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Am J Physiol Gastrointest Liver Physiol 295: G895-G903, 2008. First published September 4, 2008; doi:10.1152/ajpgi.90378.2008
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LIVER AND BILIARY TRACT

The proteome of rodent mesenteric lymph

Anubhav Mittal,1 Martin Middleditch,2 Katya Ruggiero,2 Christina M. Buchanan,2,3 Mia Jullig,2,3 Benjamin Loveday,1 Garth J. S. Cooper,2,3 John. A. Windsor,1 and Anthony R. J. Phillips1,2,3

1Department of Surgery, Faculty of Medicine and Health Sciences, 2School of Biological Sciences, and 3Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland, New Zealand

Submitted 16 June 2008 ; accepted in final form 1 September 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mesenteric lymph contributes to normal homeostasis and has an emerging role in the pathogenesis of multiple organ dysfunction syndrome. The aim of this study was to define the proteome of normal rodent mesenteric lymph in the fasted and fed states. Eight male Wistar rats fed a standard rodent diet were randomized to two groups. Group 1 (fasted, n = 4) were fasted for 24 h before anesthetized collection of mesenteric lymph. Group 2 (fed, n = 4) were allowed ad libitum access to food before lymph collection. Mesenteric lymph was subjected to proteomic analysis using iTRAQ and liquid chromatography-tandem mass spectrometry (LC-MS/MS). One hundred fifty proteins, including 26 hypothetical proteins, were identified in this study. All proteins were identified in lymph from both the fasted and fed states. The relative distribution profiles of protein functional classes in the mesenteric lymph differed significantly from that reported for plasma. The most abundant classes identified in lymph were protease inhibitors (16%) and proteins related to innate immunity (12%). In conclusion, this study provides the first detailed description of the normal mesenteric lymph proteome in the fed and fasted states using iTRAQ and LC-MS/MS.

rat; fed; fasted

MESENTERIC LYMPH VESSELS in the small intestine were first described in the 4th century BC in the writings of the Alexandrian school (13), and investigations into lymphatic structure and function have subsequently progressed slowly over the centuries (33). The role of mesenteric lymph in maintaining fluid homeostasis by returning interstitial fluid to the systemic circulation and transportation of macromolecules such as lipids is now well documented (13). Although various studies have looked to define specific aspects of its composition, there are no reports of the detailed proteome for normal mesenteric lymph (13, 26). This proteomic information would be particularly relevant given that evidence is now emerging to support the importance of the intestine and mesenteric lymph in the pathogenesis of multiple organ dysfunction syndrome (MODS) and other inflammatory diseases (8, 10, 13).

The conventional view has been that lymph is just a simple plasma filtrate and a necessary means to recycle excess interstitial fluid (32). However, several recent rodent studies in models of shock have demonstrated that disease-conditioned mesenteric lymph can contribute to the pathogenesis of MODS. For example, mesenteric lymph obtained after insults induced by trauma or hemorrhagic shock has been shown to cause neutrophil dysfunction (2, 6), bone marrow suppression (12), and damage to endothelial cells of the pulmonary microvasculature (11) and pulmonary epithelial cells (24). It is now thought that biologically active factors produced by the intestine during critical illness and transported by the aqueous and protein fraction of mesenteric lymph (8) contribute more than translocated bacteria to distant organ failure (1, 9). The observation that postshock portal vein blood does not elicit these toxic effects on neutrophils or endothelial cells strengthens the argument that mesenteric lymph may be a hitherto underrecognized link between the intestine and distant organ dysfunction (37).

Thus the evidence collectively points to an important underlying role for mesenteric lymph in critical illness as well as normal homeostasis. It is therefore surprising that unlike just about every other body fluid, and despite the growing interest in mesenteric lymph, its proteome is unavailable in the literature (13, 19, 26, 30). Likewise, it is unknown whether the compositional proteins of mesenteric lymph from the fed and fasted states differ in any marked manner. The aim of this study was therefore to provide the first comprehensive description of the normal rodent mesenteric lymph proteome in the fasted and fed states by using the simultaneous sample measurement method of isobaric tags (iTRAQ) (38) together with liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification of the component proteins.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. This study was approved by the University of Auckland Animal Ethics Committee. Eight inbred male Wistar rats (446 ± 4.1 g, mean ± SE) fed a standard 18% protein plant-derived rodent diet (Harlan Teklad 2018; Madison, WI), were randomized to two groups. Group 1 (fasted, n = 4) were fasted with ad libitum access to tap water for 24 h before the laparotomy for collection of mesenteric lymph. Group 2 (fed, n = 4) were allowed ad libitum access to food and water until immediately before the same collection procedure. In each case, the surgery commenced at the same time each day (0900).

Collection of mesenteric lymph. General anesthesia was induced by isoflurane (2–5%; 2 l/min O2 via nasal cone). A tracheostomy was inserted (modified 14-gauge angiocath) and connected to a small animal ventilator (Kent Scientific, Torrington, CT). Balanced general anesthesia was maintained with isoflurane (2–3.5%) and buprenorphine (0.05 µg/kg sc Temgesic; Reckitt and Coleman, Hull, UK). The fraction of inspired oxygen/air was kept at 40%, the respiratory rate was 50–80 breaths per minute, and the peak inspiratory pressures were 11–15 cmH2O, which kept the expired CO2 at 35–45 ml/l as measured with a capnograph (Pryon, Menomonee Falls, WI). Maintenance fluid (0.9% NaCl) was infused at 2 ml/h for the duration of the experiment via a femoral intravenous line. Mean arterial pressure was maintained above 80 mmHg and monitored using a solid-state 2-French pressure transducer (Millar Instruments, Houston, TX) placed in the right femoral artery.

A subcostal transverse laparotomy was performed under sterile conditions. The duodenum and intestines were reflected to the left, thus exposing the base of the mesentery. The mesenteric lymph duct was then cleared of surface peritoneum and fat. Silastic tubing [0.96-mm internal diameter, presoaked in 70% (vol/vol) ethanol, rinsed with MQ water, 18 M{Omega}] was drawn through the right posterolateral abdominal wall using a 14-gauge angiocath. The mesenteric lymph duct was cannulated with the Silastic tube and secured in place with a drop of cyanoacrylate tissue glue (Aesculap, Center Valley, PA). The intestines were then returned to their original position and the abdomen closed. Lymphatic flow was promoted by infusing 1 ml/h normal saline via an orogastric tube placed in the stomach and its tip in the proximal duodenum. mesenteric lymph was allowed to drain for 15 min before experimental collection commenced and continued for the following 3 h. Collection was directly into sterile ice-cold siliconized Eppendorf tubes preloaded with protease inhibitors (final: 16.7 µM bestatin, 8.3 µM pepstatin, and 5 mM EGTA; Sigma Aldrich, Castle Hill, Australia). At the end of the experiment, the mesenteric lymph was centrifuged (1,700 g, 4°C, 10 min) to remove cellular debris and then immediately stored at –80°C until analysis.

Before the protein depletion steps and proteomic analysis, mesenteric lymph was pooled from two of the four rats in the fed group to give a MLFED1 sample and from the remaining two fed rats to give a MLFED2 sample. The MLFASTED1 sample was similarly derived by combining the lymph from two of the fasted rats, and the MLFASTED2 sample was derived from the remaining two fasted rats.

Depletion of the major proteins. IgY immunoaffinity columns were used to deplete the most abundant proteins and enhance the detection of lower abundance proteins (17). In this study, the 12 expected major abundant proteins of mesenteric lymph were depleted using ProteomeLab IgY-12 affinity spin columns (Beckman Coulter, Fullerton, CA). These spin columns deplete albumin, IgG, fibrinogen, transferrin, IgA, IgM, apolipoprotein (apo)A-I, apoA-II, haptoglobin, {alpha}1-antitrypsin, anti-{alpha}1-acid glycoprotein, and anti-{alpha}2-macroglobulin. Each of the four samples (MLFED1, MLFED2, MLFASTED1, and MLFASTED2) were individually depleted as outlined in Table 1. Protein measurement was carried out using the EZQ protein assay (Molecular Probes, Eugene, OR). The depleted samples were concentrated by ultrafiltration using Vivaspin 4 concentrators with a 5-kDa polyethersulfone filter (Sartorius, Goettingen, Germany).


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  		Table 1. Depletion and LC-MS/MS protocols

 
LC-MS/MS based proteomics. After the depletion protocol, samples (50 µg of protein) underwent reduction (incubation with 10 mM DTT at 56°C for 1 h) and alkylation (incubation with 20 mM iodoacetamide at pH 8.0 in the dark for 1 h). Protein was then digested by incubation with 1 µl of trypsin (Promega, Madison, WI) at 1 mg/ml and incubated at 37°C overnight. The peptides were then desalted on 10-mg Oasis SPE cartridges (Waters, MA), eluted with 70% acetonitrile, and completely dried using a speed vacuum concentrator (Thermo Savant, Holbrook, NY).

To identify the proteome more cost-effectively, we used iTRAQ labeling, which enabled us to independently and uniquely label each sample's protein components and then mix them together to simultaneously run them in the LC-MS/MS. This reduced the total number of relatively expensive mass spectrometry runs but preserved our ability to resolve the composition of the samples. The iTRAQ approach has also been validated previously by ourselves and others as a method of tracking relative concentrations of proteins in four different samples (19, 39) (although in this instance, we were not using it in this way). Dried protein digests were reconstituted with 30 µl of dissolution buffer from the iTRAQ reagent multiplex kit (Applied Biosystems, Foster City, CA) and labeled with iTRAQ reagents according to the manufacturer's instructions. Labeled material from the four depleted sample groups (MLFED1, MLFED2, MLFASTED1, and MLFASTED2) was then combined, acidified by addition of 10% (vol/vol) formic acid, concentrated to ~200 µl, and then diluted to 2 ml with 0.1% formic acid. This sample was desalted as described above, and the eluate was then concentrated to 100 µl and, finally, diluted to 270 µl with 0.1% (vol/vol) formic acid.

Samples were then fractionated online on a BioSCX II 0.3 x 35-mm column (Agilent Technologies, Santa Clara, CA). Runs 1 (R1) and 2 (R2) used 4 KCl salt steps (40, 60, 100, and 200 mM KCl), whereas runs 3 (R3) and 4 (R4) used 20 salt-steps (10, 15, 20, 40, 50, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 400, and 500 mM KCl) as outlined in Table 1. Peptides were captured on a 0.3 x 5-mm PepMap cartridge (LC Packings; Dionex, Sunnyvale, CA) before being separated on a C18 300SB 0.3 x-100 mm Zorbax column (Agilent). The HPLC gradient between buffer A (0.1% formic acid in water) and buffer B (0.1% formic acid in acetonitrile) was formed at 6 µl/min as follows: 10% buffer B for the first 3 min, increasing to 35% buffer B by 80 min, increasing to 95% buffer B by 83 min, held at 95% until 91 min, back to 10% buffer B at 91.5 min, and held there until 100 min. The LC effluent was directed into the ion spray source of a QSTAR XL hybrid mass spectrometer (Applied Biosystems) scanning from 300 to 1,600 m/z. The three most abundant, multiply charged peptides were selected for MS/MS analysis (80–1,600 m/z). The mass spectrometer and HPLC system were under the control of the Analyst QS software package (Applied Biosystems).

Sequence database searches. ProteinPilot (version 1.0; Applied Biosystems) was used to search the MS/MS data against the International Protein Index (IPI) rat database (version 3.27) with the following search parameters: Cys alkylation, iodoacetamide; digestion, trypsin; and instrument, QSTAR ESI. The data were also searched against the above database with the use of Mascot 2.0.5 software (Matrix Science, London, UK), and a similar set of protein hits were obtained (data not shown). Proteins that were identified as "hypothetical" by the ProteinPilot IPI rat database search were then subjected to a National Center for Biotechnology Information (NCBI) BLASTP search against the UniProt Clusters 100% database.

Validation of protein identifications. A search of the IPI rat database with the reversed amino acid sequence of each entry was carried out to determine the minimum required ProteinPilot score for the proteins that would yield an overall confidence >97%. Protein matches were considered valid if their ProteinPilot scores were equal to or above the minimum required score for each run (Table 1). The reverse search enabled calculation of the false-positive rates for each run (Table 1).


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
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Fed-fasted model. All animals survived the procedures. The fed and fasted status was confirmed in each case with the intraoperative observation of semidigested food throughout the small intestine in the fed state and absence of this in the fasted animals. There was no significant difference in the flow rate of mesenteric lymph between the fasted group at 0.85 ± 0.11 ml/h (mean ± SE) and the fed group at 0.94 ± 0.09 ml/h (Mann-Whitney U-test, P = 0.68).

Effect of depletion. The proteome of the nondepleted mesenteric lymph (R1) showed that albumin was the most abundant protein in lymph, accounting for an average of 53% of the total iTRAQ reporter ion signal. This dropped to 21.6% of the total signal after one cycle of IgY depletion (R2) and to 0.85% after two cycles of depletion (R4). The other proteins depleted by the IgY column comprised variably transferrin at 8% and IgG at 2.8%, down to apoA-I at 0.06% of the signal in R1. The combined total contribution of depleted proteins was reduced from ~66 to ~2% after two cycles of depletion (data not shown). This process enabled a dramatic increase in the number of identified proteins from 46 in nondepleted R1 to 129 in depleted R4. The comparative redundancy of identifications within each protocol is summarized in Fig. 1.


Figure 1
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  		Fig. 1. Summary of protein identifications obtained from the 4 different analytical runs (R1–R4) (see Table 1 for run descriptions). Numbers are the total sum of mutually identified proteins for the stated overlapping protocols. Numbers within parentheses are the number of "hypothetical" proteins.

 
R3 to R4 each had two cycles of depletion followed by 20-salt step LC-MS/MS; however, we improved our yield from 87 protein identifications in R3 to 129 protein identifications in R4 by increasing the amount of protein we loaded onto the depletion columns by 50% (Table 1). The nondepleted R1 approach was necessary because it enabled comment on the ratios of the proteins otherwise depleted by the IgY columns. The R2 protocol allowed optimization of the depletion method and added some additional identifications.

Description of the mesenteric lymph proteome. Combining the results of all four MS/MS runs (R1–R4) produced a nonredundant list of 150 proteins. There were 124 known proteins (Table 2) and 26 hypothetical proteins (Table 3) listed according to the IPI rat database. Of these 150 proteins, 147 (94.8%) were identified using two or more unique peptides. The 26 hypothetical proteins were subjected to a NCBI BLASTP search to ascertain their similarity to other known proteins (Table 3). These hypothetical proteins were also classified by their theoretical function when available, and the main groups were complement component (n = 4), immunoglobulin (n = 3), coagulation (n = 2), and protease inhibitor (n = 2).


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  		Table 2. Nonredundant list of identified proteins

 

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  		Table 3. Nonredundant list of hypothetical proteins identifications

 
All of the 150 proteins were identified in all of the individual (n = 4) lymph samples derived from either fasted or fed states, respectively. Figure 2 shows the breakdown of the relative numbers of the known proteins according to their general functional classification. Of the 124 known proteins, the largest single grouping was protease inhibitors (16%), followed by proteins related to innate immunity (12%), carrier proteins (9%), and proteins related to the clotting system (6%) and lipid transport or metabolism (7%).


Figure 2
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  		Fig. 2. Pie chart of the nonredundant proteins based on their functional groups.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study provides the first detailed description of the mesenteric lymph proteome in the stable fasted and fed states using LC-MS/MS. In addition, we employed iTRAQ, a relatively new but validated proteomic technique (38) that permitted the simultaneous and compositional analysis of four different lymph sample mixtures. Using stringent criteria, we identified with >97% confidence a total of 150 unique proteins, including 26 hypothetical proteins. All of the proteins identified were in all the individual fasted and fed samples that were analyzed, confirming the robust and ubiquitous nature of the identifications.

Lymph is usually referred to as a filtrate of plasma (32); however, lymph proteins are derived not only from filtered plasma but also from direct cell secretion into the interstitial fluid (20). The ratio of plasma volume to interstitial fluid volume has been reported at 0.22 in healthy male subjects (35), meaning that interstitial fluid comprises a volume roughly four times greater than the intravascular volume (22). It is not surprising then that contributions directly from interstitial cells may alter lymphatic composition relative to plasma. Lindena et al. (21, 22) have shown that lymph recycles substances from the larger interstitial volume and influences the composition of plasma. For example, {gamma}-glutamyl transpeptidase from the small intestine is transported via mesenteric lymph and can alter the concentration in plasma (18). In plasma, the major functional protein groups are reportedly signaling proteins, proteases, and transport-related proteins in order of decreasing abundance (4). In contrast, we found that the major functional groups in mesenteric lymph were protease inhibitors, immune-related proteins (particularly those implicated in innate immunity), and carrier proteins (Fig. 2). This specific mesenteric lymph compositional profile may reflect the body's need to inhibit multiple proteases in the intestinal interstitium and provide a first line of defense against potential gut-derived pathogens, respectively. It also demonstrates that mesenteric lymph has a unique profile compared with plasma and thus represents more than a simple filtrate.

Lymph is a complex protein mixture. Its protein content is dominated by albumin, IgG, and fibrinogen. Immunodepletion of the most abundant proteins in the mesenteric lymph greatly improved our ability to identify more of the lower abundance proteins. An example of this increased resolution was the ability to identify proteins in the picomolar range, such as β-enolase, which has a reported normal plasma concentration of 6 ng/ml (3, 27). Figure 2 shows that multiple proteins representing many functional classes were present in the mesenteric lymph. The only previous published attempt at a proteomic analysis of lymph used a two-dimensional gel electrophoresis (2DGE) approach on thoracic duct lymph in sheep without immunodepletion and identified only 50 proteins (20). There are definite limitations with this gel approach given that 2DGE struggles with extremes of isoelectric point and proteins of high hydrophobicity. 2DGE also has difficulty providing conclusive data for changes in specific protein levels, since numerous proteins may be represented in the same spots or overlap with other spots, and single proteins may be distributed across several gel spots. The present study using iTRAQ and LC MS/MS avoided many of these issues and consequently identified an additional 115 proteins (89 known and 26 hypothetical) in mesenteric lymph to that shown by 2DGE in ovine thoracic lymph duct. Of the 124 known proteins in our study, 104 also have been identified in a comprehensive proteomic analyses of human plasma (3, 4, 29). This suggests that the 20 proteins not identified in human plasma (Table 2) are either characteristic of lymph or may be due to species-specific differences.

Perhaps most surprising was our finding that 26 (17%) of the proteins identified in mesenteric lymph in the current study were listed as hypothetical in the IPI rat database version 3.27. The proportion of hypothetical proteins from comparable proteomic studies of other body fluids, such as urine, bile, amniotic fluid, and colostrum, is variable but ranges from 0 to only 6.5% (19, 30, 31, 34). Of the 26 hypothetical proteins in our series, only three identifications were based on one unique peptide, but the total protein score for these three still met our predefined false-positive cutoff scores (Table 1). A NCBI BLASTP search (Table 3) showed that of the 26 possible hypothetical proteins, two (IPI00764461 and IPI00765011) had 100% similarity to human actin but had not previously been identified in rodents. The majority of the novel proteins identified fall into functional groups (complement component, immunoglobulin, coagulation, and protease inhibitor) that closely mirror the major functional groups seen in mesenteric lymph for the known protein identifications. This would support the notion that mesenteric lymph is likely to be a rewarding target body fluid in which to seek new protein identifications, especially in these functional groups.

Mesenteric lymph also has been suggested as a route by which the intestine influences distant organ function in critical illness and chronic disease states (10, 13). It appears that the aqueous and presumably protein containing fraction of disease-conditioned mesenteric lymph is more toxic than the lipid-based fraction (8) or the contributions from bacteria, endotoxin, and cytokines (1). This current study would support application of an iTRAQ- and LC MS/MS-based proteomic analysis of changes in the mesenteric lymph proteome during various critical and chronic illnesses as a method to reveal proteins of potential relevance to the diagnosis, severity assessment, monitoring, and treatment in this fluid.

This study successfully identified a large number of proteins; however, the masking of many lower abundance proteins by the few highly abundant proteins continues to be a challenge in proteomics. Depletion of these highly abundant proteins has been validated as a necessary step in being able to penetrate deeper into the proteome and identify proteins of medium to low abundance (5, 16, 17, 23, 28). It is acknowledged that the unintentional loss of proteins during depletion remains a potential issue, but this must be balanced against the increased protein identifications that can occur by this approach. Protein loss may occur due to nonspecific binding to the column, specific binding to column Ig due to structural homology to the proteins being depleted, or binding to the proteins that are depleted (5, 17). These are the current limitations of this technology, and until the field of "Seppromics" evolves further, we are bound by these constraints (17).

There have been a number of different surgical animal models that have been described in the literature for the collection of mesenteric lymph (7). The two models that have been employed most often are the anesthetized animal model (15, 36) and the conscious lymphatic fistula model (14, 25). For the purpose of this first attempt to describe the mesenteric lymph proteome, we chose the anesthetized rat model because it offers shorter experimental time, no postsurgery recovery effects, and standardized collection with controlled blood pressure and intravenous fluid regimen, along with a high collection success rate (7). In the future, it would be interesting to complement the anesthetized model with data from the conscious lymphatic fistula model, which would allow collection of mesenteric lymph within the same animal over a longer time period and under different physiological states.

Conclusions. The detailed proteome for many body fluids have been reported to date (19, 26, 29–31, 34), but that of mesenteric lymph appears to have been overlooked. This study has therefore defined the mesenteric lymph proteome in the fed and fasted states for the first time. The application of iTRAQ with LC-MS/MS enabled simultaneous tracking of mass spectrometry results from multiple mesenteric lymph samples and resulted in the successful identification of 150 proteins, including a surprising 26 hypothetical proteins over a wide range of functional classes. The iTRAQ- and LC-MS/MS-based protein identification approach can now be applied to defining the changes that occur in the mesenteric lymph proteome in other normal states or in critical and chronic illness.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
These studies were supported by funding from Royal Australasian College of Surgeons, the Health Research Council of New Zealand, the University of Auckland Research Committee, and the Maurice & Phyllis Paykel Trust.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. R. J. Phillips, Level 4, Thomas Bldg., School of Biological Sciences, Univ. of Auckland, Auckland 1023, New Zealand (e-mail: a.phillips{at}auckland.ac.nz)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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