Proteomic Analysis for Tissues and Liquid from Bonghan Ducts on Rabbit Intestinal Surfaces

Article Outline

• Abstract
• 1.. Introduction
• 2.. Materials and Methods
  • 2.1.. Sample preparation
  • 2.2.. Tryptic digestion and LC-MS/MS analysis
• 3.. Results
  • 3.1.. Proteomic analysis of the Bonghan liquid
  • 3.2.. Proteomic analysis of BHD
• 4.. Discussion
• Acknowledgments
• Tables of Data
  • Database analysis
• References
• Copyright

Abstract 

Research on the Bonghan system has recently prompted great interest in the theory proposed by Bong Han Kimin in the early 1960s. In order to study the biochemical characteristics of the Bonghan system, we analyzed Bonghan ducts (BHD) on the surface of rabbit intestines and characterized the liquid in the BHD at the level of the proteome. Proteomic analysis was performed using nano LC-ESI MS/MS. Using a solution digestion technique, we identified 70 different proteins in the liquid of the BHD. We used gel-based digestion to analyze the BHD itself and our results showed the presence of 207 proteins. We used these proteins to analyze gene ontology (GO) to yield insights into biological processes, molecular functions and cellular compartmentalization. Remarkably, GO clustering showed high concentrations of proteins involved in metabolism. These proteins are not usually found in blood, lymph or blood vessels, and thus can be useful for characterizing BHD. It is worth studying their association with stem cells, especially mesenchymal stem cells, cancer cells and myeloid cells.

Key words:  cancer , mass spectrometry , proteomics , stem cell

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1. Introduction 

In the early 1960s, Bonghan Kim claimed to have discovered the anatomical structure corresponding to acupuncture meridians [1, 2]. Despite the potential significance in both Western and Eastern medicines, his work has been ignored for many years due to the absence of verification from other researchers. Only one Japanese anatomist, Fujiwara, has ever managed to replicate his results [3].

With modern fluorescence and microscopy technologies, it has become possible to rediscover the Bonghan system and this has led to a number of new research efforts. Scientists have explored the intravascular Bonghan duct (BHD) and Bonghan corpuscles (BHC) in blood vessels [4, 5, 6] and lymphatic vessels [7, 8, 9], and organ-surface BHD and BHC [10, 11]. A series of investigations to elucidate the details of BHC and BHD anatomy and morphology have been performed using confocal laser scanning microscopy [12], various electron microscopy techniques [13], x-ray microtomography [14], and immunohistochemical techniques [15]. Measurement of the flow speed of Bonghan liquid in BHD was performed by injecting Alcian Blue and the speed of travel was found to be 0.3±0.1 mm/sec [16]. Researchers also confirmed that BHC has chromaffin cells that produce and store catecholamine, suggesting a medical significance of the BHD as a hormonal pathway [17].

Our work describes an initial step towards functionally characterizing the Bonghan system in the rabbit using proteomic analysis. In order to identify BHC proteins, we used electrospray ionization (ESI) that featured a linear ion-trap mass spectrometer coupled with nano liquid chromatography (LC). The proteins identified were clustered using GO according to their involvement in biological processes, molecular function and cellular compartmentalization. Our proteomic analysis of Bonghan liquid and BHD showed remarkably high levels in carbohydrate metabolic derivatives. We compared the chemical composition of Bonghan liquid with that observed in blood [18], lymph [19] or blood vessels [20], but found similarity in composition to that more usually associated with stem cells [21, 22], cancer cells [23] and differentiated myeloid cells [24]. In particular, we identified several proteins more normally associated with mesenchymal stem cells [25, 26, 27].

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2. Materials and Methods 

2.1. Sample preparation 

New Zealand white rabbits weighing about 1.8 kg were used for this study. The animals were housed in a temperature-controlled environment (23°C) with 60% relative humidity and a 12 hour light/dark cycle. The animals had free access to food and water and were fasted overnight before abdominal dissection. All procedures were conducted in accordance with institutional research animal care and use guidelines. The rabbits were anesthetized with intraperitoneal urethane (1.5 g/kg) and all surgical procedures were performed under general anesthesia.

We searched for BHDs on organ surfaces with the help of a stereoscopic microscope (SZX12, Olympus, Japan). The liquid in each BHD was extracted using a capillary needle and the remaining BHDs were subsequently isolated for proteomic analysis.

2.2. Tryptic digestion and LC-MS/MS analysis 

The isolated BHDs were homogenized and sonicated. Then, 10 μg of tissue was loaded onto a 4–12% gradient Tris-Glycine Gel (Invitrogen, Carlsbad, CA). The PAGE-gel of the BHDs was manually segmented into 10 pieces. In-gel digestion of the gel pieces was carried out using 10 ng/μL sequencing grade modified trypsin (Promega, Madison, WI) in 50 μL of 50 mM NH₄HCO₄ buffer (pH 8.0) at 37°C overnight as described in the literature [28]. The liquid from the BHDs was in-sol digested directly under the same conditions as described above. The tryptic peptides were then loaded onto a fused silica microcapillary C18 column (75 μm × 10 cm).

LC separation was conducted under a linear gradient as follows: 0 min, 3% B; 5 min, 3% B; 75 min, 40% B; 80 min, 90% B; 90 min, 90% B; 91 min, 3% B; 110 min, 3% B. The initial solvent condition was 3% solvent B and the flow rate was 200 nL/min. Solvent A was 0.1% formic acid in H₂O and solvent B was 0.1% formic acid in acetonitrile. The separated peptides were subsequently analyzed using a linear ion-trap mass spectrometer, LTQ (ThermoFinnigan, San Jose, CA). The electrospray voltage was set at 2.0 kV, and the threshold for switching from MS to MS/MS was 250. Each full MS scan was followed by three MS/MS scans that focused on the three most pronounced peaks of the full MS scan.

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3. Results 

Figure 1 shows a stereomicroscopic image of the BHD on the surface of a rabbit intestine from which the Bonghan liquid (BHL) had previously been extracted with a capillary needle.

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• Figure 1. 

  A glass capillary was inserted into a Bonghan duct, held by microforceps, above the large intestine of rabbit using in situ and in vivo stereomicroscopy (SZX12, Olympus, Japan). The capillary tip (dotted circle) was correctly inserted into the Bonghan duct in order to extract its liquid. The scale bar, located in the bottom right, is 5 mm.

3.1. Proteomic analysis of the Bonghan liquid 

We investigated the BHL using ESI-MS/MS coupled with nano LC. The acquired MS/MS spectra were searched using SEQUEST's rabbit database, which listed 7490 proteins. Proteomic analysis of BHL identified 70 proteins (Table 1) and the criteria of the SEQUEST search were as mentioned in the Materials and Methods section.

Table 1. Protein list identified in Bonghan liquid

Orthologous conversion was necessary to translate rabbit proteins into human proteins. This introduced a set of well-known limitations. The orthologous conversion scheme was as shown in Figure 2. For the orthologous conversion, we attempted a blast similarity operation that translated rabbit proteins in Table 1 into human proteins. The corresponding human proteins are listed in Table 2. Further data on the BHD are provided in the Supplementary Information on the JAMS web server.

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• Figure 2. 

  Protein conversion using the blast similarity operation. The process consisted of three steps: (1) obtaining the amino acid sequence of a given rabbit protein; (2) running the blast similarity operation; and (3) selecting the corresponding human protein that exhibited the lowest E-value. For the blast similarity operation, the human ‘RefSeq protein’ was used as the database for blast searching and ‘BLASTP’ was applied to compare protein sequences. The protein with the lowest E-value was selected as the best match. If two or more proteins with the same lowest E-value were identified, the upper listed protein was arbitrarily chosen.

Table 2. Full conversion list from Bonghan liquid proteins of rabbit onto correspondent human proteins

After the conversion, the corresponding human proteins were clustered according to their involvement with biological processes, molecular functions and cellular compartmentalization in GO using DAVID and Cytoscape (Figure 3). In the cases of biological processes, the categories of ‘localization,’ ‘response to stimuli’ and ‘metabolism’ accounted for the majority of the proteins.

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• Figure 3. 

  Clustering of proteins identified in Bonghan liquid. The clustering was executed consistent with the three GO domains of biological processes (A), molecular functions (B) and cellular compartmentalization (C). The pie chart values indicate the numbers of proteins included in each category. Certain proteins belonged to more than two categories and as a result the total number of proteins in the pie chart is different from the number of proteins identified in Table 1.

‘Localization’ refers to any process by which a cell or a cellular entity, such as a protein complex or organelle, is transported to and/or maintained in a specific location. Proteins in this category included annexin AI, ATPase class VI, caldesmon, vitamin D binding protein, hemoglobin, hemopexin, transferrin and more. ‘Response to stimulus’ refers to a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression etc.) that occurs as a result of exposure to a stimulus. The ‘Response to stimulus’ category included albumin, annexin AI, bleomycin hydrase, clusterin, crystalline and others. Some proteins, such as annexin AI, belong to two classes. We note that many proteins were related to metabolic processes such as carbohydrate, alcohol and other cellular catabolism.

In the context of molecular function, ‘binding’ and ‘transporter activity’ accounted for almost all of the BHL proteins. With respect to the cellular compartmentalization category, ‘extracellular region’ and ‘protein complex’ were dominant and this could be evidence that our analyses were appropriate. In Table 3, GO clustering of human BHL proteins are presented in terms of biological processes, molecular function and cellular compartmentalization.

Table 3. List of gene ontology clustering of Bonghan liquid proteins in terms of biological process, molecular function and cellular compartment

3.2. Proteomic analysis of BHD 

We also performed proteomic analyses of the BHD. The number of proteins identified in BHD was 207 (Table 4). The rabbit proteins identified in the BHD and their corresponding human proteins are shown in Table 5. GO clustering of the BHD proteins in Table 6 was achieved as in the case of BHL (Figure 4). Tables 4–6 are Supplementary Information.

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• Figure 4. 

  Clustering of proteins identified in Bonghan duct. Note the similarity with Figure 3.

For biological processes, we identified several categories: (1) ‘metabolic process’ (annexin Al, ATP synthase-H⁺ transporting, ATPase-Na⁺/K⁺ transporting, carbonic anhydrase II, cytochrome b5 type A, fructose-1, 6-bisphosphatasae, lactate dehydrogenase A, etc.), (2) ‘localization’ (annexin Al, calreticulin, cytochrome b5 type A, vitamin D binding protein, hemoglobin, hemopexin, transferrin, vimentin, etc.), (3) ‘response to stimulus’ (albumin, aldo-keto reductase family 1, annexin Al, annexin All, arginase type II, clusterin, glucose phosphate isomerase, isocitrate dehydrogenase 1, protein phosphatase 2, etc.), and (4) ‘cell development’ (actin alpha1, actin beta, albumin, annexin Al, apolipoprotein E, calreticulin, clusterin, protein disulfide isomerase family A, protein kinase C, protein phosphatase 2, etc.).

With regard to molecular function, ‘binding,’ ‘catalytic activity’ and ‘transporter activity’ were prominent. In the case of cellular compartmentalization, the ‘intracellular part’ was dominant and other categories such as ‘extracellular region,’ ‘macromolecular complex’ and ‘cell fraction’ were also evidenced. The GO clustering of human BHD proteins is shown in Table 6, categorized again in terms of biological processes, molecular functions and cellular compartmentalization.

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4. Discussion 

Our study used proteomics to conduct molecular investigations into the BHD. The rabbit was selected to achieve our minimum required sample volume, but the database for functional clustering of proteins was incomplete. Therefore, the rabbit proteins identified in BHL and the BHD had to be associated with corresponding human proteins. A blast similarity operation was used to achieve this association. This operation uses a search strategy to match a given rabbit protein with the exact or closest amino acid sequence in a human protein database, as described in Figure 2. We note that this process is limited because of the possibility of protein conversions that do not maintain cross-species functionality. However, orthologous conversions have been successfully used in other cases where species-specific protein databases were unavailable [29]. In this study, almost all the corresponding human proteins were identical or similar to the original rabbit proteins when we conducted a manual survey of their functionalities.

The human proteins used in the blast similarity search were clustered into three domains, namely, biological processes, molecular functions and cellular compartmentalization. In this search we used Cytoscape, a free software program (www.cyto-scape.org). In the “biological processes” category, it was clear that metabolic processes, especially carbohydrate-based ones, were very prominent categories in both BHL and the BHD. Other scientists have reported proteomic analyses of blood and lymph that do not show these remarkable carbohydrate-based processes [18, 19]. Another study concluded that the proteome of blood vessels also included few carbohydrate-based processes [20]. Our findings imply that either BHL or the BHD must require an efficient energy supply. Proteomic analyses of certain cell types such as stem cells, cancer cells and differentiated myeloid cells, all of which show vigorous proliferation or differentiation, have shown a similar abundance of carbohydrate- or energy-related processes [21, 22, 23, 24].

We note the existence of proteins related to (1) the recruitment of mesenchymal stem cells (MSC) [25], (2) the cell processes in MSCs (Ezrin, Actinin, myosin) [26], and (3) the differentiation of MSC/myofibroblasts (alpha-smooth muscle actin, CD147) [27]. These protein profiles suggest that BHDs located on the organ surface has a role as a temporary depot and point of differentiation of stem cells for tissue regeneration.

In conclusion, our proteomic analysis of rabbit BHL and BHDs suggests that proteins can be categorized in terms of their involvement with biological processes, molecular functions and cellular compartmentalization following orthologous conversion to human proteins. The abundance of carbohydrate-based processes was surprising. This fact distinguished the proteomic fingerprint of the Bonghan system from that of blood, lymph or blood vessel physiology, but was similar to that of stem cells, especially mesenchymal stem cells, cancer cells and differentiated myeloid cells.

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Acknowledgments 

This research was supported by a NRL (No.R0A-2003-000-10371-0) from the Korean Ministry of Education, Science and Technology and by a “Systems Biology Infrastructure Establishment Grant” from the Gwangju Institute of Science and Technology.

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Tables of Data 

Database analysis 

All MS/MS spectra recorded were searched on rabbit database, obtained from the taxonomy site in NCBI (www.ncbi.nlm.nih.gov/sites/entrez?db=taxonomy), by using the SEQUEST algorithm. Dynamic modifications were permitted for oxidized methio-nine (+16Da), carboxyamidomethylated cysteine (+57Da). SEQUEST criteria for peptide selection were Xcorr, which must be greater than 1.8, 2.3 and 3.5 for +1, +2 and +3 charge state respectively, and delta Cn above 0.1. The parameter for selection of identified proteins was a protein consensus score which was above 10.1. Due to the poorness of the functional DB for rabbits, it was needed to orthologously convert the rabbit proteins into corresponding human proteins. Blast similarity operation, provided by NCBI, was used for this purpose. The resulting human proteins were clustered according to biological process, molecular function and cellular compartment with the help of Cytoscape (www.cytoscape.org) which was freely obtained from the web.

Table 1 List of proteins identified in Bonghan liquid. The accession column refers to GI accession number, the score refers to the consensus score from SEQUEST, MW is molecular weight and peptide is the number of peptides identified by proteomic analysis. The × correlation value (greater than 1.8, 2.3 and 3.5 for +1, +2 and +3 charges, respectively), delta Cn (greater than 0.1), and number of top matches (only 1) were used as criteria for peptide selection. The consensus score was utilized for filtering proteins. Proteomic analysis of Bonghan liquid was conducted twice and this table shows results from just one experiment. Only part of the protein conversion results are listed in this table. The total protein conversion list is shown in Table 2, in the supplementary material.

Table 2 Conversion of Bonghan liquid proteins from rabbit into corresponding human proteins. E-value is a parameter that describes the number of hits one can “expect” to see by chance when searching a database of a particular size. The lower E-value indicates the more valid result. ‘Positive’ means positive matrix score which is expressed as the ratio of the number of identified or conserved amino acids to the total number of amino acids of each protein. In the process of protein conversion, one protein from rabbits (part of fibrinogen alpha chain; GI number, 120095) was missed because the short peptide consists of only 16 amino acids.

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