Enrichment of the basic/cationic urinary proteome using ion exchange chromatography and batch adsorption

Anionic or acidic proteins are the main compositions of normal urinary proteome. Efforts to characterize human urinary proteome, thus, have focused mainly on the anionic compartment. The information of cationic or basic proteins present in the normal urine is virtually unknown. In the present study, we applied different methods to enrich cationic urinary proteome. Efficacies of these methods were compared using equal volume (1 L) of urine samples from the same pool obtained from 8 normal healthy individuals. Cation exchange chromatography using RESOURCE-S column provided the least amount of the recovered proteins, whereas batch adsorption using SP Sepharose 4 Fast Flow beads equilibrated with acetic acid (pH 4.8) provided the greatest yield of protein recovery. The recovered proteins were then resolved with 2-DE (pI 7-11) and identified by peptide mass fingerprinting using MALDI-TOF MS. There were several isoforms of immunoglobulin heavy and light chains enriched by these methods. In addition, three isoforms of interferon alpha-3 (IFNalpha3) and six isoforms of eosinophil-derived neurotoxin (EDN), were also enriched. The enrichment of IFNalpha3 and EDN was particularly effective by batch adsorption using SP Sepharose 4 Fast Flow beads equilibrated with acetic acid (pH 6.0). Initial depletion of anionic components using DEAE batch adsorption reduced the recovery yield of these two proteins and did not improve recovery of any other cationic urinary proteins. We conclude that batch adsorption using SP Sepharose Fast Flow beads equilibrated with acetic acid (pH 6.0) is the method of choice to examine the basic/cationic urinary proteome, as this protocol provided the satisfactory yield of protein recovery and provided the greatest amount as well as maximal number of IFNalpha3 and EDN isoforms. Our data will be useful for further highly focused study targeting on cationic/basic urinary proteins. Moreover, the techniques described herein may be applicable for enrichment of cationic proteomes in other body fluids, cells, and tissues.     

Simple urinary sample preparation for proteomic analysis

Since the completion of the human genome sequence, attention has now focused on establishing reference maps of body fluids such as plasma and urine for detecting diagnostic markers of disease. Although some progress has been made, challenges still remain in the development of an optimal sample preparation method for proteomic analysis of urine. We have developed a simple and efficient urine preparation method for two-dimensional (2-D) gel electrophoresis which involves precipitation of proteins with simultaneous desalting. Acetonitrile precipitation produced 2-D gel separations with the highest resolution and the greatest number of protein spots compared to precipitation by other organic solvents. The method was applied to observe changes in the urinary proteome over a 6 week period and to establish a reference map of a healthy subject. A total of 339 proteins from 159 genes was identified from healthy male urine by peptide mass fingerprinting. The profiles of the urinary proteome at three times in 1 day and on four different days were compared and were found to vary in number and spatial location of the proteins on the map. The method was also shown to be applicable to the higher concentrations of protein found in the urine of an ovarian cancer subject. We have developed a facile and robust method for preparing urine for 2-D gels that will encourage further use of urine.     

Evaluation of three different protocols of protein extraction for Arabidopsis thaliana leaf proteome analysis by two-dimensional electrophoresis

This work was performed to compare three precipitation protocols of protein extraction for 2-DE proteomic analysis using Arabidopsis leaf tissue: TCA-acetone, phenol, and TCA-acetone-phenol. There were no statistically significant differences in protein yield between the three methods. Samples were subjected to 2-DE in the 5 to 8 pH and 14-80 kDa ranges. The TCA-acetone-phenol protocol provided the best results in terms of spot focusing, resolved spots, spot intensity, unique spots detected, and reproducibility. In all, 93 qualitative or quantitative statistically significant differential spots were found between the three protocols. The 2-DE map of TCA-acetone-phenol extracts presented more resolved spots above 40 kDa, with no pI-dependent differences observed between the three protocols. 54 spots were selected for trypsin digestion, and the peptides were analyzed by MALDI-TOF-TOF MS. After database search using peptide mass fingerprinting, and MS/MS combined search, 30 proteins were identified, the proteins from chloroplastic photosynthetic and carbohydrate metabolism being those most highly represented. From these data, we were able to conclude that each extraction protocol had its main features. Considering this, the workflow of any standard comparative proteomic experiment should include the optimization and adaptation of the protein extraction protocol to the plant tissue and to the particular objective pursued.     

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.     

Variation in protein levels obtained from human blood cells and biofluids for platelet, peripheral blood mononuclear cell, plasma, urine and saliva proteomics

Blood cells and biofluid proteomics are emerging as a valuable tool to assess effects of interventions on health and disease. This study is aimed to assess the amount and variability of proteins from platelets, peripheral blood mononuclear cells (PBMC), plasma, urine and saliva from ten healthy volunteers for proteomics analysis, and whether protein yield is affected by prolonged fasting. Volunteers provided blood, saliva and morning urine samples once a week for 4 weeks after an overnight fast. Volunteers were fasted for a further 24 h after the fourth sampling before providing their final samples. Each 10 mL whole blood provided 400–1,500 μg protein from platelets, and 100–600 μg from PBMC. 30 μL plasma depleted of albumin and IgG provided 350–650 μg protein. A sample of morning urine provided 0.9–8.6 mg protein/dL, and a sample of saliva provided 70–950 μg protein/mL. None of these yields were influenced by the degree of fasting (overnight or 36 h). In conclusion, in contrast to the yields from plasma, platelets and PBMC, the protein yields of urine and saliva samples were highly variable within and between subjects. Certain disease conditions may cause higher or lower PBMC counts and thus protein yields, or increased urinary protein levels.     

Different techniques for urinary protein analysis of normal and lung cancer patients

Many components in urine are useful in clinical diagnosis and urinary proteins are known as important components to define many diseases such as proteinuria, kidney, bladder and urinary tract diseases. In this study, we focused on the comparison of different sample preparation methods for isolating urinary proteins prior to protein analysis of pooled healthy and lung cancer patient samples. Selective method was used for preliminary investigation of some putative urinary protein markers. Urine samples were passed first through a gel filtration column (PD-10 desalting column) to remove high salts and subsequently concentrated. Remaining interferences were removed by ultrafiltration or four precipitation methods. The analysis of urinary proteins by high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed many similarities in profiles among preparation methods and a few profiles were different between normal and lung cancer patients. In contrast, the results of two-dimensional gel electrophoresis (2-DE) showed more distinctly different protein patterns. Our finding showed that the sequential preparation of urinary proteins by gel filtration and ultrafiltration could retain most urinary proteins which demonstrated the highest protein spots on 2-D gels and able to identify preliminary urinary protein markers related to cancer. Although sequential preparation of urine samples by gel filtration and protein precipitation resulted in low amounts of proteins on 2-D gels, high Mr proteins were easily detected. Therefore, there are alternative choices for urine sample preparation for studying the urinary proteome and identifying urinary protein markers important for further preclinical diagnostic and therapeutic applications.     

Evaluation of sample extraction methods for proteomic analysis of coniferous seeds

In this study, three methods of protein extraction from the seeds of the Chinese fir were compared by examining the quality (including the number of protein spots observed) in the two-dimensional gel electrophoresis (2-DE), obtained by isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis of the total released protein. Three protein extraction methods were: TCA-acetone precipitation, SDS extraction/acetone precipitation, and phenol extraction methanol/ammonium acetate precipitation. The results showed that TCA-acetone precipitation was the most effective method for protein extraction; it gave the highest yield of total protein (8.9 mg protein per g seed weight) and the greatest number of proteins spots (1,034 spots) on the 2-DE gel. Further, several proteins were identified by liquid chromatography mass spectrometry (LC MS/MS), which are legumin-like storage protein, similar to AMP binding/acetate-CoA ligase, similar to 40S ribosomal protein S20, actin, ascorbate peroxidase, Similar to cysteine synthase, and unknown protein. These data demonstrates that TCA-acetone precipitation followed by 2-DE and LC MS/MS is a suitable method for proteomic analysis of coniferous species, such as Chinese fir and provides a valuable starting point for similar proteomic analysis of other coniferous tree species.     

Variation in the monocyte proteome

We have determined the variability of the monocyte proteome and identified those proteins that demonstrate the greatest variation in the general control population. Monocytes were isolated from 18 healthy (9 male and 9 female) donors ages 18-50 and with no known genetic or blood disorder. A combination of Ficoll-Paque PLUS density centrifugation of cells found in the buffy coat and positive selection with monoclonal antibodies against CD14, coupled to magnetic beads, led to >98% purity of monocytes. A 100,000 g microsomal membrane fraction or 100,000 g supernatant fraction from a control subject was compared to the equivalent fractions from a distinct control subject by two-dimensional differential gel electrophoresis (2D DIGE). Those protein spots that demonstrated Cy3-/Cy5- ratios greater than 2.5-fold in at least one experiment were selected for further statistical analysis. We determined variability for 31 cytosolic and 12 membrane protein spots. Proteins have been identified for 27 of the cytosolic protein spots and 9 of the microsomal membrane protein spots by in-gel digestion with trypsin followed by reverse-phase high-performance liquid chromatography in line with tandem mass spectrometry. We identified 24 distinct monocyte proteins that demonstrated the greatest variability in this general control population. The proteins demonstrating the greatest variance in the cytosolic fraction were enolase-1 and WD (tryptophan-aspartate) repeat-containing protein 1, and in the membrane fraction they were lamin B1 and L-plastin. This study demonstrates the importance of considering variance in the control population when performing future protein profiling comparisons of monocytes derived from disease versus control populations.     

Comparison of methods for sample preparation of individual rat cerebrospinal fluid samples prior to two-dimensional polyacrylamide gel electrophoresis

Different pre-treatment methods have been compared for two-dimensional mapping of individual rat cerebrospinal fluid samples based on acetone, trichloroacetic acid/acetone and methanol/acetone precipitation of proteins. Acetone precipitation following incubation with DTT gave the highest protein recovery (72%) and the largest number of protein spots (92 +/- 4) as well as minimizing the time taken.     

Proteome analysis of rat hippocampal neurons by multiple large gel two-dimensional electrophoresis

The goal of the present study was to detect as many protein spots as possible in mammalian cells using two-dimensional gel electrophoresis (2-DE). For proteome analysis, it is of importance to reveal as many proteins as possible. A single standard 2-DE gel (pH 3-10, 18 cm x 20 cm, 13.5% gel) could detect 853 spots from proteins of cultured rat hippocampal neurons when visualized by silver staining. To increase the resolution of the separation and the number of detectable proteins by 2-DE, we utilized seven different narrow pH range immobilized pH gradients in the first dimension. In the second dimension, fourteen long SDS polyacrylamide gels were used: seven 7.5% gels for the separation of high molecular mass proteins (> or = 40 kDa) and seven 13.5% gels for the separation of low molecular mass proteins (< or = 40 kDa). Three hundred and sixty microg of proteins from cultured hippocampal neurons were loaded on to individual gels and visualized by silver staining. All 14 gel images were assembled into a 70 cm x 67 cm cybergel that contained 6677 protein spots, thereby indicating that the utilization of the present strategy led to a 783% increase in the number of detected spots in comparison to the standard procedure. Loading double the amount (720 microg) of proteins on to a 13.5% gel led to a 184% increase in the number of detected spots, thereby indicating that the present strategy has a potential to display more protein spots in the cybergels.     

基于信息熵的新疆降水时空变异特征研究

基于信息熵理论对新疆降水序列的时空变异性进行研究。利用边际熵研究不同时间尺度降水序列的变化特征。利用分配熵和强度熵分别研究降水量和降水天数年内和年代际(10a)分配情况。利用改进的Mann-Kendall趋势检验法分析新疆降水过程不确定性变化趋势。研究表明:(1)新疆降水量与降水天数年内分配不均匀性主要表现为由南向北减小的空间分布特征;(2)新疆不同尺度的降水序列不确定性具有明显空间特征;(3)越降水越稀少的地区,降水量与降水天数变异性就越大。本研究对该区域降水时空变异研究与水资源规划具有重要意义。     

Analysis of grape berry cell wall proteome: a comparative evaluation of extraction methods

Different methods were tested for the extraction of proteins from the cell wall-enriched fraction (CWEf) obtained from a sample formed by skin and seeds of ripe berries of Vitis vinifera L. cv. Cabernet Sauvignon. The CWEf was isolated using a disruptive approach that involves tissue homogenization and precipitation by centrifugation. To extract proteins, the CWEf was treated with CaCl(2) and LiCl in two successive steps or, alternatively, with phenol. The efficiency of the protocols was evaluated by measuring protein yield and by analyzing two-dimensional gel electrophoresis (2-DE) gels for the highest detectable spot number and the greatest spot resolution. The phenol method was also adopted for the extraction of proteins from the cytosolic fraction (CYf). The comparison of 2-DE reference maps of protein extracts from CWEf and CYf indicated the presence of both common traits and unique characteristics. To survey this aspect some spots detected in both fractions or present in only one fraction were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Of the 47 spots identified, some were found to be cell wall proteins, while others were proteins not traditionally considered as localized in the apoplastic space. The data presented here provide initial information regarding the apoplastic proteome of grape berry tissues, but also raise the issue of the technical problems that characterize the isolation of cell wall proteins from these very hardy tissues.     

Uncovering the unfoldome: enriching cell extracts for unstructured proteins by acid treatment

A method to enrich cell extracts in totally unfolded proteins was investigated. A literature search revealed that 14 of 29 proteins isolated by their failure to precipitate during perchloric acid (PCA) or trichloroacetic acid (TCA) treatment where also shown experimentally to be totally disordered. A near 100 000-fold reduction in yield was observed after 5% or 9% PCA treatment of total soluble E. coli protein. Despite this huge reduction, 158 and 142 spots were observed from the 5% and the 9% treated samples, respectively, on silver-stained 2-D SDS-PAGE gels loaded with 10 microg of protein. Treatment with 1% PCA was less selective with more visible spots and a greater than 3-fold higher yield. A substantial yield of unprecipitated protein was obtained after 3% TCA treatment, suggesting that the common use of TCA precipitation prior to 2-D gel analysis may result in loss of unstructured protein due to their failure to precipitate. Our preliminary analysis suggests that treating total protein extracts with 3-5% PCA and determining the identities of soluble proteins could be the starting point for uncovering unfoldomes (the complement of unstructured proteins in a given proteome). The 100 000-fold reduction in yield and concomitant reduction in number of proteins achieved by 5% PCA treatment produced a fraction suitable for analysis in its entirety using standard proteomic techniques. In this way, large numbers of totally unstructured proteins could be identified with minimal effort.     

Establishment of a 2-D human urinary proteomic map in IgA nephropathy

Immunoglobulin A nephropathy (IgAN) is the most common form of immune complex-mediated glomerulonephritis worldwide. Although chronic renal failure develops in considerable numbers of IgAN patients, the exact etiology has not yet been clearly elucidated. To establish the urinary protein map of IgAN, we performed a urinary proteomic analysis. Thirteen patients with IgAN and 12 normal controls were recruited. Morning midstream spot urine samples were used with Centriprep ultrafiltration for concentration and desalting. 2-DE was performed and compared between IgAN and normal control, and urinary proteins were identified by MALDI-TOF MS. A large number of protein spots were identified in IgAN and normal control samples, with means of 311 spots and 174 spots, respectively. Approximately 216 protein spots were detected as differentially expressed in IgAN. Among these, 82 spots were over-expressed, and 134 spots were under-expressed compared to normal controls. A total of 84 differentially expressed spots, representing 59 different proteins, were finally identified in IgAN. We have established a urinary proteomic map of IgAN and this result helps in the identification. Further study is needed to determine the potential pathogenic role of these proteins.     

Syringe-push membrane absorption as a simple rapid method of urine preparation for clinical proteomics

Background

The analysis of urinary proteome might reveal biomarkers of clinical value. However, current methods of urine preparation for down-stream proteomic analysis are complicated, time-consuming, and/or expensive. This study aims to develop a robust, simple, inexpensive and readily accessible urine preparation method to facilitate clinical proteomic workflow.

Result

Syringe-push membrane absorption (SPMA) was successfully developed by a combination of 5-ml medical syringe and protein-absorbable membrane. Comparing three membranes i.e., nitrocellulose, polyvinylidene difluoride (PVDF) and Whatman no.1, nitrocellulose combined with SPMA (nitrocellulose-SPMA) provided the greatest quality of proteome profile as demonstrated by 2-DE. The quality of the proteome profile and the performance of nitrocellulose-SPMA were systematically compared with three current methods of urine preparation (i.e., ultrafiltration, dialysis/lyophilization and precipitation). While different methods of urine preparation provided comparable proteome quality, nitrocellulose-SPMA had better working performance due to acceptable recovery yield, less workload, short working time, high accessibility and low unit cost. In addition, protein absorbed on nitrocellulose harvested from the SPMA procedure could be stored as a dried membrane at room temperature for at least 1-month without protein degradation or modification.

Conclusions

SPMA is a simple rapid method of preparing urine for downstream proteomic analysis. Because of it is highly accessible and has long storage duration, this technique holds potential benefit for large-scale multi-center research and future development of clinical investigation based upon urinary proteomic analysis.

Electronic supplementary material

The online version of this article (doi:10.1186/s12014-015-9087-4) contains supplementary material, which is available to authorized users.     

Investigating the intra-individual variability in the human metabolic profile of urinary selenium

Selenium is an essential micronutrient widely present in our diet. It plays its role through the selenoproteins. Previous reports have shown marked variation between individuals in the excretion of this trace element, but the intra-individual variability in selenium excretion has not been specifically investigated. The present study investigates the intra-individual variation in the urinary excretion of selenium in a group of healthy volunteers. We also discuss inter-individual variability trends. Urine samples were collected from healthy volunteers without selenium supplementation twice a day for 7 days and then once a week for an additional 7 weeks. A total of 168 urine samples were collected and analyzed for total selenium and individual selenium species using elemental mass spectrometry and HPLC/mass spectrometry, respectively. We found only modest day-to-day and week-to-week intra-individual variation of selenium excretion. Two commonly reported urine metabolites, selenosugar 1 and selenosugar 3, were detected in all urine samples, and our data suggest that selenosugar 3 is a deacetylated product of selenosugar 1 produced in a manner dependent on selenium intake. Trimethylselenonium displayed no intra-individual variability but considerable inter-individual variability in agreement with the involvement of genetic polymorphisms, as recently reported. Se-methylselenoneine was consistently detected in the urine of all volunteers and was a significant metabolite in one volunteer contributing up to 24% of total urinary selenium. Our data indicate that selenium urinary excretion is consistent within an individual, and that intra-individual variation in selenium excretion is unlikely to complicate future inter-individual variation studies.     

Protein extraction from the earthworm Eisenia fetida for 2‐DE

We identified an efficient protocol for extracting proteins from whole earthworm, Eisenia fetida, for 2‐DE. Sample preparation is a critical step in a 2‐DE proteome approach and is absolutely essential for obtaining good results. Six protein extraction protocols based on different protein precipitation agents were tested and evaluated using 2‐DE. The methods generated remarkably different 2‐DE protein spot patterns. We conclude that trichloroacetic acid (TCA)‐A eliminates interfering compounds, thus allowing for the efficient resolubilization of proteins. TCA‐A gives good distinction, more bands in 1‐DE gels, and the most number of protein spots in 2‐DE gels. It is also rapid, provides the higher protein yield, and has the less number of steps. To demonstrate the quality of the extracted proteins, we cut several protein spots that were common to four methods from 2‐DE gels, analyzed them using MALDI‐TOF/TOF MS, and tentatively identified them. The classic TCA‐A method proved to be most useful as a standard method of extracting proteins from E. fetida.     

Proteomic methodological recommendations for studies involving human plasma, platelets, and peripheral blood mononuclear cells

This study was designed to develop, optimize and validate protocols for blood processing prior to proteomic analysis of plasma, platelets and peripheral blood mononuclear cells (PBMC) and to determine analytical variation of a single sample of depleted plasma, platelet and PBMC proteins within and between four laboratories each using their own standard operating protocols for 2D gel electrophoresis. Plasma depleted either using the Beckman Coulter IgY-12 proteome partitioning kit or the Amersham albumin and IgG depletion columns gave good quality gels, but reproducibility appeared better with the single-use immuno-affinity column. The use of the Millipore Filter Device for protein concentration gave a 16% ( p < 0.005) higher recovery of protein in flow-through sample compared with acetone precipitation. The use of OptiPrep gave the lowest level of platelet contamination (1:0.8) during the isolation of PBMC from blood. Several proteins (among which are alpha-tropomyosin, fibrinogen and coagulation factor XIII A) were identified that may be used as biomarkers of platelet contamination in future studies. When identifying preselected spots, at least three out of the four centers found similar identities for 10 out of the 10 plasma proteins, 8 out of the 10 platelet proteins and 8 out of the 10 PBMC proteins. The discrepancy in spot identifications has been described before and may be explained by the mis-selection of spots due to laboratory-to-laboratory variation in gel formats, low scores on the peptide analysis leading to no or only tentative identifications, or incomplete resolution of different proteins in what appears as a single abundant spot. The average within-laboratory coefficient of variation (CV) for each of the matched spots after automatic matching using either PDQuest or ProteomWeaver software ranged between 18 and 69% for depleted plasma proteins, between 21 and 55% for platelet proteins, and between 22 and 38% for PBMC proteins. Subsequent manual matching improved the CV with on average between 1 and 16%. The average between laboratory CV for each of the matched spots after automatic matching ranged between 4 and 54% for depleted plasma proteins, between 5 and 60% for platelet proteins, and between 18 and 70% for PBMC proteins. This variation must be considered when designing sufficiently powered studies that use proteomics tools for biomarker discovery. The use of tricine in the running buffer for the second dimension appears to enhance the resolution of proteins especially in the high molecular weight range.     

The extraction of proteins from eukaryotic ribosomes and ribosomal subunits

Proteins were extracted from rat liver ribosomes and ribosomal subunits: with 67% acetic acid (in the presence of 3.3 mM, 33 mM, or 67 mM Mg) with 2 M LiCL in 4 M urea; with 0.25 N HCI; with 1% SDS; and after RNase digestion. The most efficient extraction and the best recovery were either with acetic acid in the presence of 33 mM or 67 mM Mg, or with LiCI-urea. Protein extracted with acetic acid, LiCi-urea, or with HCI had little or no contamination with RNA. The ribosomal proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis: the proteins extracted with acetic acid were the most soluble in the sample gel solution; their electrophoretograms displayed the maximum number of spots and the smallest number of derivatives or altered proteins. Preparations of protein extracted with SDS or RNase were relatively insoluble in the sample gel solution, and proteins extracted with HCI showed a large number of derivatives. All things considered, the most satisfactory method for the extraction of protein from eukaryotic ribosomes is with 67% acetic acid in the presence of 33 mM MgCl2.     

不同提取方法及上样量对牡丹试管苗茎基部蛋白双向电泳的影响

为建立一套适合于牡丹试管苗茎基部蛋白的双向电泳技术,以便更好地利用蛋白质组技术研究牡丹试管苗不定根的发生机理,本研究比较了三种不同蛋白质提取方法对双向电泳结果的影响,并在蛋白质上样量方面进行了比较。结果表明,乙酸铵/甲醇酚提取法所得2-DE图谱的蛋白点很少,仅检测到45个,且较模糊,有明显的拖尾现象,分辨率很低;乙醇/乙醚丙酮法所得的蛋白点也较少(101个),较模糊,且横竖纹干扰较大;三氯乙酸/丙酮法所得蛋白点数较多,可检测到434个清晰的蛋白点,且形状规则,重复性好,适合后续分析,操作也较为简便。用三氯乙酸/丙酮法提取蛋白,采用800μg、1000μg和1200μg三个不同的上样量进行双向电泳,在上样量为1200μg时(IPGpH3～10,24cm),蛋白质在12%SDS-PAGE胶上得到了较好的分离,在2-DE图谱上可分辨出562个蛋白点。因此,三氯乙酸/丙酮法是较适合于牡丹试管苗茎基部蛋白质提取的方法,1200μg是较为合适的上样量。