A method for the rapid depletion of albumin and immunoglobulin from human plasma

In studies of the plasma proteome, the high abundance of proteins such as albumin and immunoglobulin impedes the investigation of lower abundance proteins that may be more suitable as biomarkers of disease. We report the specific removal of 98% of albumin and 80% of immunoglobulin heavy chain from human plasma by affinity chromatography, and the subsequent improvement in the number of spots detected and their resolution following two-dimensional gel electrophoresis.     

Identification of hydrophobic proteins as biomarker candidates for colorectal cancer

Nowadays, colorectal cancer is one of the major causes of cancer death in Western countries. Due to the lack of biomarkers with clinical utility for this pathology, and considering that membrane and hydrophobic proteins have not been studied in depth, we performed a prefractionation of colorectal tissues prior to two-dimensional gel electrophoresis in order to identify hydrophobic proteins differentially expressed in colorectal cancer patients. Fractions enriched in hydrophobic proteins were obtained from healthy mucosa and tumor tissue by a specific extraction method based on temperature-dependent phase partitioning with Triton X-114. Proteins were separated by two-dimensional gel electrophoresis and gels were silver-stained, scanned and compared using the PDQuest software. Those spots presenting significantly different abundance were submitted to mass spectrometry for protein identification. Alterations in the expression of cytoskeletal proteins, including a decrease of vimentin and the absence of desmin, were found. We also detected alterations in antioxidant and transport proteins, chaperones, and in two isoforms of the calcium-binding protein S100A6. On the other hand, vimentin was chosen to corroborate the electrophoretic results by specific immunodetection. Most of the altered proteins have been related to cellular membranes, many of them to lipid rafts microdomains in the plasma membrane, and they have also been implicated in the control of cell proliferation, apoptosis, or metastasis. In conclusion, all the proteins found altered in colorectal tumor samples could be considered as candidates for future studies focused on their utility as markers for colorectal diagnosis and prognosis, or as targets for colorectal cancer therapy.     

Plasma proteomics of lung cancer by a linkage of multi-dimensional liquid chromatography and two-dimensional difference gel electrophoresis

To investigate aberrant plasma proteins in lung cancer, we compared the proteomic profiles of serum from five lung cancer patients and from four healthy volunteers. Immuno-affinity chromatography was used to deplete highly abundant plasma proteins, and the resulting plasma samples were separated into eight fractions by anion-exchange chromatography. Quantitative protein profiles of the fractionated samples were generated by two-dimensional difference gel electrophoresis, in which the experimental samples and the internal control samples were labeled with different dyes and co-separated by two-dimensional polyacrylamide gel electrophoresis. This approach succeeded in resolving 3890 protein spots. For 364 of the protein spots, the expression level in lung cancer was more than twofold different from that in the healthy volunteers. These differences were statistically significant (Student's t-test, p-value less than 0.05). Mass spectrometric protein identification revealed that the 364 protein spots corresponded to 58 gene products, including the classical plasma proteins and the tissue-leakage proteins catalase, clusterin, ficolin, gelsolin, lumican, tetranectin, triosephosphate isomerase and vitronectin. The combination of multi-dimensional liquid chromatography and two-dimensional difference gel electrophoresis provides a valuable tool for serum proteomics in lung cancer.     

New approaches for biomarker discovery: the search for liver fibrosis markers in hepatitis C patients

Despite many shortcomings, liver biopsy is regarded as the gold standard for assessing liver fibrosis. A less invasive and equally or more reliable approach would constitute a major advancement in the field. Proteomics can aid discovery of novel serological markers and these proteins can be measured in patient blood. A major challenge of discovering biomarkers in serum is the presence of highly abundant serum proteins, which restricts the levels of total protein loaded onto gels and limits the detection of low abundance features. To overcome this problem, we used two-dimensional gel electrophoresis (2-DE) over a narrow pH 3-5.6 range since this lies outside the range of highly abundant albumin, transferrin and immunoglobulins. In addition, we used in-solution isoelectric focusing followed by SDS-PAGE to find biomarkers in hepatitis C induced liver cirrhosis. Using the pH 3-5.6 range for 2-DE, we achieved improved representation of low abundance features and enhanced separation. We found in-solution isoelectric focusing to be beneficial for analyzing basic, high molecular weight proteins. Using this method, the beta chains of both complement C3 and C4 were found to decrease in serum from hepatitis C patients with cirrhosis, a change not observed previously by 2-DE. We present two proteomics approaches that can aid in the discovery of clinical biomarkers in various diseases and discuss how these approaches have helped to identify 23 novel biomarkers for hepatic fibrosis.     

Quantitative protein profiling using two-dimensional gel electrophoresis,isotope-coded affinity tag labeling,and mass spectrometry

Quantitative protein profiling is an essential part of proteomics and requires new technologies that accurately, reproducibly, and comprehensively identify and quantify the proteins contained in biological samples. We describe a new strategy for quantitative protein profiling that is based on the separation of proteins labeled with isotope-coded affinity tag reagents by two-dimensional gel electrophoresis and their identification and quantification by mass spectrometry. The method is based on the observation that proteins labeled with isotopically different isotope-coded affinity tag reagents precisely co-migrate during two-dimensional gel electrophoresis and that therefore two or more isotopically encoded samples can be separated concurrently in the same gel. By analyzing changes in the proteome of yeast (Saccharomyces cerevisiae) induced by a metabolic shift we show that this simple method accurately quantifies changes in protein abundance even in cases in which multiple proteins migrate to the same gel coordinates. The method is particularly useful for the quantitative analysis and structural characterization of differentially processed or post-translationally modified forms of a protein and is therefore expected to find wide application in proteomics research.     

Serum protein profiling to identify biomarkers for small renal cell carcinoma

Diagnostic biomarkers for early detection of renal cell carcinoma (RCC) are in great need. In the present study, we compared the serum protein profiles of patients with small RCC to those of healthy individuals to identify the differentially expressed proteins with potential to serve as biomarkers. Serum samples were collected from 10 patients with small RCC and 10 healthy individuals. The serum protein expression profiles were analyzed by two-dimensional (2-D) gel electrophoresis. Twenty-seven proteins with differences in expression levels between RCC patients and healthy volunteers were identified. Of these, 19 were expressed at different levels and eight were expressed in serum from the RCC group, but not from the control group. Six differentially expressed proteins identified by using mass spectrometry included coagulation factor XIII B, complement C3 and its precursor, misato homolog 1 (isoform CRA_b), hemopexin, and alpha-1-B-glycoprotein. Some of these serum proteins are known regulators of tumor progression in human malignancies. In conclusion, we successfully applied 2-D gel electrophoresis and identified six serum proteins differentially expressed between patients with small RCC and healthy volunteers. These proteins may provide novel biomarkers for early detection and diagnosis of human RCC.     

Proteomic analyses of amniotic fluid: potential applications in health and diseases

Amniotic fluid (AF) is a potential source of biomarkers for many disorders which may occur during pregnancy. The purpose of this study was to evaluate the place of two-dimensional gel electrophoresis (2-DE) technologies to compare AF in both normal and pathological situations. Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE; Ettan DIGE) as well as two-dimensional gel electrophoresis and silver staining followed by image analysis were used. Differentially expressed proteins were identified by mass spectrometry. This approach was used to study electrophoregrams of normal AF obtained at 17 weeks of gestation and at term, as well as AF from fetuses presenting with congenital diaphragmatic hernia. Finally, the potential of two-dimensional electrophoresis was assessed by studying the protein profile of plasma containing AF proteins in a model of premature rupture of the membranes (PROM). Our results clearly show that two-dimensional electrophoresis technologies still have place for analyzing biological fluids such as AF.     

Proteomic identification of plasma biomarkers in uterine leiomyoma

Recent progresses in quantitative proteomics have offered opportunities to discover plasma proteins as biomarkers for tracking the progression and for understanding the molecular mechanisms of uterine leiomyomas. In the present study, plasma samples were analyzed by fluorescence two-dimensional differential gel electrophoresis (2D-DIGE) and differentially expressed proteins were identified by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). In total, 20 proteins have been firmly identified representing 13 unique gene products. These proteins mainly functioned in transportation (such as apolipoprotein A-I) and coagulation (such as fibrinogen gamma chain). Additionally, our quantitative proteomic approach has identified numerous previous reported plasma markers of uterine leiomyomas such as alpha-1-antitrypsin. On the contrary, we have presented several putative uterine leiomyomas biomarkers including afamin, apolipoprotein A-I, carbonic anhydrase 1, fibrinogen beta chain, fibrinogen gamma chain, gelsolin, hemopexin, leucine-rich alpha-2-glycoprotein, serotransferrin and vitamin D-binding protein which have not been reported and may be associated with the progression and development of the disease. In summary, we report a comprehensive patient-based proteomic approach for the identification of potential plasma biomarkers for uterine leiomyomas. The potential of utilizing these markers for screening and treating uterine leiomyomas warrants further investigations.     

An immunoproteomic approach for identification of clinical biomarkers for monitoring disease: application to cystic fibrosis

Circulating antibodies can be used to probe protein arrays of body fluids, prepared by two-dimensional gel electrophoresis, for antigenic biomarker detection. However, detected proteins, particularly low abundance antigens, often remain unidentifiable due to proteome complexity and limiting sample amounts. Using a novel enrichment approach exploiting patient antibodies for isolation of antigenic biomarkers, we demonstrate how immunoproteomic strategies can accelerate biomarker discovery. Application of this approach as a means of identifying biomarkers was demonstrated for cystic fibrosis (CF) lung disease by isolation and identification of inflammatory-associated autoantigens, including myeloperoxidase and calgranulin B from sputum of subjects with CF. The approach was also exploited for isolation of proteins expressed by the Pseudomonas aeruginosa strain PA01. Capture of PA01 antigens using circulating antibodies from CF subjects implicated in vivo expression of Pseudomonas proteins. All CF subjects screened, but not controls, were immunoreactive against immunocaptured Pseudomonas proteins, representing stress (GroES and ferric iron-binding protein HitA), immunosuppressive (thioredoxin), and alginate synthetase pathway (nucleoside-diphosphate kinase) proteins, implicating their clinical relevance as biomarkers of infection.     

Characterization of the human urinary proteome: a method for high-resolution display of urinary proteins on two-dimensional electrophoresis gels with a yield of nearly 1400 distinct protein spots

The abundance profile of the human urinary proteome is known to change as a result of diseases or drug toxicities, particularly of those affecting the kidney and the urogenital tract. A consequence of such insults is the ability to identify proteins in urine, which may be useful as quantitative biomarkers. To succeed in discovering them, reproducible urine sample preparation methods and good protein resolution in two-dimensional electrophoresis (2-DE) gels for parallel semiquantitative protein measurements are desirable. Here, we describe a protein fractionation strategy enriching proteins of molecular masses (M(r)) lower than 30 kDa in a fraction separate from larger proteins. The fraction containing proteins with M(r)s higher than 30 kDa was subsequently subjected to immunoaffinity subtraction chromatography removing most of the highly abundant albumin and immunoglobulin G. Following 2-DE display, superior protein spot resolution was observed. Subsequent high-throughput mass spectrometry analysis of ca. 1400 distinct spots using matrix-assisted laser desorption/ionization-time of flight peptide mass fingerprinting and liquid chromatography-electrospray ionization tandem mass spectrometry lead to the successful identification of 30% of the proteins. As expected from high levels of post-translational modifications in most urinary proteins and the presence of proteolytic products, ca. 420 identified spots collapsed into 150 unique protein annotations. Only a third of the proteins identified in this study are described as classical plasma proteins in circulation, which are known to be relatively abundant in urine despite their retention to a large extent in the glomerular blood filtration process. As a proof of principle that our urinary proteome display effort holds promise for biomarker discovery, proteins isolated from the urine of a renal cell carcinoma patient were profiled prior to and after nephrectomy. Particularly, the decrease in abundance of the kininogen 2-DE gel spot train in urine after surgery was striking.     

An altered pattern of circulating apolipoprotein E3 isoforms is implicated in preeclampsia

Preeclampsia is a common pregnancy complication that is an important cause of preterm birth and fetal growth restriction. Because there is no diagnostic test yet available for preeclampsia, we used a proteomic approach to identify novel serum/plasma biomarkers for this condition. We conducted case control studies comparing nulliparous women who developed preeclampsia at 36-38 weeks of gestation with healthy nulliparous women matched by gestational age at sampling. Serum/plasma was depleted of six abundant proteins and analyzed by two-dimensional gel electrophoresis (n = 12 per group) and difference gel electrophoresis (n = 12 per group). Differences in abundance of protein spots were detected by univariate and multivariate statistical analyses. Proteins were identified by mass spectrometry and expression of selected proteins was validated by immunoblotting. Proteins whose concentrations were selectively associated with preeclampsia included apolipoprotein E (apoE), apoC-II, complement factor C3c, fibrinogen, transthyretin, and complement factor H-related protein 2. An increase in a deglycosylated isoform of apoE3 and concomitantly decreased amounts of one apoE3 glycoisoform were identified in preeclamptic plasma and confirmed by immunoblotting. Altered production of these preeclampsia-related apoE3 isoforms might impair reverse cholesterol transport, contributing to arterial damage. These findings point to a novel mechanistic link between preeclampsia and subsequent cardiovascular disease.     

Biomarker discovery for kidney diseases by mass spectrometry

By the development of soft ionization such as matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI), mass spectrometry (MS) has become an indispensable technique to analyze proteins. The combination of protein separation and identification such as two-dimensional gel electrophoresis and MS, surface-enhanced laser desorption/ionization-MS, liquid chromatography/MS, and capillary electrophoresis/MS has been successfully applied for proteome analysis of urine and plasma to discover biomarkers of kidney diseases. Some urinary proteins and their proteolytic fragments have been identified as biomarker candidates for kidney diseases. This article reviews recent advances in the application of proteomics using MS to discover biomarkers for kidney diseases.     

Release of tissue-specific proteins into coronary perfusate as a model for biomarker discovery in myocardial ischemia/reperfusion injury

Diagnosis of acute coronary syndromes is based on protein biomarkers, such as the cardiac troponins (cTnI/cTnT) and creatine kinase (CK-MB) that are released into the circulation. Biomarker discovery is focused on identifying very low abundance tissue-derived analytes from within albumin-rich plasma, in which the wide dynamic range of the native protein complement hinders classical proteomic investigations. We employed an ex vivo rabbit model of myocardial ischemia/reperfusion (I/R) injury using Langendorff buffer perfusion. Nonrecirculating perfusate was collected over a temporal profile of 60 min reperfusion following brief, reversible ischemia (15 min; 15I/60R) for comparison with irreversible I/R (60I/60R). Perfusate proteins were separated using two-dimensional gel electrophoresis (2-DE) and identified by mass spectrometry (MS), revealing 26 tissue-specific proteins released during reperfusion post-15I. Proteins released during irreversible I/R (60I/60R) were profiled using gel-based (2-DE and one-dimensional gel electrophoresis coupled to liquid chromatography and tandem mass spectrometry; geLC-MS) and gel-free (LC-MS/MS) methods. A total of 192 tissue-specific proteins were identified during reperfusion post-60I. Identified proteins included those previously associated with I/R (myoglobin, CK-MB, cTnI, and cTnT), in addition to examples currently under investigation in large cohort studies (heart-type fatty acid binding protein; FABPH). The postischemic release profile of a novel cardiac-specific protein, cysteine and glycine-rich protein 3 (Csrp3; cardiac LIM domain protein) was validated by Western blot analysis. We also identified Csrp3 in serum from 6 of 8 patients postreperfusion following acute myocardial infarction. These studies indicate that animal modeling of biomarker release using ex vivo buffer perfused tissue to limit the presence of obfuscating plasma proteins may identify candidates for further study in humans.     

Qualitative and quantitative proteomics by two-dimensional gel electrophoresis, peptide mass fingerprint and a chemically-coded affinity tag (CCAT)

The chemically-coded affinity tag (CCAT) method combines standard electrophoresis protocols with MALDI-TOF-MS analysis to identify and quantify protein abundances in complex samples in one step. This method is designed to fit into the workflow of SDS-PAGE or two-dimensional electrophoresis (2-DE) only requiring basic proteome laboratory equipment. Prior to electrophoresis two protein samples are separately labelled with a heavy or a light version of the CCAT reagent via reduced cysteines in the proteins. Equal amounts are then combined and electrophoretically separated. Proteins can then be excised from the gel to obtain their peptide mass fingerprint by mass spectrometry. This fingerprint enabled not only identification, but also quantification by comparing relative peak intensities of CCAT-labelled peptides. In this article, we display how the CCAT method can be used to analyse two protein samples in one gel and that the peak intensities of labelled peptides reflect the abundance of a protein in it.     

Comprehensive proteomic analysis of the human amniotic fluid proteome: gestational age-dependent changes

Amniotic fluid (AF) is a significant contributor to fetal health and constitutes a potential rich source of biomarkers for diagnosis of maternal and fetal disorders. In this study, we performed a comprehensive survey of the proteins expressed in AF, combining gel and liquid-based fractionation approaches coupled with LC-MS/MS analysis. Two-dimensional Liquid Chromatography (2D-LC) analysis identified 118 nonredundant proteins with high confidence. One- and two-dimensional gel electrophoresis and in-gel digestion identified 101 proteins. Combining both sets resulted in 219 proteins, of which 96 are unique to AF; 70, 18, and 35 proteins are present in serum, cervico-vaginal fluid, and all three fluids, respectively. Fluorescence two-dimensional differential in-gel electrophoresis (2D-DIGE) comparison of first-, second-, and third-trimester AF samples revealed that maximal differences in the relative abundance of AF proteins occur between the first and second trimesters. A systematic analysis of proteins present both in AF and maternal serum could lead to the development of new noninvasive diagnostic procedures to monitor fetal status.     

Identification and validation of mannose 6-phosphate glycoproteins in human plasma reveal a wide range of lysosomal and non-lysosomal proteins

Acid hydrolase activities are normally confined within the cell to the lysosome, a membrane-delimited cytoplasmic organelle primarily responsible for the degradation of macromolecules. However, lysosomal proteins are also present in human plasma, and a proportion of these retain mannose 6-phosphate (Man-6-P), a modification on N-linked glycans that is recognized by Man-6-P receptors (MPRs) that normally direct the targeting of these proteins to the lysosome. In this study, we purified the Man-6-P glycoforms of proteins from human plasma by affinity chromatography on immobilized MPRs and characterized this subproteome by two-dimensional gel electrophoresis and by tandem mass spectrometry. As expected, we identified many known and potential candidate lysosomal proteins. In addition, we also identified a number of abundant classical plasma proteins that were retained even after two consecutive rounds of affinity purification. Given their abundance in plasma, we initially considered these proteins to be likely contaminants, but a mass spectrometric study of Man-6-phosphorylation sites using MPR-purified glycopeptides revealed that some proportion of these classical plasma proteins contained the Man-6-P modification. We propose that these glycoproteins are phosphorylated at low levels by the lysosomal enzyme phosphotransferase, but their high abundance results in detection of Man-6-P glycoforms in plasma. These results may provide useful insights into the molecular processes underlying Man-6-phosphorylation and highlight circumstances under which the presence of Man-6-P may not be indicative of lysosomal function. In addition, characterization of the plasma Man-6-P glycoproteome should facilitate development of mass spectrometry-based tools for the diagnosis of lysosomal storage diseases and for investigating the involvement of Man-6-P-containing glycoproteins in more widespread human diseases and their potential utility as biomarkers.     

Optimizing Human Synovial Fluid Preparation for Two-Dimensional Gel Electrophoresis

Background

Prenatal screening for Down Syndrome (DS) would benefit from an increased number of biomarkers to improve sensitivity and specificity. Improving sensitivity and specificity would decrease the need for potentially risky invasive diagnostic procedures.

Results

We have performed an in depth two-dimensional difference gel electrophoresis (2D DIGE) study to identify potential biomarkers. We have used maternal plasma samples obtained from first and second trimesters from mothers carrying DS affected fetuses compared with mothers carrying normal fetuses. Plasma samples were albumin/IgG depleted and expanded pH ranges of pH 4.5 - 5.5, pH 5.3 - 6.5 and pH 6 - 9 were used for two-dimensional gel electrophoresis (2DE). We found no differentially expressed proteins in the first trimester between the two groups. Significant up-regulation of ceruloplasmin, inter-alpha-trypsin inhibitor heavy chain H4, complement proteins C1s subcomponent, C4-A, C5, and C9 and kininogen 1 were detected in the second trimester in maternal plasma samples where a DS affected fetus was being carried. However, ceruloplasmin could not be confirmed as being consistently up-regulated in DS affected pregnancies by Western blotting.

Conclusions

Despite the in depth 2DE approach used in this study the results underline the deficiencies of gel-based proteomics for detection of plasma biomarkers. Gel-free approaches may be more productive to increase the number of plasma biomarkers for DS for non-invasive prenatal screening and diagnosis.     

Effects of cigarette smoking on the human urinary proteome

In this pilot study we used a proteomic approach to compare the urinary protein patterns of healthy smokers and non-smokers. Proteins were resolved by two-dimensional gel electrophoresis and identified by mass spectrometry. The relative abundance of three inflammatory proteins (S100A8, inter-alpha-trypsin inhibitor heavy chain 4, CD59) and that of two isoforms of pancreatic alpha amylase was significantly higher in smokers. Zinc-alpha-2-glycoprotein was the only protein down-regulated in smokers. Its abundance was significantly correlated with urinary glucocorticoids. Most of the proteins identified may be non-specific biomarkers of tobacco effects, since they are involved in inflammatory responses associated with several diseases. Of greater interest are the changes in abundance of pancreatic alpha amylase and zinc-alpha-2-glycoprotein, which after proper validation, might be candidate biomarkers of diseases resulting from exposure to tobacco smoke. The data also show for the first time that smoking can affect the expression profile of urinary proteins.     

Enhanced detection of CNS cell secretome in plasma protein-depleted cerebrospinal fluid

Human cerebrospinal fluid (CSF) proteome is actively investigated to identify relevant biomarkers and therapeutic targets for neurological disorders. Approximately 80% of CSF proteome originate from plasma, yielding a high dynamic range in CSF protein concentration and precluding identification of potential biomarkers originating from CNS cells. Here, we have adapted the most complete multiaffinity depletion method available to remove 20 abundant plasma proteins from a CSF pool originating from patients with various cognitive disorders. We identified 622 unique CSF proteins in immunodepleted plus retained fractions versus 299 in native CSF, including 22 proteins hitherto not identified in CSF. Parallel analysis of neuronal secretome identified 34 major proteins secreted by cultured cortical neurons (cell adhesion molecules, proteins involved in neurite outgrowth and axonal guidance, modulators of synaptic transmission, proteases and protease inhibitors) of which 76% were detected with a high confidence in immunodepleted CSF versus 50% in native CSF. Moreover, a majority of proteins previously identified as secretory products of choroid plexus cells or astrocytes were detected in immunodepleted CSF. Hence, removal of 20 major plasma proteins from CSF improves detection of brain cell-derived proteins in CSF and should facilitate identification of relevant biomarkers in CSF proteome profiling analyses.