Variability of urine proteome of healthy persons during 105-daily isolation

Human proteome is very plastic, it changes under the influence of various biological factors. It is of big interest to find out how specific factors of an environment, including a long-term isolation affect on urine proteome. The study was conducted during the experiment with 105-day isolation. In the present investigation we collected urine samples from 6 healthy volunteers (26-41 years old). The physical activity, daily rhythms and diet were controlled. Urine samples were fractionated on magnetic beads MB-HIC C8 (MB - hydrophobic-interaction chromatography) with ClinProt robot (Bruker Daltonics) prior to MALDI-TOF mass spectrometer analysis with Autoflex III TOF/TOF (Bruker Daltonics), working in a positive linear mode. 117 peaks have been obtained in each spectrum of urine. We have shown that even during isolation and under controlled conditions of life a high variability of urine proteome of healthy personas (36 protein MC-peaks in the urine, on average) are revealed.     

Variability of low-molecular-weight serum subproteome in healthy humans under the conditions of normal vital activity

To study interindividual variability of the low-molecular-weight serum subproteome in healthy humans, the proteome profiles of blood sera were studied in subjects divided into three age groups: from 20 to 30 years (36 subjects), from 30 to 40 years (11 subjects), and from 40 to 50 years (11 subjects). Serum samples were fractionated by MB WCX magnetic beads using a ClintProt robot prior to the mass spectrometry based profiling. The mass spectra have been obtained using an Autoflex III time-of-flight mass spectrometer (Bruker Daltonics) in the automated mode. The low-molecular-weight serum subproteome in healthy humans was found to be characterized by significant interindividual variability: 21% of all the peaks in the proteome profiles had a coefficient of variation of more than 50%, and 29% of all the peaks had a low variance (CV < 30%). Therefore, the majority of the peaks in the proteome profile had a moderate group variation (the CV was in the interval from 30 to 50%). Fragments of high-molecular-weight kininogen, inter-α-trypsin inhibitor, C3 and C4a complement components, CI apolipoprotein, platelet factor IV, β2-microglobulin, and C cystatin were shown to display a wide variation among the tested groups of healthy humans. The peak area variance of high-molecular-weight kininogen, inter-α-trypsin inhibitor, AII and CIII apolipoproteins increased with age.     

Variability in low-molecular subproteome of blood serum of healthy man in normal life conditions

For analysis of inter-individual variability in low-molecular serum subproteome proteome profiles of healthy men at the age of 20-30 years (36 subjects), 30-40 years (11 subjects) and 40-50 years (11 subjects) were obtained. Serum samples were fractionated on magnetic beads MB WCX using ClinProt robot prior to mass-spectrometry based profiling. Mass-spectra were obtained with time-of-flight mass-spectrometer Autoflex III ("Bruker Daltonics") in automatic mode. It was shown that low-molecular serum subproteome of healthy humans was characterized by significant inter-individual variability. 21% of all peaks in proteome profiles had coefficient of variation more than 50% and 29% of all peaks had low dispersion (CV < 30%).Therefore majority of peaks in proteome profile were peaks with moderate inter-individual variability (CV from 30% to 50%). Fragments of high-molecular kininogen, inter-alpha-trypsin inhibitor, complement components C3 and C4a, apolipoprotein CI, platelet factor IV, beta2-microglobulin and cystatin C showed wide variation among examined groups of healthy men. Dispersion of high-molecular kininogen, inter-alpha-trypsin inhibitor, apolipoproteins AII and CIII peaks increased with age.     

Urine sex steroid profile in healthy volunteers during isolation in a pressurized compartment

In order to study the time course of the parameters of urine sex steroid profile and its potential boundary changes, the quantitative determination of a number of endogenous steroids and their metabolites in healthy human urine has been carried out by the gas chromatography method using mass-selective detection. The samples were obtained from six volunteers under the conditions of total monitoring of vital activity factors affecting urine steroid profile (diet, water consumption, physical activity, temperature and air composition, day-night rhythm, and the psychoemotional state) in an experimental study using a pressurized compartment. The healthy human profile parameters of urine steroids were found, which were affected by conditions of controlled vital activity in a pressurized compartment. Parameters of the individual and group variability of the parameters of steroid profile and their dependence on experimental factors, salt consumption mode, and autonomous vital activity, were found.     

Profile of sex steroids in healthy volunteers' urine during experiment in isolated object

The quantitative determination a number of endogenous steroids and their metabolites in urine of healthy volunteers by means of gas chromatography - mass spectrometry was performed. The dynamic of steroid profile of healthy individuals as well as possible ranges of several endogenous steroid parameters have been investigated. Samples were obtained during 105-days experiment with 6 volunteers in isolated on ground modules where were modeling the main life conditions which could influence the steroid profile: meal volume and composition, water consumption, motion activity, air composition and temperature, rate sleep - wakefulness and emotional tension. The parameters of urine steroid profile of healthy volunteer which were affected by life conditions in isolated object were revealed. The parameters of individual and group variability of steroid profile and its dependence from definite experiment conditions - change of salt consumption periods, autonomy of vital activity were detected.     

Dynamic changes of urinary proteins in a rat model of acute hypercoagulable state induced by tranexamic acid

The hypercoagulable state leads to the development of thrombotic diseases, but it is difficult to diagnose due to the lack of available biomarkers. This study aimed to investigate systematic changes of the urinary proteome in the acute hypercoagulable state. A rat model of the acute hypercoagulable state was induced by an antifibrinolytic agent tranexamic acid and urine samples were collected for proteomic analysis by liquid chromatography-tandem mass spectrometry. A total of 28 differential proteins were detected in the urinary proteome of the model rats, of which 12 had been previously considered as candidate biomarkers such as myoglobin, and 10 had been considered stable in healthy human urine. Of the 28 differentially expressed proteins 18 had counterparts in humans. Of these 18 proteins, 10 were members of the human core urinary proteome distributed in a variety of human tissues but concentrated in the urinary and digestive systems. Fumarylacetoacetase was verified as a potential marker of the acute hypercoagulable state by Western blot analysis. In conclusion, urine proteome analysis is a powerful approach to identify potential biomarkers of acute hypercoagulable state.     

Analysis of the variability of human normal urine by 2D-GE reveals a "public" and a "private" proteome

The characterization of the normal urinary proteome is steadily progressing and represents a major interest in the assessment of clinical urinary biomarkers. To estimate quantitatively the variability of the normal urinary proteome, urines of 20 healthy people were collected. We first evaluated the impact of the sample conservation temperature on urine proteome integrity. Keeping the urine sample at RT or at +4°C until storage at -80°C seems the best way for long-term storage of samples for 2D-GE analysis. The quantitative variability of the normal urinary proteome was estimated on the 20 urines mapped by 2D-GE. The occurrence of the 910 identified spots was analysed throughout the gels and represented in a virtual 2D gel. Sixteen percent of the spots were found to occur in all samples and 23% occurred in at least 90% of urines. About 13% of the protein spots were present only in 10% or less of the samples, thus representing the most variable part of the normal urinary proteome. Twenty proteins corresponding to a fraction of the fully conserved spots were identified by mass spectrometry. In conclusion, a "public" urinary proteome, common to healthy individuals, seems to coexist with a "private" urinary proteome, which is more specific to each individual.     

The Urine Proteome Profile Is Different in Neuromyelitis Optica Compared to Multiple Sclerosis: A Clinical Proteome Study

Objectives

Inflammatory demyelinating diseases of the CNS comprise a broad spectrum of diseases like neuromyelitis optica (NMO), NMO spectrum disorders (NMO-SD) and multiple sclerosis (MS). Despite clear classification criteria, differentiation can be difficult. We hypothesized that the urine proteome may differentiate NMO from MS.

Methods

The proteins in urine samples from anti-aquaporin 4 (AQP4) seropositive NMO/NMO-SD patients (n = 32), patients with MS (n = 46) and healthy subjects (HS, n = 31) were examined by quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) after trypsin digestion and iTRAQ labelling. Immunoglobulins (Ig) in the urine were validated by nephelometry in an independent cohort (n = 9–10 pr. groups).

Results

The analysis identified a total of 1112 different proteins of which 333 were shared by all 109 subjects. Cluster analysis revealed differences in the urine proteome of NMO/NMO-SD compared to HS and MS. Principal component analysis also suggested that the NMO/NMO-SD proteome profile was useful for classification. Multivariate regression analysis revealed a 3-protein profile for the NMO/NMO-SD versus HS discrimination, a 6-protein profile for NMO/NMO-SD versus MS discrimination and an 11-protein profile for MS versus HS discrimination. All protein panels yielded highly significant ROC curves (AUC in all cases >0.85, p≤0.0002). Nephelometry confirmed the presence of increased Ig-light chains in the urine of patients with NMO/NMO-SD.

Conclusion

The urine proteome profile of patients with NMO/NMO-SD is different from MS and HS. This may reflect differences in the pathogenesis of NMO/NMO-SD versus MS and suggests that urine may be a potential source of biomarkers differentiating NMO/NMO-SD from MS.     

Selection of the peptide mass tolerance value for the protein identification with peptide mass fingerprinting

Peptide mass-fingerprint is widely used for protein identification while studying proteome with the use of 1D or 2D electrophoresis. Peptide mass tolerance indicates the fit of theoretical peptide mass with the experimental measurements, and choice of this parameter sufficiently influences the protein identification. The role of peptide mass tolerance was estimated by counting the number of identified proteins for the reference set of mass-spectra. The reference set of 400 Ultraflex (Bruker Daltonics, Germany) mass-spectra was obtained for the slices of 1D gel of liver microsomes. Using Mascot server for protein identification, the peptide mass tolerance value was varied in the range from 0.02 to 0.40 Da with a step 0.01 Da. Depending on the tolerance the number of identified protein changes up to 10 times. Maximal number of identified proteins was reported for the tolerance value of 0.15 Da (120 ppm), which is 1.5 - 2 times higher than the recommended values for such type of mass-spectrometers. The software program PMFScan was developed to obtain the dependence of number of identified proteins of the tolerance values.     

Constant urinary proteins in healthy humans in a 520-day isolation experiment

The aim of this study was the search of permanent proteins of the urinary proteome during a 520-day isolation experiment at the Institute of Biomedical Problems (IBMP) Ground-Based Test Facility in controlled conditions, using an autonomous life support system. The object of the study was urine sampled from 6 normal male subjects aged 25 to 37. The biological material samples (the second morning urine fractions) were collected for proteomic investigations against the background, on the 50th, 93rd, 124th, 153rd, 180th, 251st, 274th, 303rd, 330th, 371st, 400th, and 427th days of isolation, and on the 7th day after its completion. The samples were analyzed using chromatography–mass spectrometry, while the obtained results were analyzed using bioinformatics resources. The following seven permanent proteins were identified and observed during the entire period of urine investigations: epidermal growth factor, polymeric immunoglobulin receptor, plasma serine protease inhibitor, Alpha 1 microglobulin/bikunin precursor (AMBP), keratin (type II cytoskeletal 1), collagen alpha-1 (VI) chain, and serum albumin.     

Selection of the peptide mass tolerance value for protein identification with peptide mass fingerprinting

Peptide mass fingerprinting (PMF) is widely used for protein identification while studying proteome via time-of-flight mass spectrometer or via 1D or 2D electrophoresis. Peptide mass tolerance indicating the fit of theoretical peptide mass to an experimental one signifcantly influences protein identification. The role of peptide mass tolerance could be estimated by counting the number of correctly identified proteins for the reference set of mass spectra. The reference set of 400 Ultraflex (Bruker Daltonics, Germany) protein mass spectra was obtained for liver microsomes slices hydrolyzed via 1D gel electrophoresis. Using a Mascot server for protein identification, the peptide mass tolerance value varied within 0.02–0.40 Da with a step of 0.01 Da. The number of identified proteins changed up to 10 times depending on the tolerance. The maximal number of identified proteins was reported for the tolerance value of 0.15 Da (120 ppm) known to be 1.5–2-fold higher than the recommended values for such a type of mass spectrometer. The software program PMFScan was developed to obtain the dependence between the number of identified proteins and the tolerance values.     

Reconstruction of associative protein networks connected with processes of sodium exchange regulation and sodium deposition in healthy volunteers based on urine proteome analysis

The study was conducted during the experiment with a 105-day isolation in an experimental complex. Urine samples were collected from six healthy volunteers. The physical activity, diurnal rhythm, temperature parameters, and levels of oxygen and carbon dioxide were controlled during the experiment. According to the program, food intake (electrolysis, water, calories, fat, carbohydrates, protein, vitamins, etc.) at each stage of the experiment was normalized to the body weight of each subject. All samples were analyzed using an LTQ FT MS ionic cyclotron resonance mass spectrometer with Fourier transform (Thermo) on the basis of the accurate mass and time (AMT) tag approach. Among more than 20 000 peptides, 690 proteotypical proteins were found. A total of 600 urine proteins were identified to be included in the database of healthy human urine proteins. For physiological interpretation of the proteome data, computer ANDCell and AND-Viso systems were used. Clustering of proteins and the application of these systems revealed proteins that were most closely associated with the regime of sodium intake and allowed building the network of their interactions.     

Reconstruction of associative protein networks connected with processes of sodium exchange' regulation and sodium deposition in healthy volunteers by urine proteome analysis

The study was conducted during the experiment with 105-day isolation in experimental complex. In the present investigation we collected urine samples from 6 healthy volunteers. The physical activity, diurnal rhythm, temperature parameters and level of oxygen and carbon dioxide were controlled during the experiment. According to the program, food intake (electrolytes, water, calories, fat, carbohydrates, protein, vitamins, etc.) on each stage of experiment was normalized. All samples were analyzed using mass spectrometer of an ionic cyclotron resonance with transformation of Fure LTQ FT MS (Thermo) on the basis of the AMT-tags (accurate mass and time tags) approach. Among more than 20 000 we found out 690 proteotypical proteins and we identified about 600 urine proteins. For physiological interpretation of the proteome data we used computer systems ANDCell and ANDVisio. Clustering of proteins and application of these systems revealed proteins that are most closely associated with the regime of sodium intake, as well as build the network of their interactions.     

Detection of renal and urinary tract proteins in urine before and after space flight

The urine protein composition (proteome) of healthy humans was analyzed using proteomic techniques to obtain data under normal physiological conditions and after six-month space flights. It was shown that, after long space flights, specific minor proteins are revealed in cosmonauts’ urine that can be identified as proteins coming from kidneys and urinary tracts.     

Rapid MALDI biotyper-based identification and cluster analysis of <Emphasis Type="Italic">Streptococcus iniae</Emphasis>

Streptococcus iniae causes severe mortalities among cultured marine species, especially in the olive flounder (Paralichthys olivaceus), which is economically important in Korea and Japan. Recently, there has been growing concern regarding the emergence of S. iniae as a zoonotic pathogen. Here, 89 S. iniae isolates obtained from diseased olive flounders collected from 2003 to 2008 in Jeju Island, South Korea, were characterized using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The results were aligned both with the available Bruker Daltonics data-base and with a new set of S. iniae data entries developed in our laboratory, and the results were compared. When we used the Bruker Daltonics database, the 89 isolates yielded either “no reliable identification” or were incorrectly identified as Streptococcus pyogenes at the genus level. When we used the new data entries from our laboratory, in contrast, all of the isolates were correctly identified as S. iniae at the genus (100%) and species (96.6%) levels. We performed proteomic analysis, divided the 89 isolates into cluster I (51.7%), cluster II (20.2%), and cluster III (28.1%), and then used the MALDI Biotyper software to identify specific mass peaks that enabled discrimination between clusters and between Streptococcus species. Our results suggest that the use of MALDI TOF MS could outperform the conventional methods, proving easier, faster, cheaper and more efficient in properly identifying S. iniae. This strategy could facilitate the epidemiological and taxonomical study of this important fish pathogen.     

2014蛋白质组学专刊序言

蛋白质组学研究是后基因组学时代最重要的功能基因组学研究之一,与医学生物学、化学、物理学、信息学以及现代技术等关系十分密切。为了检阅近年来国内外蛋白质组学某些重要研究进展,探索其可能的应用范围,讨论其存在的问题,展望其发展前景,特组织出版"蛋白质组学专刊"。本期专刊包括综述和研究论文两部分,内容主要涉及不同物种(包括人类、哺乳类动物、原核生物、放线菌等)蛋白质组学研究、蛋白质组学重要方法学与技术研究(包括串联质谱分析、尿蛋白膜保存法、定量蛋白质组学分折、meta分析等)和蛋白质组功能与应用研究(包括蜘蛛毒素蛋白质组、磷酸化蛋白质组、卵母细胞和早期胚胎蛋白质组、肝脏纤维化蛋白质组、分枝杆菌耐药的蛋白质组等)。     

Optimization of Plasma Sample Pretreatment for Quantitative Analysis Using iTRAQ Labeling and LC-MALDI-TOF/TOF

Shotgun proteomic methods involving iTRAQ (isobaric tags for relative and absolute quantitation) peptide labeling facilitate quantitative analyses of proteomes and searches for useful biomarkers. However, the plasma proteome''s complexity and the highly dynamic plasma protein concentration range limit the ability of conventional approaches to analyze and identify a large number of proteins, including useful biomarkers. The goal of this paper is to elucidate the best approach for plasma sample pretreatment for MS- and iTRAQ-based analyses. Here, we systematically compared four approaches, which include centrifugal ultrafiltration, SCX chromatography with fractionation, affinity depletion, and plasma without fractionation, to reduce plasma sample complexity. We generated an optimized protocol for quantitative protein analysis using iTRAQ reagents and an UltrafleXtreme (Bruker Daltonics) MALDI TOF/TOF mass spectrometer. Moreover, we used a simple, rapid, efficient, but inexpensive sample pretreatment technique that generated an optimal opportunity for biomarker discovery. We discuss the results from the four sample pretreatment approaches and conclude that SCX chromatography without affinity depletion is the best plasma sample preparation pretreatment method for proteome analysis. Using this technique, we identified 1,780 unique proteins, including 1,427 that were quantified by iTRAQ with high reproducibility and accuracy.     

Mealtime,Temporal, and Daily Variability of the Human Urinary and Plasma Metabolomes in a Tightly Controlled Environment

While metabolomics has tremendous potential for diagnostic biomarker and therapeutic target discovery, its utility may be diminished by the variability that occurs due to environmental exposures including diet and the influences of the human circadian rhythm. For successful translation of metabolomics findings into the clinical setting, it is necessary to exhaustively define the sources of metabolome variation. To address these issues and to measure the variability of urinary and plasma metabolomes throughout the day, we have undertaken a comprehensive inpatient study in which we have performed non-targeted metabolomics analysis of blood and urine in 26 volunteers (13 healthy subjects with no known disease and 13 healthy subjects with autosomal dominant polycystic kidney disease not taking medication). These individuals were evaluated in a clinical research facility on two separate occasions, over three days, while on a standardized, weight-based diet. Subjects provided pre- and post-prandial blood and urine samples at the same time of day, and all samples were analyzed by “fast lane” LC-MS-based global metabolomics. The largest source of variability in blood and urine metabolomes was attributable to technical issues such as sample preparation and analysis, and less variability was due to biological variables, meals, and time of day. Higher metabolome variability was observed after the morning as compared to the evening meal, yet day-to-day variability was minimal and urine metabolome variability was greater than that of blood. Thus we suggest that blood and urine are suitable biofluids for metabolomics studies, though nontargeted mass spectrometry alone may not offer sufficient precision to reveal subtle changes in the metabolome. Additional targeted analyses may be needed to support the data from nontargeted mass spectrometric analyses. In light of these findings, future metabolomics studies should consider these sources of variability to allow for appropriate metabolomics testing and reliable clinical translation of metabolomics data.     

Proteomic profiling of urine for the detection of colon cancer

Background

Colorectal cancer is the second most common cause of cancer related death in the developed world. To date, no blood or stool biomarkers with both high sensitivity and specificity for potentially curable early stage disease have been validated for clinical use. SELDI and MALDI profiling are being used increasingly to search for biomarkers in both blood and urine. Both techniques provide information predominantly on the low molecular weight proteome (<15 kDa). There have been several reports that colorectal cancer is associated with changes in the serum proteome that are detectable by SELDI and we hypothesised that proteomic changes would also be detectable in urine.

Results

We collected urine from 67 patients with colorectal cancer and 72 non-cancer control subjects, diluted to a constant protein concentration and generated MALDI and SELDI spectra. The intensities of 19 peaks differed significantly between cancer and non-cancer patients by both t-tests and after adjusting for confounders using multiple linear regressions. Logistic regression classifiers based on peak intensities identified colorectal cancer with up to 78% sensitivity at 87% specificity. We identified and independently quantified 3 of the discriminatory peaks using synthetic stable isotope peptides (an 1885 Da fragment of fibrinogen and hepcidin-20) or ELISA (β2-microglobulin).

Conclusion

Changes in the urine proteome may aid in the early detection of colorectal cancer.     

Establishment of a near-standard two-dimensional human urine proteomic map

A proteomic map for human urine on two-dimensional (2-D) gels has been developed. Initial studies demonstrated that the urine proteins prepared by conventional methods showed interference and poor reproducibility in 2-D electrophoresis (2-DE). To address this issue, urine samples were dialyzed to remove any interfering molecules. The dialysis of urine proteins and the concentration by lyophilization without fractionation significantly improved the reproducibility and resolution and likely represents the total urine proteins on a 2-D gel. In addition, removing albumin from urine using Affi-Gel Blue helped to identify the low-abundant proteins. Using the developed method, we prepared proteins from urine collected from healthy females and males. The large inter- and intra-subject variation in protein profiles on 2-D gels made it difficult to establish a normal human urine proteomic 2-D map. To resolve this problem, urinary proteins were prepared from the pooled urine collected from 20 healthy females and males, respectively. The established male and female urine proteomes separated on 2-D gels were almost identical except for some potential sex-dependent protein spots. We have annotated 113 different proteins on the 2-D gel by peptide mass fingerprinting (PMF). We propose that the established total urine proteome can be used for 2-DE analysis, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and identification of novel disease-specific biomarkers.