancer metastasis cells.A total of 1528 protein groups were identified,among which 1159 protein groups were quantified and 249 protein groups were observed as differentially expressed proteins(86 proteins up-regulated and 163 down-regulated).This method provides a commendable solution for the identification and quantification of differentially expressed proteins in biological samples.     

Isoelectric focusing of human parotid salivary proteins in hybrid carrier ampholyte-immobilized pH gradient polyacrylamide gels.

Isoelectric focusing of human salivary proteins with carrier ampholyte-isoelectric focusing systems requires prior desalting and concentration of samples, a procedure which is time-consuming and requires relatively large volumes of samples. By contrast, immobilized pH gradient gels are more tolerant to salt loads. Thus pretreatment of samples consists only of centrifugation prior to isoelectric focusing. If larger loads (greater than 50 micrograms) are required, the samples may be concentrated by lyophilization and reconstitution in a smaller volume of water or by dialysis against 30% w/v polyethylene glycol. Immobilized pH gradient polyacrylamide gels (incorporating a hybrid carrier ampholyte system) of two pH ranges (pH 4-9 and pH 3.5-5.0) have been used to separate the proteins in human parotid saliva. The effects of urea on focused patterns were studied; in pH 4-9 gels it gave improved resolution of protein bands, whereas in pH 3.5-5.0 gels it prevented protein precipitation. The salivary proteins were then visualized by staining with Coomassie Brilliant Blue G-250 or a silver procedure. Using the latter, 25-30 well-resolved bands were formed on a pH 4-9 gel loaded with 20 micrograms of proteins. The method offers considerable advantages compared with carrier ampholyte-isoelectric focusing.     

Proteins involved in neuronal differentiation of neuroblastoma cell line N1E-115

Neuronal differentiation (ND) represents a well-defined phenomenon in biological terms but proteins involved have not been studied systematically. We therefore aimed to study ND by retinoic acid (RA) in a widely used neuroblastoma cell line by comparative proteomics. The ND was induced in the N1E-115 cell line by serum deprivation and RA treatment. Undifferentiated cells and cells undergoing serum deprivation served as controls. Protein extracts were run on 2-DE followed by MALDI-TOF or MALDI-TOF-TOF analysis. Quantification was carried out using specific software and stringent statistical analysis was performed. Tubulin beta 5, cat eye syndrome critical region protein 5 homolog, putative GTP-binding protein PTD004 homolog, and the metabolic proteins glyceraldehyde-3-phosphate dehydrogenase and transketolase were differentially regulated. Differential protein levels of cytoskeleton proteins including tubulins and metabolic proteins have been reported to be regulated by ND. Herein, specific signaling differences as reflected by putative GTP-binding protein PTD004 changes in differentiated cells are shown and a possible role for the Cat eye syndrome critical region protein 5 homolog is proposed. The protein disulfide isomerase associated 3 protein fits the already proposed findings of chaperon regulation by ND. The study forms the molecular basis for further evaluation of the functional roles of the differentially expressed proteins in ND.     

Serum Proteomic Analysis of Cannabis Use Disorder in Male Patients

Cannabis use has been growing recently and it is legally consumed in many countries. Cannabis has a variety of phytochemicals including cannabinoids, which might impair the peripheral systems responses affecting inflammatory and immunological pathways. However, the exact signaling pathways that induce these effects need further understanding. The objective of this study is to investigate the serum proteomic profiling in patients diagnosed with cannabis use disorder (CUD) as compared with healthy control subjects. The novelty of our study is to highlight the differentially changes proteins in the serum of CUD patients. Certain proteins can be targeted in the future to attenuate the toxicological effects of cannabis. Blood samples were collected from 20 male individuals: 10 healthy controls and 10 CUD patients. An untargeted proteomic technique employing two-dimensional difference in gel electrophoresis coupled with mass spectrometry was employed in this study to assess the differentially expressed proteins. The proteomic analysis identified a total of 121 proteins that showed significant changes in protein expression between CUD patients (experimental group) and healthy individuals (control group). For instance, the serum expression of inactive tyrosine protein kinase PEAK1 and tumor necrosis factor alpha-induced protein 3 were increased in CUD group. In contrast, the serum expression of transthyretin and serotransferrin were reduced in CUD group. Among these proteins, 55 proteins were significantly upregulated and 66 proteins significantly downregulated in CUD patients as compared with healthy control group. Ingenuity pathway analysis (IPA) found that these differentially expressed proteins are linked to p38MAPK, interleukin 12 complex, nuclear factor-κB, and other signaling pathways. Our work indicates that the differentially expressed serum proteins between CUD and control groups are correlated to liver X receptor/retinoid X receptor (RXR), farnesoid X receptor/RXR activation, and acute phase response signaling.     

基于凝集素的唾液糖蛋白分离、分析及应用

唾液中的糖蛋白丰度偏低,给分离、分析带来挑战。该文采用麦胚素（WGA）和橙黄网胞盘菌凝集素（AAL）分别富集糖蛋白,考察了高丰度蛋白质去除和不同酶解方式对糖蛋白分离、分析的影响。结果显示,WGA和AAL提取的唾液糖蛋白经胶内酶解可鉴定到的糖蛋白数量显著多于溶液内酶解的结果,也优于去除高丰度蛋白质后的鉴定结果。选择WGA结合胶内酶解进一步对比分析肺癌患者与健康人唾液糖蛋白的差异,通过免标记定量分析共鉴定到139个蛋白质,其中102个蛋白质存在糖基化位点,包括14个在癌症组和正常组之间存在显著差异（p<0.05）的糖蛋白,表明该策略可用于唾液糖蛋白的有效分离、分析和癌症标志物的发现。     

Biomarker discovery in breast cancer serum using 2-D differential gel electrophoresis/ MALDI-TOF/TOF and data validation by routine clinical assays

In the present study, we used 2-D differential gel electrophoresis (2-D DIGE) and MS to screen biomarker candidates in serum samples obtained from 39 patients with breast cancer and 35 controls. First, we pooled the serum samples matched with age and menopausal status. Then, we depleted the two most abundant proteins albumin and IgG by immunoaffinity chromatography under partly denaturing conditions in order to enrich low-abundance proteins and proteins with low molecular weight. Concentrated and desalted samples were labeled with three different CyDyes including one internal standard, pooled from all the samples, and separated with 2-D DIGE in triplicate experiments. Biological variations of the protein expression level were analyzed with DeCyder software and evaluated for reproducibility and statistical significance. The profile of differentially expressed protein spots between patients and controls revealed proapolipoprotein A-I, transferrin, and hemoglobin as up-regulated and three spots, apolipoprotein A-I, apolipoprotein C-III, and haptoglobin alpha2 as down-regulated in patients. Finally, routine clinical immunochemical reactions were used to validate selected candidate biomarkers by quantitative determination of specific proteins in all individual serum samples. The serum level of transferrin correlated well with the 2-D-DIGE results. However, the serum levels of apolipoprotein A-I and haptoglobin could not be detected with the clinical routine diagnostic tests. This demonstrated an advantage 2-D DIGE still has over other techniques. 2-D DIGE can distinguish between isoforms of proteins, where the overall immunochemical quantification does fail due to a lack of isoform-special antibodies.     

Evaluation of Anti-Biofilm Activity of Mouthrinses Containing Tannic Acid or Chitosan on Dentin In Situ

In contrast to enamel, dentin surfaces have been rarely used as substrates for studies evaluating the effects of experimental rinsing solutions on oral biofilm formation. The aim of the present in situ study was to investigate the effects of tannic acid and chitosan on 48-h biofilm formation on dentin surfaces. Biofilm was formed intraorally on dentin specimens, while six subjects rinsed with experimental solutions containing tannic acid, chitosan and water as negative or chlorhexidine as positive control. After 48 h of biofilm formation, specimens were evaluated for biofilm coverage and for viability of bacteria by fluorescence and scanning electron microscopy. In addition, saliva samples were collected after rinsing and analyzed by fluorescence (five subjects) and transmission electron microscopy (two subjects) in order to investigate the antibacterial effect on bacteria in a planktonic state and to visualize effects of the rinsing agents on salivary proteins. After rinsing with water, dentin specimens were covered by a multiple-layered biofilm with predominantly vital bacteria. In contrast, chlorhexidine led to dentin surfaces covered only by few and avital bacteria. By rinsing with tannic acid both strong anti-adherent and antibacterial effects were observed, but the effects declined in a time-dependent manner. Transmission electron micrographs of salivary samples indicated that aggregation of proteins and bacteria might explain the antiadhesion effects of tannic acid. Chitosan showed antibacterial effects on bacteria in saliva, while biofilm viability was only slightly reduced and no effects on bacterial adherence on dentin were observed, despite proteins being aggregated in saliva after rinsing with chitosan. Tannic acid is a promising anti-biofilm agent even on dentin surfaces, while rinsing with chitosan could not sufficiently prevent biofilm formation on dentin.     

How Different Snacks Produce a Distinct Effect in Salivary Protein Composition

Saliva secretion changes in response to different stimulation. Studies performed in animals and humans suggest that dietary constituents may influence saliva composition, although the dynamics of these changes, and how they are specific for each type of food, are little known. The objective of the present study was to access the short-term effects of different foods in salivation and salivary protein composition. Twelve participants were tested for four snacks (yoghurt, bread, apple and walnuts). Non-stimulated saliva was collected before and at 0′, 5′ and 30′ after each snack intake. Flow rate, total protein, alpha-amylase enzymatic activity and salivary protein profile were analyzed. Yoghurt and apple were the snacks resulting in higher salivary changes, with higher increases in flow rate and alpha-amylase activity immediately after intake. The expression levels of immunoglobulin chains decreased after the intake of all snacks, whereas cystatins and one pink band (proline-rich proteins—PRPs) increased only after yoghurt intake. Walnut’s snack was the one resulting in lower changes, probably due to lower amounts eaten. Even so, it resulted in the increase in one PRPs band. In conclusion, changes in saliva composition varies with foods, with variable changes in proteins related to oral food processing and perception.     

Incorporating protein flexibility in structure-based drug discovery: using HIV-1 protease as a test case

We have developed a receptor-based pharmacophore method which utilizes a collection of protein structures to account for inherent protein flexibility in structure-based drug design. Several procedures were systematically evaluated to derive the most general protocol for using multiple protein structures. Most notably, incorporating more protein flexibility improved the performance of the method. The pharmacophore models successfully discriminate known inhibitors from drug-like non-inhibitors. Furthermore, the models correctly identify the bound conformations of some ligands. We used unliganded HIV-1 protease to develop and validate this method. Drug design is always initiated with a protein-ligand structure, and such success with unbound protein structures is remarkable - particularly in the case of HIV-1 protease, which has a large conformational change upon binding. This technique holds the promise of successful computer-based drug design before bound crystal structures are even discovered, which can mean a jump-start of 1-3 years in tackling some medically relevant systems with computational methods.     

分散液-液微萃取结合离子迁移谱测定人体唾液中的苯海拉明

建立一种结合便携式高分辨离子迁移谱(IMS)快速简便萃取唾液中的苯海拉明(DPH)的分散液-液微萃取法(DLLME).取2 mL唾液,采用10％(w:V)硫酸锌沉淀蛋白,以2 mL二氯甲烷为萃取剂对唾液中的苯海拉明进行萃取,萃取时间5 min,离心(3500 r/min,20 min),取二氯甲烷层,45℃真空干燥,甲...     

Application of thiophilic chromatography to deplete serum immunoglobulins in sample preparation for bidimensional electrophoresis

Serum is a typical sample for non-invasive studies in clinical research. Its proteome characterization is challenging, since requires extensive protein depletion. Methods used nowadays for removal of high-abundance proteins are expensive or show quite often a low loading capacity, which has strong repercussions on the number of samples and replicates per analysis.In order to deplete immunoglobulins (Igs) and albumin (HSA) from 1 mL serum samples, we have developed a protocol based on a combination of thiophilic chromatography, not previously used in clinical proteomics, and a HSA-specific resin. Ig/HSA-depleted samples, immunoglobulinome and albuminone were analyzed by 2-DE. Thiophilic chromatography, coupled with HSA-depletion, allows a good 2-DE resolution as well as the visualization of new spots. Moreover, it yields enough protein to evaluate technical variability and facilitate subsequent protein identification. To validate the protocol, we carried out a preliminary comparative study between triplicate Igs/HSA-depleted serum samples from healthy control individuals and recently diagnosed/untreated rheumatoid arthritis (RA) patients. RA patients showed several acute phase proteins, as well as additional serum proteins, differentially and significantly regulated.Therefore, thiophilic chromatography can be used as an efficient and economical method in 2-DE to deplete immunoglobulins from large human serum samples before a more extensive fractioning.