Abundant Serum Proteins Research Articles

Discovery of cancer biomarkers through the use of mouse models

Although our understanding of the molecular pathogenesis of common types of cancer has improved considerably, the development of effective strategies for cancer diagnosis and treatment have lagged behind. Mouse models of cancer potentially represent an efficient means for uncovering diagnostic markers as genetic alterations associated with human tumors can be engineered in mice. In addition, defined stages of tumor development, breeding conditions, and blood sampling can all be controlled and standardized to limit heterogeneity. Alternatively human cancer cells can be injected into mice and tumor development monitored in xenotransplants. Mouse-based studies promise to elucidate a repertoire of protein changes that occur in blood and biological fluids during tumor development. This is illustrated in a study in which we have applied a three-dimensional intact protein analysis system (IPAS) to elucidate detectable protein changes in serum from immunodeficient mice with lung xenografts from orthotopically implanted human A549 lung adenocarcinoma cells. With sufficiently detailed protein sequence identifications, the observed protein changes can be attributed to either the host mouse or the human tumor cells. It is noteworthy that the majority of increases identified have corresponded to relatively abundant serum proteins, some of which have previously been reported as increased in the sera of cancer patients. Proteomic studies of mouse models of cancer allow assessment of the range of changes in plasma proteins that occur with tumor development and may lead to the identification of potential cancer markers applicable to humans.

Individuals homozygous for the age-related macular degeneration risk-conferring variant of complement factor H have elevated levels of CRP in the choroid

Polymorphisms in the complement factor H gene (CFH) are associated with a significantly increased risk for, or protection against, the development of age-related macular degeneration (AMD). The most documented risk-conferring single-nucleotide polymorphism results in a tyrosine-to-histidine substitution at position 402 (Y402H) of the CFH protein. In this work, we examined the ocular distributions and relative abundance of CFH, several CFH-binding proteins, and abundant serum proteins in the retinal pigmented epithelium (RPE), Bruch's membrane, and choroid (RPE-choroid) in CFH homozygotes possessing either the 402HH or normal 402YY variants. Although CFH immunoreactivity is high in the choroid and in drusen, no differences in CFH-labeling patterns between genotypes are apparent. In contrast, at-risk individuals have significantly higher levels of the CFH-binding protein, C-reactive protein (CRP), in the choroidal stroma. Immunoblots confirm that at-risk individuals have approximately 2.5-fold higher levels of CRP in the RPE-choroid; no significant differences in the levels of CFH or other serum proteins are detected. Similarly, we find no differences in CFH transcription levels in the RPE-choroid nor evidence for local ocular CRP transcription. Increased levels of CRP in the choroid may reflect a state of chronic inflammation that is a by-product of attenuated CFH complement-inhibitory activity in those who possess the CFH at-risk allele. Because the CRP-binding site in CFH lies within the domain containing the Y402H polymorphism, it is also possible that the AMD risk-conferring allele alters the binding properties of CFH, thereby leading to choroidal CRP deposition, contributing to AMD pathogenesis.

Tandem Affinity Monolithic Microcolumns with Immobilized Protein A, Protein G‘, and Antibodies for Depletion of High Abundance Proteins from Serum Samples: Integrated Microcolumn-Based Fluidic System for Simultaneous Depletion and Tryptic Digestion

Affinity monolithic microcolumns with immobilized affinity ligands including protein A, protein G' and polyclonal antibodies were developed for the microscale depletion of the top eight most abundant proteins in human serum. These various affinity microcolumns were evaluated for their sample loading capacities with the standard protein substrates. In general, the sample loading capacity of protein A and protein G' was about 7-25 fold higher than that of the antibody-based affinity columns. The macroporous nature of the monolithic columns, which offers high permeability in pressure-driven flow, allowed the design of long tandem affinity columns for the simultaneous depletion of the top eight most abundant proteins in a single run. The tandem format could be extended to include additional affinity monolithic columns to deplete other proteins for which specific antibodies are available without running into high inlet pressure. Furthermore, the tandem affinity columns were integrated with immobilized trypsin monolithic columns to achieve the simultaneous depletion and digestion of proteins. The various formats investigated in this study could be down scaled to achieve nanoLC or up scaled to perform conventional HPLC depending on the size of the proteomic samples.

Measurements of Proteases or Protease System Components in Blood to Enhance Prediction of Disease Risk or Outcome in Possible Cancer

There is a growing body of evidence that proteases can act as molecular markers for cancer. Indeed, prostate-specific antigen (PSA or KLK3), one of the few molecular markers that is routinely used in all stages of clinical cancer care, is a protease. Moreover, attempts to improve on the diagnostic and prognostic value of PSA have often involved measuring other kallikrein-like proteases, such as KLK2 (hK2) or KLK11. Proteomic and peptidomic analyses have also highlighted the importance of the proteases. For example, in a recent report, Tempst et al showed that matrix-assisted laser desorption/ionization–time of flight mass spectrometry (MALDI-TOF) can be used to identify certain cancer-associated signature combinations of small-sized peptides known to be released from several of the most abundant human serum proteins by cancer-associated exopeptidases. This allows discrimination among malignancies (including prostate cancer) and between the malignant and cancer-free state. The findings of Tempst et al may also relate to the manifestation and regulation of proteolytic activity associated with extracellular matrix degradation and tissue remodeling during invasion and metastasis in malignancy. This includes plasminand trypsin-facilitated activation of matrix metalloproteases, capable of degrading major basal membrane components, such as collagen type IV, which may be key in allowing invading cancer cells to escape and seed metastases outside the primary organ. The components of the urokinase-type plasminogen activator system have been subject to particular attention in this context. The urokinase-type plasminogen activator (uPA) is secreted as inactive proenzyme (pro-uPA), which binds to a specific receptor, urokinase plasminogen activator receptor (uPAR), at the cell surface. Acting as a proximal catalytic template, plasminogen enhances conversion of uPAR-bound pro-uPA to active uPA. In vitro, trypsin, hK2, and other proteases have been found capable of converting pro-uPA to active uPA, while active uPA catalyzes activation of plasminogen to plasmin. Further, uPA is inactivated by formation of stable complexes with various plasminogen activator inhibitors (PAI-1, PAI-2, or PAI-3/PCI), whereas plasmin, uPA, and several other proteases cleave and release uPAR from the cell surface. uPAR is a glycolipid anchored receptor with three homologous domains; the amino terminal (domain I) is needed to bind uPA, while high affinity binding requires intact uPAR(IIII). Several proteases (including active uPA and plasmin) liberate uPAR(I) by cleaving uPAR(I-III) between domains I and II, while uPAR(II-III) is retained at the cell surface by the glycolipid anchor. uPAR can also be shed from the cell surface due to hydrolysis of the lipid anchor by either phospholipases or proteases. These mechanisms provide a basis for the occurrence of detectable levels of soluble uPAR forms in plasma from healthy individuals. It has previously been shown that uPAR levels are elevated in the blood of patients with non–small-cell lung cancer, colorectal cancer, and breast cancer. Cleaved uPAR(II-III) has been identified in cystic fluid from ovarian cancer patients. Preoperative uPAR levels in plasma or serum are associated with the prognosis of patients with colorectal cancer. It has also been proposed that levels of cleaved uPAR forms in blood or tumor tissue more closely reflect the degree of uPA-catalyzed plasminogen activation, and hence, may be more closely associated with patient prognosis than measurements of the sum of all uPAR forms. Applying measurements of the urokinase system components to patients with prostate cancer, Visakorpi et al showed that amplification of the uPA gene was frequent in metastatic lesions of castrated prostate cancer patients. Cozzi et al recently reported on the use of tissue microarrays to show that the expression of both uPA and UPAR is frequently upregulated in high-grade primary tumors, whereas a study by Piironen et al suggested that measurements of certain UPAR forms in the blood may enhance discrimination of men with positive compared to those with negative prostate biopsy findings. Shariat et al report in this issue of the Journal of Clinical Oncology that plasma levels of uPA and uPAR are higher in men with prostate cancer than in healthy controls, and increase with disease progression. Although these results are intriguing, we would do well to remember that research on molecular markers, like that on anticancer agents, passes through several phases. Demonstration of a statistically significant association between a marker and a clinical state, the aim of this article, is the equivalent of a phase I trial of a cytotoxic agent; showing that a biomarker is not independent of outcome, like identifying a dose of an agent that can be taken safely is necessary but clearly insufficient for clinical implementation. An essential next step would be to show that adding data on urokinase levels to currently available clinical JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 25 NUMBER 4 FEBRUARY 1 2007

Head-to-Head Comparison of Serum Fractionation Techniques

Multiple approaches for simplifying the serum proteome have been described. These techniques are generally developed across different laboratories, samples, mass spectrometry platforms, and analysis tools. Hence, comparing the available schemes is impossible from the existing literature because of confounding variables. We describe a head-to-head comparison of several serum fractionation schemes, including N-linked glycopeptide enrichment, cysteinyl-peptide enrichment, magnetic bead separation (C3, C8, and WCX), size fractionation, protein A/G depletion, and immunoaffinity column depletion of abundant serum proteins. Each technique was compared to results obtained from unfractionated human serum. The results show immunoaffinity subtraction is the most effective means for simplifying the serum proteome while maintaining reasonable sample throughput. The reported dataset is publicly available and provides a standard against which emergent technologies can be compared and evaluated for their contribution to serum-based biomarker discovery.

A Multienzyme Network Functions in Intestinal Protein Digestion by a Platyhelminth Parasite

Proteases frequently function not only as individual enzymes but also in cascades or networks. A notable evolutionary switch occurred in one such protease network that is involved in protein digestion in the intestine. In vertebrates, this is largely the work of trypsin family serine proteases, whereas in invertebrates, cysteine proteases of the papain family and aspartic proteases assume the role. Utilizing a combination of protease class-specific inhibitors and RNA interference, we deconvoluted such a network of major endopeptidases functioning in invertebrate intestinal protein digestion, using the parasitic helminth, Schistosoma mansoni as an experimental model. We show that initial degradation of host blood proteins is ordered, occasionally redundant, and substrate-specific. Although inhibition of parasite cathepsin D had a greater effect on primary cleavage of hemoglobin, inhibition of cathepsin B predominated in albumin degradation. Nevertheless, in both cases, inhibitor combinations were synergistic. An asparaginyl endopeptidase (legumain) also synergized with cathepsin B and L in protein digestion, either by zymogen activation or facilitating substrate cleavage. This protease network operates optimally in acidic pH compartments either in the gut lumen or in vacuoles of the intestinal lining cells. Defining the role of each of these major enzymes now provides a clearer understanding of the function of a complex protease network that is conserved throughout invertebrate evolution. It also provides insights into which of these proteases are logical targets for development of chemotherapy for schistosomiasis, a major global health problem.

Individuals homozygous for the age-related macular degeneration risk-conferring variant of complement factor H have elevated levels of CRP in the choroid

Polymorphisms in the complement factor H gene (CFH) are associated with a significantly increased risk for, or protection against, the development of age-related macular degeneration (AMD). The most documented risk-conferring single-nucleotide polymorphism results in a tyrosine-to-histidine substitution at position 402 (Y402H) of the CFH protein. In this work, we examined the ocular distributions and relative abundance of CFH, several CFH-binding proteins, and abundant serum proteins in the retinal pigmented epithelium (RPE), Bruch's membrane, and choroid (RPE-choroid) in CFH homozygotes possessing either the "at-risk" 402HH or "normal" 402YY variants. Although CFH immunoreactivity is high in the choroid and in drusen, no differences in CFH-labeling patterns between genotypes are apparent. In contrast, at-risk individuals have significantly higher levels of the CFH-binding protein, C-reactive protein (CRP), in the choroidal stroma. Immunoblots confirm that at-risk individuals have approximately 2.5-fold higher levels of CRP in the RPE-choroid; no significant differences in the levels of CFH or other serum proteins are detected. Similarly, we find no differences in CFH transcription levels in the RPE-choroid nor evidence for local ocular CRP transcription. Increased levels of CRP in the choroid may reflect a state of chronic inflammation that is a by-product of attenuated CFH complement-inhibitory activity in those who possess the CFH at-risk allele. Because the CRP-binding site in CFH lies within the domain containing the Y402H polymorphism, it is also possible that the AMD risk-conferring allele alters the binding properties of CFH, thereby leading to choroidal CRP deposition, contributing to AMD pathogenesis.

Depletion of high-abundance proteins from serum by immunoaffinity chromatography: A MALDI-FT-MS study

Immunodepletion of high-abundance proteins from serum is a widely used initial step in biomarker discovery studies. In the present work we have investigated the reproducibility of the depletion step by comparing 250 serum samples from prostate cancer patients. All samples were depleted on a single immunoaffinity column over a time period of 6 weeks with automated peak detection and fraction collection. Reproducibility in terms of surface area of the depleted serum protein peak at 280 nm was below 7% relative standard deviation (R.S.D.) and the collected volume of the relevant fraction was 0.97 mL (4.5% R.S.D.). Proteins in the depleted serum fraction were subsequently digested with trypsin and analyzed by MALDI-FT-MS. The degree of the depletion of albumin, transferrin and alpha-1-antitrypsin was determined by comparing the intensity of peptide peaks before and after depletion of 11 samples taken at regular time intervals from amongst the 250 depleted, randomized samples. As a positive control we evaluated peaks of apolipoprotein A1 (the most abundant serum protein remaining after depleteion) showing a clear increase in intensity of these peaks in the depleted samples. From this study we conclude that the depletion of the 250 serum samples was complete and reproducible over a period of 6 weeks.

Tg(Afp‐GFP) expression marks primitive and definitive endoderm lineages during mouse development

Alpha-fetoprotein (Afp) is the most abundant serum protein in the developing embryo. It is secreted by the visceral endoderm, its derivative yolk sac endoderm, fetal liver hepatocytes, and the developing gut epithelium. The abundance of this protein suggested that Afp gene regulatory elements might serve to effectively drive reporter gene expression in developing endodermal tissues. To this end, we generated transgenic mouse lines Tg(Afp-GFP) using an Afp promoter/enhancer to drive expression of green fluorescent protein (GFP). Bright GFP fluorescence allowed the visualization, in real time, of visceral endoderm, yolk sac endoderm, fetal liver hepatocytes, and the epithelium of the gut and pancreas. Comparison of the localization of green fluorescence with that of endogenous Afp transcripts and protein indicated that the regulatory elements used to generate these mouse lines directed transgene expression in what appeared to be all Afp-expressing cells of the embryo, but only in a subset of fetal liver cells. The bright GFP signal permitted flow cytometric analysis of fetal liver hepatocytes. These mice represent a valuable resource for live imaging as well as identification, quantitation, and isolation of cells from the primitive and definitive endoderm lineages of the developing mouse embryo.

Extraction of C-reactive protein from serum on a microfluidic chip

The first extraction of a protein, C-reactive protein (CRP), from unadulterated serum on a microfluidic chip is demonstrated. Two stationary phases were evaluated for their ability to selectively extract this protein directly from serum without the need for additional cleanup steps. The first extraction media tested was an affinity matrix containing monoclonal antibodies to CRP, however, after immobilization this media failed to bind CRP. The more efficient stationary phase, immobilized phosphocholine, extracted CRP in a Ca 2+-dependent manner. Significant non-specific binding by more abundant serum proteins was observed using typical washing protocols with this phase and these protocols were modified and optimized to yield extractions that were selective to phosphocholine-binding proteins. Extraction efficiencies were 40% for standard CRP solutions and run-to-run extraction reproducibility was 15% for 8 μL of total serum loaded. Serum concentrations of CRP determined by this method in patients who had undergone hip replacement surgery compared favorably with those values obtained by the conventional, currently utilized methods. Thus, this newly developed miniaturized method for protein purification from serum will provide a foundation for future extractions of protein biomarkers.

Extended haplotypes in the complement factor H (CFH) and CFH‐related (CFHR) family of genes protect against age‐related macular degeneration: Characterization, ethnic distribution and evolutionary implications

Background. Variants in the complement factor H gene (CFH) are associated with age‐related macular degeneration (AMD). CFH and five CFH‐related genes (CFHR1‐5) lie within the regulators of complement activation (RCA) locus on chromosome 1q32.Aims and Methods. In this study, the structural and evolutionary relationships between these genes and AMD was refined using a combined genetic, molecular and immunohistochemical approach.Results. We identify and characterize a large, common deletion that encompasses both the CFHR1 and CFHR3 genes. CFHR1, an abundant serum protein, is absent in subjects homozygous for the deletion. Genotyping analyses of AMD cases and controls from two cohorts demonstrates that deletion homozygotes comprise 1.1% of cases and 5.7% of the controls (chi‐square = 32.8; P = 1.6 E‐09). CFHR1 and CFHR3 transcripts are abundant in liver, but undetectable in the ocular retinal pigmented epithelium/choroid complex. AMD‐associated CFH/CFHR1/CFHR3 haplotypes are widespread in human populations.Conclusion. The absence of CFHR1 and/or CFHR3 may account for the protective effects conferred by some CFH haplotypes. Moreover, the high frequencies of the 402H allele and the delCFHR1/CFHR3 alleles in African populations suggest an ancient origin for these alleles. The considerable diversity accumulated at this locus may be due to selection, which is consistent with an important role for the CFHR genes in innate immunity.

Myocardial Stiffness, Cardiac Remodeling, and Diastolic Dysfunction in Calcification-Prone Fetuin-A–Deficient Mice

Accelerated atherosclerosis in dialysis patients is characterized by severe vascular calcification, and the magnitude of vascular calcification is associated with increased cardiovascular mortality. Calcification-dependent arterial stiffness is considered to be a major determinant of cardiac failure in uremia. Fetuin-A/alpha(2)-Heremans-Schmid glycoprotein is an abundant serum protein with powerful calcification inhibitory properties. Fetuin-A deficiency was recently linked to cardiovascular mortality in dialysis patients. Fetuin-A knockout (fetuin-KO) mice spontaneously develop widespread soft tissue calcification, including significant myocardial calcification, whereas larger arteries are spared. Therefore, this investigation offers the unique opportunity to study the functional role of isolated myocardial calcification independent of arterial stiffness by assessing the hemodynamics of fetuin-KO mice. Cardiac output in fetuin-KO mice was lower than in wild-type mice (fetuin-KO 1.81 +/- 0.18 versus WT 2.45 +/- 0.29 ml/min per g; P < 0.005), and fetuin-KO mice were refractory to dobutamine stimulation. Left ventricular relaxation was significantly impaired in fetuin-KO hearts with the relaxation index reduced by 23% (P < 0.005). After ischemia, fetuin-KO hearts displayed a continuous decline in left ventricular developed pressure after the initial phase of reperfusion, resulting in 77 +/- 15% of preischemic left ventricular developed pressure (P < 0.05 versus wild-type). In fetuin-KO mice, dystrophic cardiac calcification, with myocardial calcium contents increased 60-fold, was associated with profound induction of profibrotic TGF-beta and downstream collagen and fibronectin mRNA synthesis. In conclusion, independent of arterial stiffness, calcification-associated "myocardial stiffness" characterized by cardiac fibrosis, diastolic dysfunction, impaired tolerance to ischemia, and catecholamine resistance thus may constitute an underestimated cardiovascular risk factor that contributes to cardiac failure in calcification-prone states.

Effect of Immunoaffinity Depletion of Human Serum during Proteomic Investigations

Controversy exists regarding the proper mining of the human serum proteome. Because of the analytical challenges of accurately measuring samples containing a very large dynamic range of protein concentrations, current practices have employed depletion of the highly abundant housekeeping serum proteins, such as albumin and immunoglobins. There is question as to the selectivity of depletion, namely, is there loss of other non abundant serum proteins along with albumin, haptoglobin and other commonly depleted proteins. In this study, human serum was analyzed with and without immunoaffinity depletion of the six most abundant proteins by multidimensional liquid chromatography tandem mass spectrometry. Two replicates of each experiment were conducted and compared against one another. In both depleted and nondepleted replicates there was a 73% and 72% overlap of identified peptides and a 64% and 78% overlap of identified proteins, respectively. Of 262 unique proteins identified in the four experiments, 82 were found in common to all four experiments, 142 unique to the depleted serum, and 38 unique to the nondepleted serum. Although serum depletion of highly abundant proteins significantly increased the number of proteins identified, both the degree of sample complexity and this depletion method resulted in a nonselective loss of other proteins.

Distinctive serum protein profiles involving abundant proteins in lung cancer patients based upon antibody microarray analysis

BackgroundCancer serum protein profiling by mass spectrometry has uncovered mass profiles that are potentially diagnostic for several common types of cancer. However, direct mass spectrometric profiling has a limited dynamic range and difficulties in providing the identification of the distinctive proteins. We hypothesized that distinctive profiles may result from the differential expression of relatively abundant serum proteins associated with the host response.MethodsEighty-four antibodies, targeting a wide range of serum proteins, were spotted onto nitrocellulose-coated microscope slides. The abundances of the corresponding proteins were measured in 80 serum samples, from 24 newly diagnosed subjects with lung cancer, 24 healthy controls, and 32 subjects with chronic obstructive pulmonary disease (COPD). Two-color rolling-circle amplification was used to measure protein abundance.ResultsSeven of the 84 antibodies gave a significant difference (p < 0.01) for the lung cancer patients as compared to healthy controls, as well as compared to COPD patients. Proteins that exhibited higher abundances in the lung cancer samples relative to the control samples included C-reactive protein (CRP; a 13.3 fold increase), serum amyloid A (SAA; a 2.0 fold increase), mucin 1 and α-1-antitrypsin (1.4 fold increases). The increased expression levels of CRP and SAA were validated by Western blot analysis. Leave-one-out cross-validation was used to construct Diagonal Linear Discriminant Analysis (DLDA) classifiers. At a cutoff where all 56 of the non-tumor samples were correctly classified, 15/24 lung tumor patient sera were correctly classified.ConclusionOur results suggest that a distinctive serum protein profile involving abundant proteins may be observed in lung cancer patients relative to healthy subjects or patients with chronic disease and may have utility as part of strategies for detecting lung cancer.

Utility of electrophoretically derived protein mass estimates as additional constraints in proteome analysis of human serum based on MS/MS analysis

The proteome of a HUPO human serum reference sample was analyzed using multidimensional separation techniques at both the protein and the peptide levels. To eliminate false-positive identifications from the search results, we employed a data filtering method using molecular weight (MW) correlations derived from denaturing 1-DE. First, the six most abundant serum proteins were removed from the sample using immunoaffinity chromatography. 1-DE was then used to fractionate the remaining serum proteins according to the MW. Gel bands were isolated and in-gel digested with trypsin, and the resulting peptides were analyzed by 2-D LC/ESI-MS/MS. A SEQUEST search using the MS/MS results identified 494 proteins. Of these, 202 were excluded formally using protein data filtering as they were single-assignment proteins and their theoretical and electrophoretically-derived MWs did not correlate at high confidence. To evaluate this method, the results were compared with those of 1-D LC/MALDI-TOF/TOF and HUPO Plasma Proteome Project analyses. Our data filtering approach proved valuable in analysis of complex, large-scale proteomes such as human serum.

The human plasma proteome: Analysis of Chinese serum using shotgun strategy

We have investigated the serum proteome of Han-nationality Chinese by using shotgun strategy. A complete proteomics analysis was performed on two reference specimens from a total of 20 healthy donors, in which each sample was made from ten-pooled male or female serum, respectively. The methodology used encompassed (1) removal of six high-abundant proteins; (2) tryptic digestion of low- and high-abundant proteins of serum; (3) separation of peptide mixture by RP-HPLC followed by ESI-MS/MS identification. A total of 944 nonredundant proteins were identified under a stringent filter condition (X(corr) > or = 1.9, > or = 2.2, and > or = 3.75, < or = C(n) > or = 0.1, and R(sp) > or = 4.0) in both pooled male and female samples, in which 594 and 622 entire proteins were found, respectively. Compared with the total 3020 protein identifications confirmed by more than one laboratory or more than one specimen in HUPO Plasma Proteome Project (PPP) participating laboratories recently, 206 proteins were identified with at least two distinct peptides per protein and 185 proteins were considered as high-confidence identification. Moreover, some lower abundance serum proteins (ng/mL range) were detected, such as complement C5 and CA125, routinely used as an ovarian cancer marker in plasma and serum. The resulting nonredundant list of serum proteins would add significant information to the knowledge base of human plasma proteome and facilitate disease markers discovery.

Chromatofocusing fractionation and two‐dimensional difference gel electrophoresis for low abundance serum proteins

The technical challenge to analysis of the serum proteome is that the serum proteins are present at unequal concentrations. A few are so dominant, such as serum albumin and immunoglobulins, that they mask detection of other proteins. Because of these high abundance proteins, current technologies, while theoretically capable of analyzing protein amounts spanning four orders of magnitude, are only able to analyze proteins ranging over two orders of magnitude and cannot analyze the lower abundance proteins that may be the next biomarkers and drug targets. To facilitate the identification of low abundance proteins, we fractionated serum samples from patients with prostate cancer and patients with benign prostate hyperplasia using anion displacement liquid chromatofocusing chromatography, which separates proteins by a pH gradient and a positively charged column. Differential expression of proteins from fractions was then determined and identified by IEF gels and 2-D DIGE. Results demonstrate improved resolution of proteins within the chosen pH gradient when compared to the unfractionated samples. Several proteins that were differentially expressed in serum from patients with prostate cancer were identified in the fractionated serum. Three of these proteins, squamous cell carcinoma antigen 1 (SCCA1), calgranulin B, and haptoglobin-related protein, are present in the serum at levels below the classical protein level of mg/mL. SCCA1 is normally expressed in serum at ng/mL levels, and calgranulin B is an intracellular protein. Our results demonstrate that the use of anion displacement liquid chromatofocusing chromatography may reduce the complexity of the serum proteome by separating proteins into distinct pH ranges, and facilitate the identification of low abundance proteins.

Investigation of the Mouse Serum Proteome

With the rapid assimilation of genomic information and the equally impressive developments in the field of proteomics, there is an unprecedented interest in biomarker discovery. Although human biofluids represent increasingly attractive samples from which new and more accurate disease biomarkers may be found, the intrinsic person-to-person variability in these samples complicates their discovery. One of the most extensively used animal models for studying human disease is mouse because, unlike humans, they represent a highly controllable experimental model system. Unfortunately, very little is known about the proteomic composition of mouse serum. In this study, a multidimensional fractionation approach on both the protein and the peptide level that does not require depletion of highly abundant serum proteins was combined with tandem mass spectrometry to characterize proteins within mouse serum. Over 12 300 unique peptides that originate from 4567 unique proteins-approximately 16% of all known mouse proteins-were identified. The results presented here represent the broadest proteome coverage in mouse serum and provide a foundation from which quantitative comparisons can be made in this important animal model.

Enrichment of low‐abundant serum proteins by albumin/immunoglobulin G immunoaffinity depletion under partly denaturing conditions

We present a simple protocol for affinity depletion to remove the two most abundant serum proteins, albumin and immunoglobulin G (IgG). Under native conditions, albumin/IgG were efficiently removed and several proteins were enriched as shown by two-dimensional electrophoresis (2-DE). Besides that, partly denaturing conditions were established by adding 5 or 20% acetonitrile (ACN) in order to disrupt the binding of low-molecular-weight (LMW) proteins to the carrier proteins albumin/IgG. 2-DE results showed that the total number of detected LMW proteins increased under denaturing conditions when compared to native conditions. Interestingly, the presence of 5% ACN in serum revealed better enrichment of LMW proteins when compared to 20% ACN condition. Seven randomly distributed spots in albumin/IgG depleted serum samples under 5% ACN condition were picked from the 2-DE gels and identified by mass spectrometry (MS). The intensity of five LMW protein spots increased under denaturing conditions when compared to native conditions. Three of the seven identified spots (serum amyloid P, vitamin D-binding protein, and transthyretin) belong to a group of relatively low-abundant proteins, which make up only 1% of all serum proteins. The method presented here improves the resolution of the serum proteome by increasing the number of visualized spots on 2-D gels and allowing the detection and MS identification of LMW proteins and proteins of lower abundance.

RENAL TUBULE ALBUMIN TRANSPORT

Albumin is the most abundant protein in serum and contributes to the maintenance of oncotic pressure as well as to transport of hydrophobic molecules. Although albumin is a large anionic protein, it is not completely retained by the glomerular filtration barrier. In order to prevent proteinuria, albumin is reabsorbed along the proximal tubules by receptor-mediated endocytosis, which involves the binding proteins megalin and cubilin. Endocytosis depends on proper vesicle acidification. Disturbance of endosomal acidification or loss of the binding proteins leads to tubular proteinuria. Furthermore, endocytosis is subject to modulation by different signaling systems, such as protein kinase A (PKA), protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3-K) and transforming growth factor beta (TGF-beta). In addition to being reabsorbed in the proximal tubule, albumin can also act as a profibrotic and proinflammatory stimulus, thereby initiating or promoting tubulo-interstitial diseases.