Formation of the urine proteome of healthy humans

The review deals with modern ideas on the processes that determine the urine protein composition of healthy people. In the past decade, the development of highly sensitive mass-spectrometric methods of protein detection stimulated studies of the protein composition of various human body fluids, including urine. Nowadays, the methods of separating complex protein mixtures and identification of individual components of these mixtures provide an opportunity to detect a significant amount of proteins and peptides of different origins in human urine. Physiological variation of the urine protein composition determined by the methods of proteomics remains a poorly studied but very important problem. Under physiological conditions, there are many factors that influence the filtering of plasma proteins in the glomeruli and reabsorption in the proximal tubules of the nephron. These are hypoxia, oxidative stress, changes in the acid-base balance and blood pressure, the effects of the parathyroid hormone, angiotensin-II, and other substances that control water and electrolyte metabolism. It is demonstrated that, because of the close structural and functional relationships between reabsorption processes in the proximal tubules of the nephron, reabsorption and modulation of sodium, water, chloride, phosphate, and bicarbonate depend on changes in various parts of the process of protein reabsorption.     

Study of the urine proteome in healthy humans

A new branch of molecular biology, proteomics, has been developed recently due to a success in genomics and informatics. Proteomics is currently solving problems of the full proteome mapping of various biological substances, e.g., body fluids, cells, and tissues in the normal state and pathology; and also search for biomarkers of pathologies, including tumors. Data on the urine proteome have been analyzed in this review. Analysis of the methods used in proteomics, including sample preparation, study strategy, as well as published data on urine proteome over the past five years are presented.     

Informatic tools for proteome profiling

In recent years, the practice of proteomics research has experienced a dramatic shift within the pharmaceutical and biotechnology industry with the widespread implementation of novel applications. The areas of interest extend all the way from discovery of novel drug, vaccine, and diagnostic targets, characterization of protein-based products, toxicology, and identification of surrogate markers of activity in clinical research, to the ability to provide information on the mechanisms of drug action. The power of two-dimensional gel electrophoresis as well as advances in mass spectrometric techniques combined with sequence database correlation have enabled speed and accuracy in identification of proteins in complex mixtures. This article surveys currently available software and informatic tools related to these methods for proteome profiling. The broad acceptance of these technologies, however, has not been accompanied by significant advances in the informatics and software tools necessary to support the analysis and management of the massive amounts of data generated in the process. In this context, this article also discusses the importance of relational databases for protein identification data management.     

Association between the plasma proteome and serum ascorbic acid concentrations in humans

Vitamin C has been associated with a reduced risk of chronic diseases, but the biological pathways regulated by vitamin C are not all known. The objective was to use a proteomics approach to identify plasma proteins associated with circulating levels of ascorbic acid. Men and women (n= 1022) 20–29 years of age from the Toronto Nutrigenomics and Health Study completed a general health and lifestyle questionnaire and a 196-item food frequency questionnaire and provided a fasting blood sample. Circulating ascorbic acid was analyzed by high-performance liquid chromatography, and a mass-spectrometry-based multiple reaction monitoring method was used to measure 54 proteins abundant in plasma that are involved in numerous physiologic pathways. Mean protein concentrations were compared across tertiles of serum ascorbic acid using analysis of covariance adjusted for sex, ethnocultural group, season of blood draw, hormonal contraceptive use among women, waist circumference and tertiles of plasma α-tocopherol. A Bonferroni significance level of P<.0009 was applied, and analyses were adjusted for multiple comparisons using the Tukey–Kramer procedure. Levels of complement C9, ceruloplasmin, alpha-1-anti-trypsin, angiotensinogen, complement C3, vitamin D binding protein and plasminogen were inversely associated with levels of ascorbic acid. The inverse association between ascorbic acid and vitamin D binding protein was highest in those with higher levels of serum 25-hydroxyvitamin D. In conclusion, several plasma proteins from various physiologic pathways are significantly associated with circulating levels of ascorbic acid. These findings suggest that vitamin C may have novel physiological effects.     

Arabinoxylan-enriched meal increases serum ghrelin levels in healthy humans.

Soluble fibre like arabinoxylan (AX) is thought to have beneficial effects on metabolism. In this study, we investigated the effect of a breakfast enriched in AX fibre on glucose, insulin and ghrelin values. AX-enriched and control breakfasts were served to fifteen young volunteers (nine female, six male). Glucose, insulin and ghrelin responses were measured after the meal. To avoid effects from differences in glucose metabolism, further analysis was restricted to those subjects with known normal glucose regulation (seven female, four male). The AX fibre-enriched breakfast did not significantly change glucose levels for two hours after breakfast, but decreased insulin levels in the entire cohort (p = 0.035). Glucose response was also not significantly different in subjects with normal glucose regulation (p = 0.367), and the insulin responses after an AX-enriched breakfast showed only a tendency towards lower values (p = 0.065). Nevertheless, plasma ghrelin two hours after AX-enriched breakfast was higher than after the control meal (396.1 +/- 36.4 pg/ml vs. 328.3 +/- 32.6 pg/ml, p < 0.001). In subjects with normal glucose regulation, the AX-enriched breakfast increased ghrelin levels without any significant difference in glucose or insulin response. This effect is therefore unlikely to be mediated by insulin, but the underlying mechanism remains to be elucidated.     

Recent advances in gel-based proteome profiling techniques

This review focuses on recent developments in gel-based proteomics techniques. By combining traditional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoretic techniques with recent advances in protein labeling using different classes of molecules (i.e., fluorescent dyes, chemical probes, radioisotopes), new technologies have been developed that allow for high-throughput studies of proteins at the whole-proteome scale.     

Development of barcode and proteome profiling of glioblastoma

High grade glioma (glioblastoma) is the most common brain tumor. Its malignancy makes it the fourth biggest cause of cancer death. In our experiments, we used several glioblastoma cell lines to obtain proteomics information specific for this disease. 2DE separation with following imaging, immunochemistry, spot picking, and mass-spectrometry allowed us to detecting more than 600 protein spots and identifying more than 130 of them. Proteome profiles in normal and glioblastoma cell lines are very similar but levels of several proteins have prominent differences between norm and cancer. Among these proteins are alpha-enolase (ENOA_HUMAN), pyruvate kinase M1/M2 (KPYM_HUMAN), cofilin 1 (COF1_HUMAN), translationally-controlled tumor protein TCTP_HUMAN, annexin 1 (ANXA1_HUMAN), PCNA (PCNA_HUMAN), p53 (TP53_HUMAN) and others. Most interesting results were obtained about protein p53. Its level was dramatically up-regulated and enriched by multiple additional isoforms in all glioblastoma cell lines. An immunological analysis (Western blot) of three hub-proteins (p53, 14-3-3, PCNA) allowed us to creating the minimal barcode of glioblastoma cell lines. These preliminary data point to this barcode as a promising diagnostic tool for testing of the biological fluids from patients.     

Variability of urine proteome in healthy humans during a 105-day isolation in a pressurized compartment

The protein composition (proteome) of the body fluids is rather flexible; it can change, responding to various factors of the external environment and changes in the internal environment. In order to study the variability of the proteome profile in healthy humans under the conditions of total control of vital rhythm, physical activity, and diet, urine samples were collected from subjects who had been selected according to special criteria and qualified as healthy by a special physical evaluation board. The subjects took part in an experiment with a 105-day-long isolation in a pressurized compartment, carried out by using an autonomous life-support system at the Institute of Biomedical Problems, Russian Academy of Sciences. The purification and concentration of proteins from the urine samples were carried out using a MB-HIC C8 magnetic bead set (Bruker Daltonics). The mass spectra have been obtained using an Autoflex III time-of-flight mass spectrometer (Bruker Daltonics) in the positive lineal mode. One hundred and seventeen peaks were obtained for each urine sample; technological errors of the method have been studied. The high variability of the urine proteome profile (36 protein MC peaks on average) was shown in healthy humans under the conditions of isolation and controlled vital activity.     

Towards a standardized human proteome database: quantitative proteome profiling of living cells

Comparative proteome profiling, performed by two-dimensional polyacrylamide gel electrophoresis or multidimensional protein identification technology, usually relies on the relative comparison of samples of interest with respect to a reference. Currently, no standardized quantitative protein expression database of human cells, facilitating data comparisons between different laboratories, exists. Recently, we have published two-dimensional polyacrylamide gel electrophoresis-based techniques to assess absolute protein data comprising protein amounts, synthesis rates and biological half-lives (Mol. Cell. Proteomics 2002, 1, 528-537). Determination of protein amounts by fluorography of two-dimensional gels was followed by the exact quantification of the amount of incorporated (35)S radiolabel. Here we demonstrate an application of this highly standardized method to quiescent human T cells, phythaemagglutinin-stimulated T cells and Jurkat cells, a human T lymphoblast cell line. While the protein composition of quiescent T cells differed significantly compared to that of Jurkat cells, it was only slightly different compared to the activated T cells. Synthesis profile analyses demonstrated that activated T cells clearly differed from the quiescent cells, performing apparently almost like lymphoblast cells. The great sensitivity of this approach was further demonstrated with human umbilical vein endothelial cells treated for six hours with vascular endothelial growth factor. While no significant alteration of protein amounts was detected at all upon activation, the synthesis rate of several proteins was found to be more than doubled.     

Variability of the healthy human proteome

The aim of this review is to analyze results of studies on characteristics of protein variability and diversity of posttranslational modifications of proteins in healthy humans. Numerous studies have demonstrated that a proteomic profile is characterized by significant intra- and inter-individual variability, and quite often natural (“normal”) variability of some proteins can be comparable to changes observed in pathological processes. Results obtained by our research group have demonstrated high intra-individual variability of serum low-molecular subproteome of healthy volunteers, certified by a special medial committee (the coefficient of variation (CV) of 42.6%). The proteins characterized by high variability under normal conditions (e.g. haptoglobin — 0–40 mg/ml; lysozyme — 0.01–0.1 mg/ml; C-reactive protein — 0.01–0.3 mg/ml) cannot be considered as potential biomarkers of diseases. On the contrary, proteins and peptides characterized by insignificant dispersion in healthy population (such as albumin ( CV = 9%); transferrin-(CV = 14%); C3c complement (CV = 17%), α-1 acid glycoprotein (CV = 21%), α-2-macroglobulin (CV = 20%); transthyretin fragment (CV = 28.3%) and α2-HS-glycoprotein βchain (CV = 29.7%)) can provide valuable information about the state of health. Thus, studies of plasticity in the proteomic profiles of healthy humans will help to correct reference intervals used in clinical proteomics.     

Systematical evaluation of the effects of sample collection procedures on low-molecular-weight serum/plasma proteome profiling

Blood is an ideal source for biomarker discovery. However, little has been done to address the effects of sampling, handling and storage procedures on serum/plasma proteomes. We used magnetic bead-based MALDI-TOF MS to systematically evaluate the influence of each procedure on low-molecular-weight serum/plasma proteome profiling on the basis of the whole spectra. We found that sampling procedures, including the selection of blood collection tubes and anticoagulants, variations in clotting time or time lag before centrifugation, and hemolysis, displayed significant effects on the proteomes. Moreover, serum and plasma were mutually incompatible for proteome comparison. By contrast, overnight fasting, handling procedures, including centrifugation speeds (1500 x g vs. 3000 x g) or time (15 min vs. 30 min), and storage conditions, such as at 4 degrees C or 25 degrees C for up to 24 h or at -80 degrees C for up to 3 months, and repeated freeze/thaw of up to ten cycles, had relatively minor effects on the proteomes based upon our analysis of about 100 peaks. We concluded that low-molecular-weight serum/plasma proteomes were diversely affected by sampling, handling and storage with most change from variations of sampling procedures. We therefore suggest the necessity of standardizing sampling procedure for proteome comparison and biomarker discovery.     

Metabolite profiling reveals new insights into the regulation of serum urate in humans

Metabolomics - Serum urate, the final breakdown product of purine metabolism, is causally involved in the pathogenesis of gout, and implicated in cardiovascular disease and type 2 diabetes. Serum...     

Quantitative proteome profiling of normal human circulating microparticles

Circulating microparticles (MPs) are produced as part of normal physiology. Their numbers, origin, and composition change in pathology. Despite this, the normal MP proteome has not yet been characterized with standardized high-resolution methods. We here quantitatively profile the normal MP proteome using nano-LC-MS/MS on an LTQ-Orbitrap with optimized sample collection, preparation, and analysis of 12 different normal samples. Analytical and procedural variation were estimated in triply processed samples analyzed in triplicate from two different donors. Label-free quantitation was validated by the correlation of cytoskeletal protein intensities with MP numbers obtained by flow cytometry. Finally, the validity of using pooled samples was evaluated using overlap protein identification numbers and multivariate data analysis. Using conservative parameters, 536 different unique proteins were quantitated. Of these, 334 (63%) were present in all samples and represent an MP core proteome. Technical triplicates showed <10% variation in intensity within a dynamic range of almost 5 decades. Differences due to variable MP numbers and losses during preparative steps could be normalized using cytoskeletal MP protein intensities. Our results establish a reproducible LC-MS/MS procedure, provide a simple and robust MP preparation method, and yield a baseline MP proteome for future studies of MPs in health and disease.     

Toward plasma proteome profiling with ion mobility-mass spectrometry

Differential, functional, and mapping proteomic analyses of complex biological mixtures suffer from a lack of component resolution. Here we describe the application of ion mobility-mass spectrometry (IMS-MS) to this problem. With this approach, components that are separated by liquid chromatography are dispersed based on differences in their mobilities through a buffer gas prior to being analyzed by MS. The inclusion of the gas-phase dispersion provides more than an order of magnitude enhancement in component resolution at no cost to data acquisition time. Additionally, the mobility separation often removes high-abundance species from spectral regions containing low-abundance species, effectively increasing measurement sensitivity and dynamic range. Finally, collision-induced dissociation of all ions can be recorded in a single experimental sequence while conventional MS methods sequentially select precursors. The approach is demonstrated in a single, rapid (3.3 h) analysis of a plasma digest sample where abundant proteins have not been removed. Protein database searches have yielded 731 high confidence peptide assignments corresponding to 438 unique proteins. Results have been compiled into an initial analytical map to be used -after further augmentation and refinement- for comparative plasma profiling studies.     

Immunoassay-based proteome profiling of 24 pancreatic cancer cell lines

Pancreatic ductal adenocarcinoma is one of the most deadly forms of cancers, with a mortality that is almost identical to incidence. The inability to predict, detect or diagnose the disease early and its resistance to all current treatment modalities but surgery are the prime challenges to changing the devastating prognosis. Also, relatively little is known about pancreatic carcinogenesis. In order to better understand relevant aspects of pathophysiology, differentiation, and transformation, we analysed the cellular proteomes of 24 pancreatic cancer cell lines and two controls using an antibody microarray that targets 741 cancer-related proteins. In this analysis, 72 distinct disease marker proteins were identified that had not been described before. Additionally, categorizing cancer cells in accordance to their original location (primary tumour, liver metastases, or ascites) was made possible. A comparison of the cells' degree of differentiation (well, moderately, or poorly differentiated) resulted in unique marker sets of high relevance. Last, 187 proteins were differentially expressed in primary versus metastatic cancer cells, of which the majority is functionally related to cellular movement.     

Quantitative proteome profiling of respiratory virus-infected lung epithelial cells

Respiratory virus infections are among the primary causes of morbidity and mortality in humans. Influenza virus, respiratory syncytial virus (RSV), parainfluenza (PIV) and human metapneumovirus (hMPV) are major causes of respiratory illness in humans. Especially young children and the elderly are susceptible to infections with these viruses. In this study we aim to gain detailed insight into the molecular pathogenesis of respiratory virus infections by studying the protein expression profiles of infected lung epithelial cells.A549 cells were exposed to a set of respiratory viruses [RSV, hMPV, PIV and Measles virus (MV)] using both live and UV-inactivated virus preparations. Cells were harvested at different time points after infection and processed for proteomics analysis by 2-dimensional difference gel electrophoresis. Samples derived from infected cells were compared to mock-infected cells to identify proteins that are differentially expressed due to infection.We show that RSV, hMPV, PIV3, and MV induced similar core host responses and that mainly proteins involved in defense against ER stress and apoptosis were affected which points towards an induction of apoptosis upon infection. By 2-D DIGE analyses we have gathered information on the induction of apoptosis by respiratory viruses in A549 cells.     

Pattern analysis of serum proteome distinguishes renal cell carcinoma from other urologic diseases and healthy persons

Despite having a relatively low incidence, renal cell carcinoma (RCC) is one of the most lethal urologic cancers. For successful treatment including surgery, early detection is essential. Currently there is no screening method such as biomarker assays for early diagnosis of RCC. Surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF) is a recent technical advance that can be used to identify biomarkers for cancers. In this study, we investigated whether SELDI protein profiling and artificial intelligence analysis of serum could distinguish RCC from healthy persons and other urologic diseases (nonRCC). The SELDI-TOF data was acquired from a total of 36 serum samples with weak cation exchange-2 protein chip arrays and filtered using ProteinChip software. We used a decision tree algorithm c4.5 to classify the three groups of sera. Five proteins were identified with masses of 3900, 4107, 4153, 5352, and 5987 Da. These biomarkers can correctly separate RCC from healthy and nonRCC samples.     

The serum proteome of Equus caballus

We constructed a reference two-dimensional protein map for horse (Equus caballus) serum. The serum proteins were separated by two-dimensional electrophoresis (2-DE); 29 different gene products were identified. Proteins represented by 25 spots/spot groups were identified by tandem nanoelectrospray mass spectrometry (MS), four by matrix-assisted laser desorption ionization time-of-flight (TOF) MS and one was sequenced by TOF-TOF technology. The identities of four proteins were deduced by similarity to the human plasma protein database. In selected cases, i.e. the immunoglobulins, immunoblotting with specific antibodies provided additional information about the respective proteins. Albumin was detected as the full-length protein and as fragments of various sizes. Spots representing products of different mass and charge were also detected for alpha1-antitrypsin, haptoglobin and transthyretin. Thus, despite the fact that the Equus caballus genome is incompletely characterized, we were able to identify almost all moderate to high abundance proteins stained in the serum 2-DE pattern.     

Analysis of the human serum proteome

Changes in serum proteins that signal histopathological states, such as cancer, are useful diagnostic and prognostic biomarkers. Unfortunately, the large dynamic concentration range of proteins in serum makes it a challenging proteome to effectively characterize. Typically, methods to deplete highly abundant proteins to decrease this dynamic protein concentration range are employed, yet such depletion results in removal of important low abundant proteins. A multi-dimensional peptide separation strategy utilizing conventional separation techniques combined with tandem mass spectrometry (MS/MS) was employed for a proteome analysis of human serum. Serum proteins were digested with trypsin and resolved into 20 fractions by ampholyte-free liquid phase isoelectric focusing. These 20 peptide fractions were further fractionated by strong cation-exchange chromatography, each of which was analyzed by microcapillary reversed-phase liquid chromatography coupled online with MS/MS analysis. This investigation resulted in the identification of 1444 unique proteins in serum. Proteins from all functional classes, cellular localization, and abundance levels were identified. This study illustrates that a majority of lower abundance proteins identified in serum are present as secreted or shed species by cells as a result of signalling, necrosis, apoptosis, and hemolysis. These findings show that the protein content of serum is quite reflective of the overall profile of the human organism and a conventional multidimensional fractionation strategy combined with MS/MS is entirely capable of characterizing a significant fraction of the serum proteome. We have constructed a publicly available human serum proteomic database (http://bpp.nci.nih.gov) to provide a reference resource to facilitate future investigations of the vast archive of pathophysiological content in serum. These authors contributed equally to this work.     

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.