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1.  Serum proteome changes in acromegalic patients following transsphenoidal surgery: novel biomarkers of disease activity 
Transsphenoidal adenomectomy is the primary treatment for acromegaly. However, assessment of the therapeutical outcome remains problematic since the existing biomarkers of disease activity frequently show discordant results.
To discover novel serum biomarkers of disease activity in acromegalic patients before and after surgery.
Serum samples of eight newly diagnosed acromegaly patients before and after transsphenoidal surgery were analyzed for proteomic changes by two-dimensional gel electrophoresis. Protein spots displaying statistically significant changes, pre- versus post-surgery, were identified by mass spectrometry (MS), tandem MS (MS/MS), and western blot analysis.
Six protein spots displaying decreased intensities after surgery were identified as transthyretin (two isoforms), haptoglobin a2, b-hemoglobin, and apolipoprotein A-1 (two isoforms). One protein spot, identified as complement C4B precursor, was increased after the surgery.
Seven serum protein spots were differentially expressed following surgery in acromegalic patients. The identified proteins represent potential novel biomarkers to assess the effectiveness of surgical treatment in acromegalic individuals. Future studies will validate the use of the identified proteins as biomarkers of disease activity after medical treatment of acromegaly.
PMCID: PMC4074021  PMID: 21059862
2.  Identification of Fibrin Clot-Bound Plasma Proteins 
PLoS ONE  2012;7(8):e41966.
Several proteins are known to bind to a fibrin network and to change clot properties or function. In this study we aimed to get an overview of fibrin clot-bound plasma proteins. A plasma clot was formed by adding thrombin, CaCl2 and aprotinin to citrated platelet-poor plasma and unbound proteins were washed away with Tris-buffered saline. Non-covalently bound proteins were extracted, separated with 2D gel electrophoresis and visualized with Sypro Ruby. Excised protein spots were analyzed with mass spectrometry. The identity of the proteins was verified by checking the mass of the protein, and, if necessary, by Western blot analysis. Next to established fibrin-binding proteins we identified several novel fibrin clot-bound plasma proteins, including α2-macroglobulin, carboxypeptidase N, α1-antitrypsin, haptoglobin, serum amyloid P, and the apolipoproteins A-I, E, J, and A-IV. The latter six proteins are associated with high-density lipoprotein particles. In addition we showed that high-density lipoprotein associated proteins were also present in fibrinogen preparations purified from plasma. Most plasma proteins in a fibrin clot can be classified into three groups according to either blood coagulation, protease inhibition or high-density lipoprotein metabolism. The presence of high-density lipoprotein in clots might point to a role in hemostasis.
PMCID: PMC3411686  PMID: 22870270
3.  Identification of human plasma proteins associated to the cell wall of the pathogenic fungus Paracoccidioides brasiliensis 
FEMS microbiology letters  2013;341(2):87-95.
Paracoccidioides brasiliensis and P. lutzii are thermodimorphic species that cause paracoccidioidomycosis. The cell wall is the outermost fungal organelle to form an interface with the host. A number of host effector compounds, including immunologically active molecules, circulate in the plasma. In the present work we extracted cell wall-associated proteins from the yeast pathogenic phase of P. brasiliensis, isolate Pb3, grown in the presence of human plasma, and analyzed bound plasma proteins by liquid chromatography-tandem mass spectrometry. Transport, complement activation/regulation and coagulation pathway were the most abundant functional groups identified. Proteins related to iron/copper acquisition, immunoglobulins, and protease inhibitors were also detected. Several human plasma proteins described here have not been previously reported as interacting with fungal components, specifically, clusterin, hemopexin, transthyretin, ceruloplasmin, alpha-1-antitrypsin, apolipoprotein A-I, and apolipoprotein B-100. Additionally, we observed increased phagocytosis by J774.16 macrophages of Pb3 grown in plasma, suggesting that plasma proteins interacting with P. brasiliensis cell wall might be interfering in the fungal relationship with the host.
PMCID: PMC3619194  PMID: 23398536
Paracoccidioides brasiliensis; cell wall; human plasma proteins
4.  Discovery of pathway biomarkers from coupled proteomics and systems biology methods 
BMC Genomics  2010;11(Suppl 2):S12.
Breast cancer is worldwide the second most common type of cancer after lung cancer. Plasma proteome profiling may have a higher chance to identify protein changes between plasma samples such as normal and breast cancer tissues. Breast cancer cell lines have long been used by researches as model system for identifying protein biomarkers. A comparison of the set of proteins which change in plasma with previously published findings from proteomic analysis of human breast cancer cell lines may identify with a higher confidence a subset of candidate protein biomarker.
In this study, we analyzed a liquid chromatography (LC) coupled tandem mass spectrometry (MS/MS) proteomics dataset from plasma samples of 40 healthy women and 40 women diagnosed with breast cancer. Using a two-sample t-statistics and permutation procedure, we identified 254 statistically significant, differentially expressed proteins, among which 208 are over-expressed and 46 are under-expressed in breast cancer plasma. We validated this result against previously published proteomic results of human breast cancer cell lines and signaling pathways to derive 25 candidate protein biomarkers in a panel. Using the pathway analysis, we observed that the 25 “activated” plasma proteins were present in several cancer pathways, including ‘Complement and coagulation cascades’, ‘Regulation of actin cytoskeleton’, and ‘Focal adhesion’, and match well with previously reported studies. Additional gene ontology analysis of the 25 proteins also showed that cellular metabolic process and response to external stimulus (especially proteolysis and acute inflammatory response) were enriched functional annotations of the proteins identified in the breast cancer plasma samples. By cross-validation using two additional proteomics studies, we obtained 86% and 83% similarities in pathway-protein matrix between the first study and the two testing studies, which is much better than the similarity we measured with proteins.
We presented a ‘systems biology’ method to identify, characterize, analyze and validate panel biomarkers in breast cancer proteomics data, which includes 1) t statistics and permutation process, 2) network, pathway and function annotation analysis, and 3) cross-validation of multiple studies. Our results showed that the systems biology approach is essential to the understanding molecular mechanisms of panel protein biomarkers.
PMCID: PMC2975409  PMID: 21047379
5.  Proteome analysis of human cerebrospinal fluid as a diagnostic biomarker in patients with meningioma 
To identify meningioma-specific proteins, cerebrospinal fluid (CSF) from 4 patients with a meningioma and 4 patients with a non-brain tumorous lesion were analyzed.
Two-dimensional electrophoresis and electrospray quadrupole time-of-flight tandem mass spectrometry analyses revealed 10 unique spots, containing 11 independent proteins (spot #2 and #4 each contained 2 proteins and spot #3 was not identified) were evident in CSF associated with human meningioma: serum albumin precursor (3 different isoforms), Apolipoprotein E (Apo E), Apolipoprotein J precursor (Apo J), Transthyretin precursor (TTR), Prostaglandin D2 synthase 21kDa (PTGDS), proapolipoprotein, Chain D hemoglobin Ypsilanti, alpha-1-antitrypsin (AAT), and beta-2-microglobulin precursor (β2M).
The contents of Apo E, Apo J and AAT were increased, while PTGDS, TTR and β2M were decreased.
The results observed by 2-dimensional electrophoresis were verified by Western blot analysis. The unique proteins may represent possible candidate biomarkers of meningioma.
PMCID: PMC3560610  PMID: 23111736
meningioma; human cerebrospinal fluid; proteome; biomarker; brain tumor
6.  Comparative Proteome Analyses of Human Plasma Following in vivo Lipopolysaccharide Administration Using Multidimensional Separations Coupled with Tandem Mass Spectrometry 
Proteomics  2005;5(2):572-584.
There is significant interest in characterization of the human plasma proteome due to its potential for providing biomarkers applicable to clinical diagnosis and treatment and for gaining a better understanding of human diseases. We describe here a strategy for the comparative proteome analyses of human plasma, which is applicable to biomarker identifications for various disease states. Multidimensional liquid chromatography-mass spectrometry has been applied to make comparative proteome analyses of plasma samples from an individual prior to and 9 h after lipopolysaccharide (LPS) administration. Peptide peak areas and the number of peptide identifications for each protein were used to evaluate the reproducibility of LC-MS/MS and to compare relative changes in protein concentration between the samples following LPS treatment. A total of 804 distinct plasma proteins (not including immunoglobulins) were confidently identified with 32 proteins observed to be significantly increased in concentration following LPS administration, including several known inflammatory response or acute-phase mediators such as C-reactive protein, serum amyloid A and A2, LPS-binding protein, LPS-responsive and beige-like anchor protein, hepatocyte growth factor activator, and von Willebrand factor, and thus constituting potential biomarkers for inflammatory response.
PMCID: PMC1781926  PMID: 15627965
human plasma; proteomics; lipopolysaccharide; comparative analysis; LC-MS/MS
7.  Carbonic Anhydrase I as a New Plasma Biomarker for Prostate Cancer 
ISRN Oncology  2012;2012:768190.
Serum prostate-specific antigen (PSA) levels ranging from 4 to 10 ng/mL is considered a diagnostic gray zone for detecting prostate cancer because biopsies reveal no evidence of cancer in 75% of these subjects. Our goal was to discover a new highly specific biomarker for prostate cancer by analyzing plasma proteins using a proteomic technique. Enriched plasma proteins from 25 prostate cancer patients and 15 healthy controls were analyzed using a label-free quantitative shotgun proteomics platform called 2DICAL (2-dimensional image converted analysis of liquid chromatography and mass spectrometry) and candidate biomarkers were searched. Among the 40,678 identified mass spectrum (MS) peaks, 117 peaks significantly differed between prostate cancer patients and healthy controls. Ten peaks matched carbonic anhydrase I (CAI) by tandem MS. Independent immunological assays revealed that plasma CAI levels in 54 prostate cancer patients were significantly higher than those in 60 healthy controls (P = 0.022, Mann-Whitney U test). In the PSA gray-zone group, the discrimination rate of prostate cancer patients increased by considering plasma CAI levels. CAI can potentially serve as a valuable plasma biomarker and the combination of PSA and CAI may have great advantages for diagnosing prostate cancer in patients with gray-zone PSA level.
PMCID: PMC3506895  PMID: 23213568
8.  Plasma and urine biomarkers in acute viral hepatitis E 
Proteome Science  2009;7:39.
Hepatitis E, caused by the hepatitis E virus (HEV), is endemic to developing countries where it manifests as waterborne outbreaks and sporadic cases. Though generally self-limited with a low mortality rate, some cases progress to fulminant hepatic failure (FHF) with high mortality. With no identified predictive or diagnostic markers, the events leading to disease exacerbation are not known. Our aim is to use proteomic tools to identify biomarkers of acute and fulminant hepatitis E.
We analyzed proteins in the plasma and urine of hepatitis E patients and healthy controls by two-dimensional Differential Imaging Gel Electrophoresis (DIGE) and mass spectrometry, and identified over 30 proteins to be differentially expressed during acute hepatitis E. The levels of one plasma protein, transthyretin, and one urine protein, alpha-1-microglobulin (α1m), were then quantitated by enzyme immunoassay (EIA) in clinical samples from a larger group of patients and controls. The results showed decreased plasma transthyretin levels (p < 0.005) and increased urine α1m levels (p < 0.001) in acute hepatitis E patients, compared to healthy controls. Preliminary results also showed lower urine zinc alpha glycoprotein levels in fulminant hepatitis E compared to acute disease; this remains to be confirmed with more fulminant cases.
Our results demonstrate the utility of characterizing plasma and urine proteomes for signatures of the host response to HEV infection. We predict that plasma transthyretin and urine α1m could be reliable biomarkers of acute hepatitis E. Besides the utility of this approach to biomarker discovery, proteome-level changes in human biofluids would also guide towards a better understanding of host-virus interaction and disease.
PMCID: PMC2773234  PMID: 19860894
9.  Proteomics, bioinformatics and targeted gene expression analysis reveals up-regulation of cochlin and identifies other potential biomarkers in the mouse model for deafness in usher syndrome type 1F 
Human Molecular Genetics  2010;19(8):1515-1527.
Proteins and protein networks associated with cochlear pathogenesis in the Ames waltzer (av) mouse, a model for deafness in Usher syndrome 1F (USH1F), were identified. Cochlear protein from wild-type and av mice at postnatal day 30, a time point in which cochlear pathology is well established, was analyzed by quantitative 2D gel electrophoresis followed by mass spectrometry (MS). The analytic gel resolved 2270 spots; 69 spots showed significant changes in intensity in the av cochlea compared with the control. The cochlin protein was identified in 20 peptide spots, most of which were up-regulated, while a few were down-regulated. Analysis of MS sequence data showed that, in the av cochlea, a set of full-length isoforms of cochlin was up-regulated, while isoforms missing the N-terminal FCH/LCCL domain were down-regulated. Protein interaction network analysis of all differentially expressed proteins was performed with Metacore software. That analysis revealed a number of statistically significant candidate protein networks predicted to be altered in the affected cochlea. Quantitative PCR (qPCR) analysis of select candidates from the proteomic and bioinformatic investigations showed up-regulation of Coch mRNA and those of p53, Brn3a and Nrf2, transcription factors linked to stress response and survival. Increased mRNA of Brn3a and Nrf2 has previously been associated with increased expression of cochlin in human glaucomatous trabecular meshwork. Our report strongly suggests that increased level of cochlin is an important etiologic factor leading to the degeneration of cochlear neuroepithelia in the USH1F model.
PMCID: PMC2846161  PMID: 20097680
10.  Plasma proteomic profile of sulfur mustard exposed lung diseases patients using 2-dimensional gel electrophoresis 
Clinical proteomics  2011;8(1):2.
Sulfur mustard "bis (2-chlroethyl) sulphide" (SM) is a chemical warfare agent that remains a threat to human health. The aim of this study was to identify protein expression signature or biomarkers that reflect chronic lung damages induced by SM exposure.
Prior to analysis, plasma was fractionated using ethanol precipitation. Using two dimensional SDS-PAGE; fractionated protein profiles of 20 healthy and 20 exposed patients with lung diseases were established. Selected protein spots were successfully identified with MALDI TOF MS/MS.
The results show that α1 haptoglobin isoforms were detected in plasma of the all lung disease patients but none of the healthy controls. Amyloid A1 isoforms was also detected in plasma of the lung disease patients but none of the healthy controls. Moreover, low molecular weight proteins were enriched in ethanol supernatant compared to ethanol precipitate.
Our present results and previous studies suggest that ongoing tissue remodeling is involved in SM exposed lung damage patients. These finding might improve patient care and suitable therapies.
PMCID: PMC3167199  PMID: 21906349
11.  Metabolite Profiling Identifies a Branched Chain Amino Acid Signature in Acute Cardioembolic Stroke 
Stroke; a journal of cerebral circulation  2013;44(5):10.1161/STROKEAHA.111.000397.
There is limited information about changes in metabolism during acute ischemic stroke. The identification of changes in circulating plasma metabolites during cerebral infarction may provide insight into disease pathogenesis and identify novel biomarkers.
We performed filament occlusion of the middle cerebral artery of Wistar rats and collected plasma and cerebrospinal fluid (CSF) two hours after the onset of ischemia. Plasma samples from control and acute stroke patients were also analyzed. All samples were examined using liquid chromatography followed by tandem mass spectrometry. Positively charged metabolites including amino acids, nucleotides and neurotransmitters were quantified using electrospray ionization followed by scheduled multiple reaction monitoring.
The concentrations of several metabolites were altered in the setting of cerebral ischemia. We detected a reduction in the branched chain amino acids (BCAA; valine, leucine, isoleucine) in rat plasma, rat CSF and human plasma compared to respective controls (16%, 23% and 17%, respectively; p<0.01 for each). In patients, lower BCAA levels also correlated with poor neurological outcome (mRS 0–2 versus 3–6, p=0.002).
BCAA are reduced in ischemic stroke, and the degree of reduction correlates with worse neurological outcome. Whether BCAA are in a causal pathway or are an epiphenomenon of ischemic stroke remains to be determined.
PMCID: PMC3816089  PMID: 23520238
12.  An Increase in Phosphorylation and Truncation of Crystallin With the Progression of Cataracts☆ 
Cataracts are the leading cause of blindness worldwide; however, there is no evidence regarding the direct formation of cataracts. At present, there is no treatment method other than surgery to prevent the formation or progression of cataracts.
Understanding the protein changes during various stages of cataracts might help realize the mechanism of the formation and progression of cataracts.
Lens materials were collected from cataract surgery. Cataracts were classified according to lens opacity using the gradation of the Lens Opacities Classification System. Lens proteins were separated by 2-dimensional polyacrylamide gel electrophoresis. Protein spots were visualized by Coomassie blue staining, and expression patterns were analyzed. Protein spots of interest were excised from 2-dimensional polyacrylamide gel electrophoresis gels, digested in situ with trypsin, and analyzed by mass spectrometry and liquid chromatographic tandem mass spectrometry.
Crystallin was the major protein in the cataract lens, and αA, βB1, αB, and βA4 were the dominant types. Crystallin αB and βA4 increased with the formation of lens opacity. Moreover, phosphorylation and truncation of these proteins increased with the progression of cataracts.
Crystallin αB and βA4 and phosphorylation and truncation of crystallin in the lens might contribute to the formation of cataracts. In contrast, acetylation was not dominant in the progression of cataracts and did not play major role in the formation of cataracts.
PMCID: PMC3862202  PMID: 24384929
cataract; crystallin; mass spectrometry; phosphorylation; truncation
13.  Prostaglandin D Synthase Isoforms from Cerebrospinal Fluid Vary with Brain Pathology 
Disease Markers  2005;22(1-2):73-81.
Glutathione independent prostaglandin D synthase (Swissprot P41222, PTGDS) has been identified in human cerebrospinal fluid and some changes in PTGDS in relation to disease have been reported. However, little is known of the extent that PTGDS isoforms fluctuate across a large range of congenital and acquired diseases. The purpose of this study was to examine changes in PTGDS isoforms in such a population. Spinal fluid from 22 healthy study participants (normal controls) with no classifiable neurological or psychiatric diagnosis was obtained and PTGDS isoforms were identified by specific immunostaining and mass spectrometry after denaturing 2D gel electrophoresis. The PTGDS isoforms in controls consisted of five charge isoforms that were always present and a small number of occasional, low abundance isoforms. A qualitative survey of 98 different people with a wide range of congenital and acquired diseases revealed striking changes. Loss of the control isoforms occurred in congenital malformations of the nervous system. Gain of additional isoforms occurred in some degenerative, most demyelinating and vasculitic diseases, as well as in Creutzfeldt-Jakob disease. A retrospective analysis of published data that quantified relative amounts of PTGDS in multiple sclerosis, schizophrenia and Parkinson’s disease compared to controls revealed significant dysregulation. It is concluded that qualitative and quantitative fluctuations of cerebrospinal fluid PTGDS isoforms reflect both major and subtle brain pathophysiology.
PMCID: PMC3851407  PMID: 16410653
14.  Characterization of the Human Skeletal Muscle Proteome by One-dimensional Gel Electrophoresis and HPLC-ESI-MS/MS*S 
Molecular & cellular proteomics : MCP  2007;7(2):10.1074/mcp.M700304-MCP200.
Changes in protein abundance in skeletal muscle are central to a large number of metabolic and other disorders, including, and perhaps most commonly, insulin resistance. Proteomics analysis of human muscle is an important approach for gaining insight into the biochemical basis for normal and pathophysiological conditions. However, to date, the number of proteins identified by this approach has been limited, with 107 different proteins being the maximum reported so far. Using a combination of one-dimensional gel electrophoresis and high performance liquid chromatography electrospray ionization tandem mass spectrometry, we identified 954 different proteins in human vastus lateralis muscle obtained from three healthy, nonobese subjects. In addition to a large number of isoforms of contractile proteins, we detected all proteins involved in the major pathways of glucose and lipid metabolism in skeletal muscle. Mitochondrial proteins accounted for 22% of all proteins identified, including 55 subunits of the respiratory complexes I–V. Moreover, a number of enzymes involved in endocrine and metabolic signaling pathways as well as calcium homeostasis were identified. These results provide the most comprehensive characterization of the human skeletal muscle proteome to date. These data hold promise for future global assessment of quantitative changes in the muscle proteome of patients affected by disorders involving skeletal muscle.
PMCID: PMC3836591  PMID: 17911086
15.  2D-DIGE proteomic characterization of head and neck squamous cell carcinoma 
Identify proteins that are differentially expressed between head and neck squamous cell cancer and patient-matched normal adjacent tissue, and validate findings in a separate patient cohort.
Study Design
Cross-sectional study of surgical specimens.
Tertiary care academic medical center.
Subjects and Methods
Laser capture microdissection and two-dimensional difference gel electrophoresis were used previously to establish proteomic profiles for tumor and normal adjacent tissue from 14 patients. Here, significance analysis of microarray was used to rank candidate biomarkers. Spots meeting statistical and biological criteria of significance were analyzed by liquid chromatography and tandem mass spectrometry to obtain protein identifications. The expression pattern of the highest-ranked candidate biomarker (cornulin) was validated in a larger, independent patient cohort (n=68) by immunohistochemical staining of a tissue microarray.
117/732 spots (15.9%) met criteria for significance. Identities were obtained for 40 spots, representing 19 different proteins. Four proteins were novel in the context of HNSCC: glutathione synthetase, which was upregulated, and cornulin (SEP53), guanylate binding protein 6, and GRP78, which were downregulated. Cornulin functions in the stress response in normal squamous epithelium, and reduced expression has been proposed as a marker of susceptibility to laryngopharyngeal reflux and other stressors. Loss of cornulin expression was confirmed in an independent HNSCC patient cohort (P<.001).
Downregulation of cornulin is a prominent feature of the molecular signature of HNSCC identified by comparative proteomics. Cornulin may represent a link between HNSCC and other pathologies arising in stratified squamous epithelium.
PMCID: PMC2847880  PMID: 19861202
16.  Proteomic profiling following immunoaffinity capture of HDL: Association of acute phase proteins and complement factors with pro-inflammatory HDL in Rheumatoid Arthritis 
Arthritis and Rheumatism  2012;64(6):1828-1837.
To utilize proteomic analysis to identify protein biomarkers associated with pro-inflammatory HDL in patients with active rheumatoid arthritis.
Liquid chromatography-mass spectrometry (LC-MS) was used to analyze proteins associated with immunoaffinity purified HDL from plasma of two sets of RA patients carrying distinct HDL (anti- or pro-) inflammatory properties. Proteins were fractionated by Offgel electrophoresis and analyzed by LC-MS/MS equipped with a high capacity high performance liquid chromatography chip (HPLC-Chip) incorporating C18 reverse phase trapping and analytical columns. Sandwich enzyme-linked immunosorbent assays were used to validate select HDL-associated proteins in a second RA cohort.
Seventy-eight proteins were identified in the HDL complexes. Twelve proteins were significantly increased in RA patients with pro-inflammatory HDL compared to RA patients with anti-inflammatory HDL. These proteins included acute phase proteins, including apolipoprotein J, fibrinogen, haptoglobin, serum amyloid A, and complement factors (B, C3, C9). Four of the proteins associated with HDL were validated in a second RA cohort.
Pro-inflammatory HDL in patients with RA contains a significantly altered proteome including increased amounts of acute phase proteins and proteins involved in the complement cascade. These findings suggest that HDL is significantly altered in the setting of chronic inflammation from active RA with resultant loss of its anti-inflammatory function. The characterization of the biomarkers reported here may identify novel molecular connections that contribute to the higher risk of CVD in RA patients.
PMCID: PMC3330163  PMID: 22231638
17.  Detection of bone and cartilage-related proteins in plasma of patients with a bone fracture using liquid chromatography–mass spectrometry 
International Orthopaedics  2007;31(6):743-751.
Following bone fracture, a large number of growth factors, cytokines, and their cognate receptors involved in the repair process are active at the fracture site. To determine whether they appear in patients’ blood as candidate biomarkers for following the outcome of healing, we analysed the plasma of 25 patients with an acute bone fracture following affinity plasma purification, SDS gel electrophoresis and liquid chromatography - tandem mass spectrometry (LC-MS/MS). Two hundred and thirteen nonredundant proteins were identified in the in-gel analysis of pooled plasma proteins. Gene ontology (GO) analysis indicated that a majority of detected proteins were of extracellular origin, whereas only a small number were of intracellular (cytosol and nucleus) origin. A significant proportion of detected proteins was involved in the cell growth and proliferation, transport and coagulation. Twelve proteins were potentially related to bone and cartilage metabolism, and several have not been previously identified in the plasma, including: TGF-β induced protein IG-H3, cartilage acidic protein 1, procollagen C proteinase enhancer protein and TGF-β receptor III.
PMCID: PMC2266674  PMID: 17602227
18.  Human plasma protein adsorption onto dextranized surfaces: a two-dimensional electrophoresis and mass spectrometry study 
Protein adsorption is fundamental to thrombosis and to the design of biocompatible materials. We report a two-dimensional electrophoresis and mass spectrometry study to characterize multiple human plasma proteins adsorbed onto four different types of model surfaces: silicon oxide, dextranized silicon, polyurethane and dextranized polyurethane. Dextran was grafted onto the surfaces of silicon and polyurethane to mimic the blood-contacting endothelial cell glycocalyx surface. Surface topography and hydrophobicity/hydrophilicity were determined and analyzed using atomic force microscopy and water contact angle measurements, respectively. Using two-dimensional electrophoresis, we show that, relative to the unmodified surfaces, dextranization significantly inhibits the adsorption of several human plasma proteins including IGHG1 protein, fibrinogen, haptoglobin, Apo A-IV, Apo A-I, immunoglobulin, serum retinal-binding protein and truncated serum albumin. We further demonstrate the selectivity of plasma protein adsorbed onto the different functionalized surfaces and the potential to control and manipulate proteins adsorption on the surfaces of medical devices, implants and microfluidic devices. This result shows that adsorption experiments using a single protein or a binary mixture of proteins are consistent with competitive protein adsorption studies. In summary, these studies indicate that coating blood-contacting biomedical applications with dextran is an effective route to reduce thrombo-inflammatory responses and to surface-direct biological activities.
PMCID: PMC3079350  PMID: 21277175
19.  Comparative Proteomic Analysis of Peritoneal Dialysate from Chronic Glomerulonephritis Patients 
BioMed Research International  2013;2013:863860.
Peritoneal dialysis (PD) frequently contributes to peritoneal damage which cannot be easily identified without invasive techniques, implying the urgent need for biomarkers and revealing mechanisms. Chronic glomerulonephritis (CGN) is one of the leading causes of receiving dialysis treatment. Here, we attempted to analyze the peritoneal dialysate collected from CGN patients when they receive continuous ambulatory peritoneal dialysis (CAPD) treatment for the first time and after a year to reveal the protein changes that resulted from PD. Proteins were displayed by two-dimensional gel electrophoresis (2DE). Altered gel spots were digested followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for protein identification. Eight proteins were found to have differential expression levels between two groups. Their differential expressions were validated by Western blots in other sets of peritoneal dialysates. Proteins identified with higher levels in the first-time dialysate suggested their dominant appearance in CGN patients, while those that showed higher levels in peritoneal dialysate collected after one year may result from initial peritoneal inflammation or changes in the permeability of the peritoneum to middle-sized proteins. All the identified proteins may provide a perceptiveness of peritoneal changes caused by PD and may function as potential biomarkers or drug targets.
PMCID: PMC3666394  PMID: 23762862
20.  Proteomic Analysis of Haptoglobin and Amyloid A Protein Levels in Patients with Vivax Malaria 
Advancements in the field of proteomics have provided great opportunities for the development of diagnostic and therapeutic tools against human diseases. In this study, we analyzed haptoglobin and amyloid A protein levels of vivax malaria patients with combinations of depletion of the abundant plasma proteins, 2-dimensional gel electrophoresis (2-DE), image analysis, and mass spectrometry in the plasma between normal healthy donors and vivax malaria patients. The results showed that the expression level of haptoglobin had become significantly lower or undetectable in the plasma of vivax malaria patients due to proteolytic cleavage when compared to healthy donors on 2-DE gels. Meanwhile, serum amyloid A protein was significantly increased in vivax malaria patient's plasma with high statistical values. These 2 proteins are common acute phase reactants and further large scale evaluation with a larger number of patient's will be necessary to establish the possible clinical meaning of the existential changes of these proteins in vivax malaria patients. However, our proteomic analysis suggests the feasible values of some plasma proteins, such as haptoglobin and serum amyloid A, as associating factor candidates for vivax malaria.
PMCID: PMC2945794  PMID: 20877498
Plasmodium vivax; vivax malaria; proteomics; associating factor candidate; haptoglobin; serum amyloid A
21.  Comparative Analysis of Serum Proteomes of Moyamoya Disease and Normal Controls 
The etiology and pathogenesis of moyamoya disease remain unclear. Furthermore, the definitive diagnostic protein-biomarkers for moyamoya disease are still unknown. The present study analyzed serum proteomes from normal controls and moyamoya patients to identify novel serological biomarkers for diagnosing moyamoya disease.
We compared the two-dimensional electrophoresis patterns of sera from moyamoya disease patients and normal controls and identified the differentially-expressed spots by matrix-assisted laser desorption/ionization-time-of flight mass spectrometry and electrospray ionization quadruple time-of-flight mass spectrometry.
We found and analyzed 22 differently-expressed proteomes. Two proteins were up-regulated. Twenty proteins were down-regulated. Complement C1 inhibitor protein and apolipoprotein C-III showed predominantly changed expressions (complement C1 inhibitor protein averaged a 7.23-fold expression in moyamoya patients as compared to controls, while apolipoprotein C-III averaged a 0.066-fold expression).
Although our study had a small sample size, our proteomic data provide serologic clue proteins for understanding moyamoya disease.
PMCID: PMC2916155  PMID: 20717506
Moyamoya disease; Proteome
22.  Comparative Proteomics of Sera From HCC Patients With Different Origins 
Hepatitis Monthly  2014;14(1):e13103.
Hepatocellular carcinoma (HCC), a major fatal cancer worldwide, is induced by different etiological factors in the liver.
To gain insight into serum protein profiling of HCC with different etiologies.
Patients and Methods:
We subjected the sera of HBV-HCC, HCV-HCC, non-B non-C-HCC patients, and healthy volunteers to two-dimensional gel electrophoresis (2-DE) and liquid chromatography tandem mass spectrometry (LC-MS/MS).
We found 30 differentially expressed protein spots (≥ 1.5 fold P < 0.05) between these two analyses; of them 17 protein spots corresponding to 8 proteins were identified by MS. Transthyretin, leucine rich α-2-glycoprotein, and ficolin 3 were differentially expressed between HBV-related HCC and non-B non-C-HCC sera. Moreover, haptoglobin α-2 isoforms were decreased in HCV-HCC compared to non-B non-CHCC.
Serum proteome analyses of HCC with different origins showed a differential protein pattern, presumably related to different hepatopathogenesis in liver induced by different agents. Further studies are required to clarify the importance of identified proteins for early diagnosis of HCC with different origins.
PMCID: PMC3909643  PMID: 24497876
Hepatitis B Virus; Hepatitis C; Carcinoma, Hepatocellular; Electrophoresis, Gel, Two-Dimensional; Biological Markers
23.  Dried Blood Spot Proteomics: Surface Extraction of Endogenous Proteins Coupled with Automated Sample Preparation and Mass Spectrometry Analysis 
Dried blood spots offer many advantages as a sample format including ease and safety of transport and handling. To date, the majority of mass spectrometry analyses of dried blood spots have focused on small molecules or hemoglobin. However, dried blood spots are a potentially rich source of protein biomarkers, an area that has been overlooked. To address this issue, we have applied an untargeted bottom-up proteomics approach to the analysis of dried blood spots. We present an automated and integrated method for extraction of endogenous proteins from the surface of dried blood spots and sample preparation via trypsin digestion by use of the Advion Biosciences Triversa Nanomate robotic platform. Liquid chromatography tandem mass spectrometry of the resulting digests enabled identification of 120 proteins from a single dried blood spot. The proteins identified cross a concentration range of four orders of magnitude. The method is evaluated and the results discussed in terms of the proteins identified and their potential use as biomarkers in screening programs.
Electronic supplementary material
The online version of this article (doi:10.1007/s13361-013-0658-1) contains supplementary material, which is available to authorized users.
PMCID: PMC3713260  PMID: 23728546
Dried blood spots; Proteomics; Direct surface sampling; Liquid extraction surface analysis; Automated sample preparation
24.  Identification of Reduced Circulating Haptoglobin Concentration as a Biomarker of the Severity of Pulmonary Embolism: A Nontargeted Proteomic Study 
PLoS ONE  2014;9(6):e100902.
Risk stratification of patients with pulmonary embolism (PE) may identify patients at high risk of early death who may benefit from more intensive surveillance or aggressive therapy. Nontargeted proteomics may identify biomarkers useful for the risk stratification of patients with acute symptomatic pulmonary embolism (PE). We studied 6 patients presenting with low-risk PE and 6 patients presenting with intermediate (n = 3) or high-risk (n = 3) PE. Two-dimensional difference gel electrophoresis was used to compare their plasma protein abundances. Candidate protein markers were identified by matrix assisted laser desorption ionization time-of-flight mass spectrometry. A panel of four biomarkers (haptoglobin, hemopexin, α2-macroglobulin, and Ig α1-chain C region) showed differences in plasma abundance among patients with acute symptomatic PE of different severity. Haptoglobin and hemopexin were decreased, whereas α2-macroglobulin and Ig α1-chain C region were increased, in patients with high or intermediate-risk PE compared with low-risk PE patient. In a separate clinical population consisting of 104 adults with acute PE, serum haptoglobin concentrations had an 85% chance of correctly identifying patients with high-risk PE according to receiving operating characteristics curve analysis. Moreover, serum haptoglobin concentrations ≤1 g/l showed an 80% sensitivity and a 96% specificity for the diagnosis of high-risk PE. Nontargeted proteomics identified protein biomarkers for the severity of PE that are involved in iron metabolism pathways and acute-phase response. Among them, reduced serum haptoglobin concentrations show a high accuracy for the biochemical detection of high-risk PE.
PMCID: PMC4076207  PMID: 24979072
25.  Differential profiling of breast cancer plasma proteome by isotope-coded affinity tagging method reveals biotinidase as a breast cancer biomarker 
BMC Cancer  2010;10:114.
Breast cancer is one of the leading causes of women's death worldwide. It is important to discover a reliable biomarker for the detection of breast cancer. Plasma is the most ideal source for cancer biomarker discovery since many cells cross-communicate through the secretion of soluble proteins into blood.
Plasma proteomes obtained from 6 breast cancer patients and 6 normal healthy women were analyzed by using the isotope-coded affinity tag (ICAT) labeling approach and tandem mass spectrometry. All the plasma samples used were depleted of highly abundant 6 plasma proteins by immune-affinity column chromatography before ICAT labeling. Several proteins showing differential abundance level were selected based on literature searches and their specificity to the commercially available antibodies, and then verified by immunoblot assays.
A total of 155 proteins were identified and quantified by ICAT method. Among them, 33 proteins showed abundance changes by more than 1.5-fold between the plasmas of breast cancer patients and healthy women. We chose 5 proteins for the follow-up confirmation in the individual plasma samples using immunoblot assay. Four proteins, α1-acid glycoprotein 2, monocyte differentiation antigen CD14, biotinidase (BTD), and glutathione peroxidase 3, showed similar abundance ratio to ICAT result. Using a blind set of plasmas obtained from 21 breast cancer patients and 21 normal healthy controls, we confirmed that BTD was significantly down-regulated in breast cancer plasma (Wilcoxon rank-sum test, p = 0.002). BTD levels were lowered in all cancer grades (I-IV) except cancer grade zero. The area under the receiver operating characteristic curve of BTD was 0.78. Estrogen receptor status (p = 0.940) and progesterone receptor status (p = 0.440) were not associated with the plasma BTD levels.
Our study suggests that BTD is a potential serological biomarker for the detection of breast cancer.
PMCID: PMC2861033  PMID: 20346108

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