ITI-H4, as a biomarker in the serum of recurrent pregnancy loss (RPL) patients

Recurrent pregnancy loss (RPL) is defined as at least three pregnancy losses in series prior to the 20-28 weeks of pregnancy. There are several etiological factors associated with immunology, anatomy, endocrinology, genetic, infection, chromosomal abnormalities, and environmental factors contributing to the condition. The aim of this study was to identify RPL associated factors in human blood using proteomics. Since it is difficult to obtain tissues or follicular fluids, we used blood samples from normal and RPL patients to conduct a comparative proteomic study. Three RPL blood samples and one cocktailed blood sample from 3 normal women were used. We performed 2-DE and selected spots were analyzed with MALDI-TOF/MS. In the three RPL blood samples, 2-DE analysis revealed 549, 563 and 533 spots to be differentially expressed, respectively. Through a comparative analysis between the control and RPL, 21 spots were shown to be differentially expressed. Of these, 5 proteins were confirmed by Western blot analysis. One of these proteins, ITI-H4 (inter-α trypsin inhibitor-heavy chain 4), was weakly expressed at a molecular weight of 120 kDa, but was highly expressed at a modified molecular weight of 36 kDa in RPL patients. These findings suggest that ITI-H4 expression may be used as a biomarker, which could facilitate the development of novel diagnostic and therapeutic tools.     

Apolipoprotein A-IV attenuates oxidant-induced apoptosis in mitotic competent, undifferentiated cells by modulating intracellular glutathione redox balance

Oxidant-mediated modulation of the intracellular redox state affects the apoptotic cascade by altering the balance between cellular signals for survival and suicide. Apolipoprotein A-IV (Apo A-IV) is known to possess antioxidant-like activity. In the present study, we tested 1) whether Apo A-IV could influence redox-dependent apoptosis and, if so, 2) whether such an effect could be mediated by modulation of intracellular redox balance. Mitotic competent, undifferentiated PC-12 cells were incubated with either tert-butyl hydroperoxide (TBH) or diamide with or without preincubation with human Apo A-IV. Apo A-IV significantly decreased apoptosis produced by both TBH and diamide, and washout of A-IV before incubation with TBH and diamide did not eliminate its protective effect. Apo A-I had no such protective effect. The Apo A-IV effect was not blocked by D,L-buthionine-[S,R]-sulfoximine, but it was reversed by both dehydroisoandrosterone and transfection with an antisense oligodeoxynucleotide to glucose-6-phosphate dehydrogenase (G6PD). Apo A-IV abolished the transient, oxidant-induced rise in glutathione disulfide (GSSG) and cellular redox imbalance previously shown to initiate the apoptotic cascade. Apo A-IV had no effect on GSSG reductase activity, but it stimulated G6PD activity 10-fold. These results suggest a novel role for Apo A-IV in the regulation of intracellular glutathione redox balance and the modulation of redox-dependent apoptosis via stimulation of G6PD activity. tert-butyl hydroperoxide; diamide; dehydroisoandrosterone; glucose-6-phosphate dehydrogenase; antisense     

Activity-dependent neuroprotector homeobox protein: A candidate protein identified in serum as diagnostic biomarker for Alzheimer's disease

Alzheimer's disease (AD) is the most common cause of dementia of late life. To enhance our understanding of AD proteome, the serum proteins were analyzed using two-dimensional gel electrophoresis (2DE) combined with nano-high performance liquid chromatography electrospray ionization tandem mass spectrometry (nano-HPLC-ESI-MS/MS) followed by peptide fragmentation patterning. In this study, six protein spots with differential expression were identified. Five up-regulated proteins were identified as actin, apolipoprotein A-IV (Apo A-IV), inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), alpha-1-antitrypsin (AAT), and antithrombin-III (AT-III); one protein, activity-dependent neuroprotector homeobox protein (ADNP) was down-regulated in AD patients. These proteins with differential expression in the serum may serve as potential indicators of AD. Our results suggested that ADNP may play an important role in slowing the progression of clinical symptoms of AD.     

Comparison of the metabolic behavior of rat apolipoproteins A-I and A-IV, isolated from both lymph chylomicrons and serum high density lipoproteins

Rat apolipoprotein (apo) A-I and A-IV, isolated from both lymph chylomicrons and serum high density lipoproteins (HDL) were analyzed by isoelectric focusing. Lymph chylomicron apo A-I consisted for 81 +/- 2% of the pro form and for 19 +/- 2% of the mature form, while apo A-I isolated from serum HDL was present for 36 +/- 4% in the pro form and for 64 +/- 4% in the mature form. Apo A-IV also showed two major protein bands after analysis by isoelectric focusing. The most prominent component is the more basic protein that amounts to 80 +/- 2% in apo A-IV isolated from lymph chylomicrons and to 60 +/- 3% in apo A-IV isolated from serum HDL. Apo A-I (or apo A-IV), isolated from both sources (lymph chylomicrons or serum HDL), was iodinated and the radioactive apolipoproteins were incorporated into rat serum lipoproteins. The resulting labeled HDL was isolated from serum by molecular sieve chromatography on 6% agarose columns and injected intravenously into rats. No difference in the fractional turnover rate or the tissue uptake of the two labeled HDL preparations was observed, neither for apo A-I nor for apo A-IV. It is concluded that the physiological significance of the extracellular pro apo A-I conversion or the post-translational modification of apo A-IV is not related to the fractional turnover rate in serum or to the rate of catabolism in liver and kidneys.     

Apolipoprotein A-IV polymorphism in Singapore ethnic groups

A total of 627 subjects comprising 455 Chinese, 127 Dravidian Indians and 45 Malays were investigated for serum Apo A-IV polymorphism. The frequency of Apo A-IV*2 was found to be significantly higher (p less than 0.001) in Indians (0.043) compared to that in the Chinese (0.010) and Malays (0.011). The frequency of A-IV*3 was found to be around 0.02 in all the ethnic groups. A low frequency of A-IV*4 (less than 0.01) was observed in the Chinese and Indians. The phenotypic distribution of Apo A-IV was at Hardy-Weinberg equilibrium in the three ethnic groups.     

Serum Immune-Related Proteins are Differentially Expressed during Hibernation in the American Black Bear

Hibernation is an adaptation to conserve energy in the face of extreme environmental conditions and low food availability that has risen in several animal phyla. This phenomenon is characterized by reduced metabolic rate (∼25% of the active basal metabolic rate in hibernating bears) and energy demand, while other physiological adjustments are far from clear. The profiling of the serum proteome of the American black bear (Ursus americanus) may reveal specific proteins that are differentially modulated by hibernation, and provide insight into the remarkable physiological adaptations that characterize ursid hibernation. In this study, we used differential gel electrophoresis (DIGE) analysis, liquid chromatography coupled to tandem mass spectrometry, and subsequent MASCOT analysis of the mass spectra to identify candidate proteins that are differentially expressed during hibernation in captive black bears. Seventy serum proteins were identified as changing by ±1.5 fold or more, out of which 34 proteins increased expression during hibernation. The majority of identified proteins are involved in immune system processes. These included α₂-macroglobulin, complement components C1s and C4, immunoglobulin μ and J chains, clusterin, haptoglobin, C4b binding protein, kininogen 1, α₂-HS-glycoprotein, and apoplipoproteins A-I and A-IV. Differential expression of a subset of these proteins identified by proteomic analysis was also confirmed by immunodetection. We propose that the observed serum protein changes contribute to the maintenance of the hibernation phenotype and health, including increased capacities for bone maintenance and wound healing during hibernation in bears.     

Hypoglycemic dipeptide cyclo (His-Pro) significantly altered plasma proteome in streptozocin-induced diabetic rats and genetically-diabetic (ob/ob) mice

The proteins in plasma perform many important functions in the body, and the protein profiles of the plasma vary under different physiological and pathological conditions. In an attempt to identify novel marker proteins for diabetes prognosis, we examined the effect of hypoglycemic dipeptide cyclo (His-Pro) (CHP) on the differential regulation of plasma proteins in streptozocin-induced diabetic rats and genetically-diabetic (ob/ob) mice. The orally-administrated CHP produced an excellent hypoglycemic effect in both animal models, lowering the average plasma glucose level by over 50 %. In the 2-DE analysis of the plasma, a total of 23 spots among 500 visualized spots were found to be differentially regulated, and they were identified by MALDI/TOF mass spectrometry. These proteins include the down-regulation of Apo E and the up-regulation of FGA, Apo A-I, Apo A-IV, and A1M in STZ-induced diabetic rats. Moreover, CHP significantly reduced the plasma protein levels of FGB, FGC, F12, C1QTNF5, and SPA3K, as well as increased the abundance of A1M, A2M, Apo E, and TTR in genetically-diabetic mice. In conclusion, alteration in the regulation of these proteins indicates that this treatment may be successful in overcoming the diabetic state. The present proteomic data can serve as the basis for the development of specific evidence-based interventions allowing for the prevention and treatment of diabetes.     

Proteomic study of plasma from moderate hypercholesterolemic patients

Proteomics is a technology to detect and identify several proteins and their isoforms in a single sample. We used proteomics to analyze modifications in the protein map of plasma after simvastatin treatment of moderate hypercholesterolemic patients. Plasma from hypercholesterolemic patients (n = 9) was compared before and after 12 weeks of simvastatin treatment (40 mg/day). Patients with similar cardiovascular risk factors were used as controls (CR group). By using two-dimensional electrophoresis and mass spectrometry, we identified the different protein isoforms. The plasma expression of three fibrinogen gamma chain isoforms (FGG) was enhanced, whereas the expression of two isoforms of the fibrinogen beta chain (FGB) was reduced in the hypercholesterolemic patients compared with the CR group. The expression of apolipoprotein A-IV and three haptoglobin isoforms was higher in hypercholesterolemic patients. Simvastatin treatment modified the plasma expression of FGG chain isoform 1, FGB chain isoforms 1 and 2, vitamin D binding protein isoform 3, apo A-IV, and haptoglobin isoform 2. The modification of FGG chain isoform 1 and FGB chain isoforms 1 and 2 was positively correlated with total plasma cholesterol level. Proteomic analysis of plasma may help to know more in depth the molecular mechanism modified by simvastatin treatment.     

Proteomic Analysis of Kidney in Rats Chronically Exposed to Monosodium Glutamate

Background

Chronic monosodium glutamate (MSG) intake causes kidney dysfunction and renal oxidative stress in the animal model. To gain insight into the renal changes induced by MSG, proteomic analysis of the kidneys was performed.

Methods

Six week old male Wistar rats were given drinking water with or without MSG (2 mg/g body weight, n = 10 per group) for 9 months. Kidneys were removed, frozen, and stored at –75°C. After protein extraction, 2-D gel electrophoresis was performed and renal proteome profiles were examined with Colloidal Coomassie Brilliant Blue staining. Statistically significant protein spots (ANOVA, p<0.05) with 1.2-fold difference were excised and analyzed by LC-MS. Proteomic data were confirmed by immunohistochemistry and Western blot analyses.

Results

The differential image analysis showed 157 changed spots, of which 71 spots were higher and 86 spots were lower in the MSG-treated group compared with those in the control group. Eight statistically significant and differentially expressed proteins were identified: glutathione S-transferase class-pi, heat shock cognate 71 kDa, phosphoserine phosphatase, phosphoglycerate kinase, cytosolic glycerol-3-phosphate dehydrogenase, 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase, α-ketoglutarate dehydrogenase and succinyl-CoA ligase.

Conclusion

The identified proteins are mainly related to oxidative stress and metabolism. They provide a valuable clue to explore the mechanism of renal handling and toxicity on chronic MSG intake.     

Time-dependent plasma protein changes in streptozotocin-induced diabetic rats before and after fungal polysaccharide treatments

Previous studies about protein modulation with chemically induced models of diabetes in animals have yielded conflicting results, in that many investigators have reported different regulation patterns for the same proteins. Therefore, it is reasonable to determine biomarkers for prognosis and diagnosis of diabetes with time profiling for the candidate proteins. In this regard, we examined the influence of hypoglycemic fungal polysaccharides (EPS) on the time-dependent plasma protein alterations in streptozotocin-induced diabetic rats. The 2-DE analysis of rat plasma demonstrated that about 50 proteins from about 900 visualized spots were found to be differentially regulated, of which 20 spots were identified as principal diabetes-associated proteins. The results of time profiling revealed that most of the identified proteins showed significant alterations in a time-dependent manner during 14 days, with notable trends. Nine out of the twenty proteins displayed very similar time profiles between normal healthy and EPS-treated diabetic rats. Interestingly, the altered profiles of several proteins by diabetes induction almost returned to control levels after EPS treatments. In particular, we found a clear distinction in differential expression of oxidative stress proteins (ceruloplasmin and transferrin) and lipid metabolism related proteins (Apo A-I, Apo A-IV, and Apo E) in the STZ-induced diabetic rats. The data presented here have identified and characterized the time-dependent changes in plasma proteins associated with EPS treatment in STZ-induced diabetic rats, thereby leading to the discovery of early-response and late-response biomarkers in diabetic and EPS-treated states.     

Lipid,Detergent, and Coomassie Blue G-250 Affect the Migration of Small Membrane Proteins in Blue Native Gels: MITOCHONDRIAL CARRIERS MIGRATE AS MONOMERS NOT DIMERS*

Blue native gel electrophoresis is a popular method for the determination of the oligomeric state of membrane proteins. Studies using this technique have reported that mitochondrial carriers are dimeric (composed of two ∼32-kDa monomers) and, in some cases, can form physiologically relevant associations with other proteins. Here, we have scrutinized the behavior of the yeast mitochondrial ADP/ATP carrier AAC3 in blue native gels. We find that the apparent mass of AAC3 varies in a detergent- and lipid-dependent manner (from ∼60 to ∼130 kDa) that is not related to changes in the oligomeric state of the protein, but reflects differences in the associated detergent-lipid micelle and Coomassie Blue G-250 used in this technique. Higher oligomeric state species are only observed under less favorable solubilization conditions, consistent with aggregation of the protein. Calibration with an artificial covalent AAC3 dimer indicates that the mass observed for solubilized AAC3 and other mitochondrial carriers corresponds to a monomer. Size exclusion chromatography of purified AAC3 in dodecyl maltoside under blue native gel-like conditions shows that the mass of the monomer is ∼120 kDa, but appears smaller on gels (∼60 kDa) due to the unusually high amount of bound negatively charged dye, which increases the electrophoretic mobility of the protein-detergent-dye micelle complex. Our results show that bound lipid, detergent, and Coomassie stain alter the behavior of mitochondrial carriers on gels, which is likely to be true for other small membrane proteins where the associated lipid-detergent micelle is large when compared with the mass of the protein.     

Effect of fungal polysaccharides on the modulation of plasma proteins in streptozotocin-induced diabetic rats

To search out novel biomarkers for monitoring diabetes prognosis, we examined the effect of hypoglycemic fungal exopolysaccharides (EPS) on the differential levels of plasma proteins in streptozotocin-induced diabetic rats. The orally administrated EPS exhibited an excellent hypoglycemic effect, lowering the average plasma glucose level, and increasing insulin secretion in diabetic rats. The 2-DE analysis of rat plasma demonstrated that about 500 visualized spots were differentially regulated, of which 20 spots were identified as principal diabetes-associated proteins. The distinct effect of diabetes induction on the pattern of rat plasma proteins includes the down-regulation of albumin, apolipoprotein E (Apo E), alpha1-inhibitor-3, fetuin beta, Gc-globulin, hemopexin, vitronectin, and transthyretin (TTR) monomer, and the up-regulation of Apo A-I, Apo A-IV, ceruloplasmin, alpha1-antitrypsin, serine protease inhibitor III, and transferrin. Those protein levels were interestingly restored to those of healthy rats by EPS treatment, although the order of magnitude of the changes differed widely. Two proteins of interest showed distinct differential expression with opposite trends: TTR tetramer was significantly down-regulated and immunoglobulin (Ig) kappa light chain was significantly up-regulated upon diabetes induction, both of which were also normalized to those of healthy groups after EPS treatment.     

Detection of SNPs in Porcine Haptoglobin and Apolipoprotein Genes

To find differentially expressed protein spots using two-dimensional electrophoresis proteomic analysis, we took blood serum samples from 40 purebred Yorkshire pigs at 12, 18, 24, and 30 weeks. Each growth stage contained 10 male pigs having half-sib pedigrees. With the pooled serum samples, two interesting spots, differentially expressed in the growth stages, were identified using MALDI-TOF-TOF MS/MS analysis as haptoglobin alpha 1S (Hp) and apolipoprotein A-IV (APOA4) gene products. The Hp was down-regulated from 12 to 30 weeks, and APOA4 was not expressed much before 18 weeks but was highly expressed in the late growth stages. There may be an inverse relationship between the Hp and APOA4 genes. Four segments for the Hp and APOA4 genes were successfully amplified with sizes around 500 bp. The porcine Hp and APOA4 genes were screened in the 40 purebred Yorkshire pigs and a random cross population (90 pigs), resulting in the location of 6 single nucleotide polymorphisms (SNPs) in the coding regions. The mutations resulted in amino acid changes in segments of Hp627, Hp742, and APOA41203. Further investigation of the function of the Hp and APOA4 genes with SNPs will be necessary to understand fully the different expression profiles and association studies.     

Dopamine Transporter Loss in 6-OHDA Parkinson’s Model Is Unmet by Parallel Reduction in Dopamine Uptake

The dopamine transporter (DAT) regulates synaptic dopamine (DA) in striatum and modulation of DAT can affect locomotor activity. Thus, in Parkinson’s disease (PD), DAT loss could affect DA clearance and locomotor activity. The locomotor benefits of L-DOPA may be mediated by transport through monoamine transporters and conversion to DA. However, its impact upon DA reuptake is unknown and may modulate synaptic DA. Using the unilateral 6-OHDA rat PD model, we examined [³H]DA uptake dynamics in relation to striatal DAT and tyrosine hydroxylase (TH) protein loss compared with contralateral intact striatum. Despite >70% striatal DAT loss, DA uptake decreased only ∼25% and increased as DAT loss approached 99%. As other monoamine transporters can transport DA, we determined if norepinephrine (NE) and serotonin (5-HT) differentially modulated DA uptake in lesioned striatum. Unlabeled DA, NE, and 5-HT were used, at a concentration that differentially inhibited DA uptake in intact striatum, to compete against [³H]DA uptake. In 6-OHDA lesioned striatum, DA was less effective, whereas NE was more effective, at inhibiting [³H]DA uptake. Furthermore, norepinephrine transporter (NET) protein levels increased and desipramine was ∼two-fold more effective at inhibiting NE uptake. Serotonin inhibited [³H]DA uptake, but without significant difference between lesioned and contralateral striatum. L-DOPA inhibited [³H]DA uptake two-fold more in lesioned striatum and inhibited NE uptake ∼five-fold more than DA uptake in naïve striatum. Consequently, DA uptake may be mediated by NET when DAT loss is at PD levels. Increased inhibition of DA uptake by L-DOPA and its preferential inhibition of NE over DA uptake, indicates that NET-mediated DA uptake may be modulated by L-DOPA when DAT loss exceeds 70%. These results indicate a novel mechanism for DA uptake during PD progression and provide new insight into how L-DOPA affects DA uptake, revealing possible mechanisms of its therapeutic and side effect potential.     

Evaluation of antibacterial activity induced by Staphylococcus aureus and Ent A in the hemolymph of Spodoptera littoralis

The problem of antibiotic resistance considers one of the most dangerous challenges facing the medical field. So, it is necessary to find substitutions to conventional antibiotics. Antimicrobial peptides (AMPs) are a bio-functional derivative that have been observed as one of the important solutions to such upcoming crisis. Owing to their role as the first line of defense against bacteria, fungi, and viruses. This study was conducted to induce the immune response of Spodoptera littoralis larvae by inoculation of sub lethal doses of Staphylococcus aureus and its enterotoxin. Since Staphylococcal enterotoxin A (SEA) considers the major causative agents of Staphylococcal food poisoning, our study oriented to purify and characterize this toxin to provoke its role in yielding AMPs with broad spectrum antimicrobial activity. A great fluctuation was recorded in the biochemical properties of immunized hemolymph not only in the total protein content but also protein banding pattern. Protein bands of ∼22 kDa (attacin-like) and ∼15 kDa (lysozyme-like) were found to be common between the AMPs induced as a result of both treatments. While protein bands of molecular weight ∼70 kDa (phenoloxidase-like) and ∼14 kDa (gloverin-like) were found specific for SEA treatment. Chromatographic analysis using HPLC for the induced AMPs showed different types of amino acids appeared with differences in their quantities and velocities. These peptides exhibited noticeable antimicrobial activity against certain Gram-positive and Gram-negative bacteria. In conclusion, the antimicrobial potential of the antimicrobial peptides (AMP) induced in the larval hemolymph of S. littoralis will be a promising molecule for the development of new therapeutic alternatives.     

Identification of a detergent-solubilized plasma membrane protein from rat liver recognizing apolipoprotein A-IV

Apolipoprotein A-IV (apo A-IV) is present in plasma associated to both HDL and as a complex with lipids that cannot be floated by ultracentrifugation at 1.21 g/ml density. Apo A-IV is likely an important molecular determinant in HDL binding to the liver. In this communication, data are presented supporting the view that a specific liver plasma membrane protein of Mr 95,000 is a constituent of the apo A-IV binding site. The protein was solubilized with CHAPS from purified rat liver plasma membranes and subjected to SDS-PAGE. Transblotted to nitrocellulose sheet could be identified as recognizing 125I-apo A-IV-DMPC by autoradiography. 125I-apo A-I-DMPC and radioiodinated rat apo E-poor HDL, also bound to the protein. Apo B-100 (as human LDL) and apo C-III did not bind. The protein identified is likely to be the same that has been previously identified by Graham and Oram [1987) J. Biol. Chem. 262, 7439-7442) as 'HDL receptor protein'.     

帕金森病患者脑脊液蛋白质组学分析

帕金森病（Parkinson′s disease,PD）是一种中枢神经系统慢性进展性疾病.本研究采用双向凝胶电泳（two-dimensional gel electrophoresis,2-DE）分离脑脊液（cerebrospinal fluid,CSF）蛋白,获得2-DE图谱,通过ImageMaster 2D Elite软件分析寻找两组的差异蛋白点.结果显示,PD患者CSF中有4个蛋白点丰度下降,22个蛋白点丰度上升.还利用电喷雾质谱（electrospray ionization-tandem mass spectrometric,ESI-MS）对差异蛋白点进行鉴定,发现丰度上升的蛋白点有电压依赖性钙通道α2/δ1亚基,结合珠蛋白,β2-微球蛋白和阿朴脂蛋白A-IV前体,丰度下降的蛋白点为转铁蛋白和转甲状腺蛋白.研究发现,PD患者与对照组CSF蛋白质表达有明显差异,对差异蛋白进行质谱鉴定并了解它们的功能,为以后进一步研究他们在PD发病机制和病程进展中的作用奠定基础.     

Serum protein profile of rheumatoid arthritis treated with anti-TNF therapy (infliximab)

We analyzed the changes in the serum protein profile by infliximab using two-dimensional gel electrophoresis and mass spectrometry. More than 50 gel spots were seen to increase or decrease in correlation with clinical improvements of RA. The spots corresponding to CRP, C3, and Apo J showed reduced staining intensity, while the spots corresponding to Apo A-I, RBP, and transthyretin were enhanced. The protein profile of RA patients treated with infliximab was mostly similar to that of normal healthy controls except for several protein spots. This suggested that infliximab normalized the serum protein profile of RA patients, leading to modification in the serum lipid profile and antioxidant status in RA.     

Identification of putative serum glycoprotein biomarkers for human lung adenocarcinoma by multilectin affinity chromatography and LC-MS/MS

Glycoproteins in human serum play fundamental roles in many biological processes, and also have clinical value as biomarkers for disease progression and treatment. In this study, we isolated glycoproteins from the sera of three healthy individuals and three lung adenocarcinoma patients using multilectin affinity chromatography. The recovered glycoproteins were subjected to treatment with peptide-N-glycosidase F (PNGase F) and in-gel digestion by trypsin. Tryptic peptides were analyzed by nano-LC coupled to ESI-MS/MS and the MS/MS spectra were processed by Bioworks 3.2 and an in-house bioinformatics tool, ProtAn. Approximately 90% of the proteins identified contained more than one potential glycosylation site. Comparison of the serum glycoproteome of healthy and adenocarcinoma individuals revealed 38 cancer-selective proteins. Among them, 60% have previously been reported as low abundance proteins in human sera. We identified several cancer-selective proteins that have been previously characterized as potential indicators of lung cancer in serum or plasma, including haptoglobin (HP), inter-alpha-trypsin inhibitor heavy chain 4 (ITI-H4), complement C3 precursor, and leucine-rich alpha-2-glycoprotein. In addition, plasma kallikrein (KLKB1) and inter-alpha-trypsin inhibitor heavy chain 3 (ITI-H3) were identified as being potentially elevated in the lung cancer group, and were validated by Western blot analysis. Furthermore, approximately 18 kDa plasma kallirein protein fragment was detected at high levels in 25 out of 28 adenocarcinoma patients, while one of the eight normal individuals showed moderate positive. The results suggest that KLKB1 represents a potential candidate serum biomarker of lung cancer.