Proteomic profiling of heterotopic heart-transplanted rats using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry: Potential biomarkers and drug targets

Abstract

Objective

Methotrexate and rapamycin demonstrate an additive effect in prolonging cardiac allograft survival in a major histocompatibility complex mismatched rat model. The present study aimed to identify functional proteins involved in the allograft-protective effects of these two agents and reveal potential diagnostic markers for treating rejection.

Methods

Serum samples from heterotopic heart-transplanted LEW(RT-1¹) rats (either without immunosuppressive treatment or treated with methotrexate alone, rapamycin alone, or methotrexate and rapamycin combined) were analysed by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. Protein profiles obtained using a weak cation exchange ProteinChip® CM-10 array were then analysed using ProteinChip® Software.

Results

Of 28 rejection-related proteins identified, isoelectric point and mass information from two potential candidate proteins matched information from the UniProtKB/Swiss-prot database, suggesting them to be complement component C3f fragment and complement component 4A (C4A, anaphylatoxin).

Conclusions

Proteomic analysis revealed 28 proteins as potential diagnostic markers of tissue rejection. Of these, 11 proteins may represent targets relating to the additive effects of methotrexate and rapamycin. Two protein peaks, with mass-to-charge ratios of 1950 Da and 8577 Da, may have potential for use in post-transplant diagnosis of rejection.

Keywords

Rejection surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS)transplant complement component C3f fragment complement component 4A (C4A anaphylatoxin)

Introduction

In solid-organ transplantation, the major challenge to survival is allograft rejection, which requires lifelong immunosuppression.¹ There are currently no known biomarkers with both high specificity and sensitivity to indicate rejection before allograft damage has occurred. In order to manage the potential rejection that occurs between almost all major histocompatibility complex (MHC) mismatched donor tissues and recipients, post-transplant immunosuppressant medication is indiscriminately administrated to recipients.¹ The majority of immunosuppressants act nonselectively and the immune system is normally oversuppressed.¹ In order to achieve a better immunosuppressive effect without potentially fatal complications, it is crucial to find biomarkers to diagnose rejection more precisely. This would facilitate post-transplant immunosuppression regimens that are tailored to each individual.

Used in combination, methotrexate and rapamycin have a synergistic effect on prolonging cardiac graft survival in an MHC-mismatched rat model.² The immune-unresponsive state induced by methotrexate was found to be related to a number of humoral factors.³ It remains unclear, however, which of these factors could be used as a biomarker (or biomarkers) to diagnose rejection.

Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) is a matrix-assisted laser desorption/ionization (MALDI) time-of-flight technique that employs a special chromatographic-like probe surface (called a protein chip array) to capture different classes of proteins; SELDI-TOF MS has superior accuracy compared with electrospray ionization and MALDI.⁴ Through SELDI-TOF MS, multiple serum samples can be analysed simultaneously, requiring small sample volumes and short testing times.⁵ SELDI-TOF MS is the preferred method (due to its high sensitivity) for detecting low molecular weight (<30 kDa) proteins,⁶ such as those involved in immune responses.

Using SELDI-TOF MS, the present study investigated factors involved in prolonged allograft survival, following treatment with methotrexate and rapamycin.

Materials and methods

Laboratory animals

The study was conducted in the Department of Surgery, Peking University Third Hospital, Peking University, Beijing, China. Laboratory animals were obtained from the Department of Laboratory Animals of Peking University Health Science Centre. Inbred 6–8-week-old male DA(RT-1^a) rats (body weight, 200–220 g) and LEW(RT-1¹) rats (body weight, 200–220 g) were used as the cardiac transplant donors and recipients, respectively. All rats ware maintained at 20–25℃ in a special pathogen-free environment with a natural day/night cycle and free access to food and water. All rats were fasted for 12 h before and after surgery.

All procedures were performed according to the Beijing Regulations for Principles of Laboratory Animal Care. The study was approved by the Ethics Committee for Laboratory Animal Care of the Health Science Centre, Peking University (No. LA2010-027; date: 29 April 2010).

Cardiac transplantation

Heterotopic murine cardiac transplantation was performed precisely, according to the method described previously.⁷ The day of transplantation was recorded as day 0 and the cardiac allograft was monitored daily by palpation. Rejection was defined as the total cessation of a heartbeat. An animal was excluded from the study if the graft heartbeat ceased within 72 h of transplantation.

Immunosuppressive agents

Methotrexate (Wyeth Lederle Company, Tokyo, Japan) was dissolved at a concentration of 0.05 mg/ml in 0.02 N NaOH before use, then administered at a dose of 0.2 mg/kg by intraperitoneal injection, once daily from day 0 to day 3 following transplantation.

Rapamycin (Wyeth Pharmaceutical Company, Philadelphia, USA) was administered using a gastric tube at a dose of 3 mg/kg body weight, once daily from 2 days prior to transplantation until the day before transplantation, then at 0.2 mg/kg body weight, once daily from day 0 to day 6 following transplantation.

Study groups

Allograft recipients (DA [RT-1^a] to LEW [RT-1¹] rats) were divided into four subgroups (A1–A4) according to the pre- or post-transplant intervention (control group, methotrexate alone, rapamycin alone and methotrexate + rapamycin). Syngraft recipients (LEW [RT-1¹] to LEW [RT-1¹] rats) were similarly divided into subgroups (S1–S4). There were 12 grafted rat recipients in each subgroup, of which six were used for graft survival rate analysis. The other six rats in each group were sacrificed 5 days after surgery, by anaesthesia followed by cardiac puncture, to collect arterial blood for serum sample collection.

SELDI-TOF MS analysis

Arterial blood samples (5 ml) were collected as described above and allowed to stand at room temperature for 1 h to permit clotting. Blood samples were centrifuged at room temperature at 1500 g for 10 min; the resulting serum samples were immediately frozen at −80℃ until analysis.

The SELDI-TOF MS profiles for all samples were recorded using a weak cation exchange ProteinChip® CM-10 array (Ciphergen Biosystems, Fremont, CA, USA), and the protein analysis was performed according to the manufacturer’s instructions. Briefly, the serum proteins were loaded onto the CM-10 chip and analysed using Protein Biology System IIc (Ciphergen Biosystems), which was calibrated for mass accuracy using the ‘all in one’ peptide standard. The laser shot sensitivity and detector sensitivity were adjusted to 220 and 8, respectively. The mass-to-charge ratio (m/z) range was optimized to between 2 and 30 kDa for this study. Protein profiles obtained using the ProteinChip® CM-10 array were analysed using ProteinChip® Software, version 3.1.1 (Ciphergen Biosystems).

The isoelectric point (pI) and mass information (including m/z and molecular weight [Mw] for the proteins identified in SELDI-TOF MS analysis) were used to search the UniProtKB/Swiss-prot database (European Bioinformatics Institute: http://www.ebi.ac.uk/uniprot/) using the TagIdent protein identification tool.

Statistical analyses

Between-group differences in peak frequencies were analysed by Student’s t-test using the SPSS® statistical software package, version 17.0 (SPSS Inc., Chicago, IL, USA) for Windows®. Two-tailed tests were used for all of the statistical analyses and a P-value <0.05 was accepted as showing statistical significance.

Results

The mean survival time for the allograft with methotrexate plus rapamycin group (A4) was significantly longer than that for all other allograft groups (P < 0.05, Table 1). The mean survival time for all syngraft groups was significantly longer than that for the allograft control and allograft methotrexate plus rapamycin groups (P < 0.05, Table 1).

Table 1.

Survival times for LEW(RT-1¹) rats who had received either heart allografts from DA(RT-1^a) rats (group A1–A4, n = 6 per group) or heart syngrafts from LEW(RT-1¹) rats (group S1–S4, n = 6 per group).

Group	Treatment		Survival time days	Mean survival time days
Group	Rapamycin	MTX	Survival time days	Mean survival time days
A1	−	−	5 (1), 6 (4), 7 (1)	6.00 ± 0.63
A2	+	−	11 (1), 12 (3), 13 (1)^a	12.00 ± 0.71^b,c
A3	−	+	11 (1), 12 (4), 13 (1)	12.00 ± 0.63^b,c
A4	+	+	16 (1), 18 (2), 19 (2)^a	18.00 ± 1.22^b
S1	−	−	>100 (6)	>100^b,c
S2	+	−	>100 (6)	>100^b,c
S3	−	+	>100 (6)	>100^b,c
S4	+	+	>100 (6)	>100^b,c

Data presented as days (n rats) or mean ± SD.

Two rats died during the observation period and were excluded: one each in groups A2 and A4.

P < 0.05 compared with A1; ^cP < 0.05 compared with A4; Student’s t-test.

MTX, methotrexate.

In the allograft control group (A1), 46 proteins had significantly altered proteomic peak intensities, compared with the syngraft control group (P < 0.05, data not shown). Of these, 28 proteins had significant differences when comparing each of the allograft treatment groups against the allograft control group (Table 2). The allograft methotrexate and rapamycin group showed 11 of 28 rejection-related proteins with significant differences in peak intensity, compared with the allograft control group (P < 0.05).

Table 2.

Proteomic peak intensities of 28 potential rejection-related proteins from surface-enhanced laser desorption/ionization time-of-flight mass spectrometry analysis of serum from LEW(RT-1¹) rats, who had received either heart allografts from DA(RT-1^a) rats or heart syngrafts from LEW(RT-1¹) rats.

m/z	Allograft control n = 6	Syngraft control n = 6	Allograft rapamycin n = 6	Allograft MTX n = 6	Allograft MTX + rapamycin n = 6
1950	3.6415 ± 1.0943	1.5008 ± 0.2237^a	1.6445 ± 0.7638^a	1.4117 ± 0.7408^a	3.9404 ± 1.8841
2881	8.0644 ± 3.7057	2.0534 ± 1.0916^a	0.2847 ± 0.7939^a	1.7830 ± 1.6635	3.2285 ± 0.9987
3376	6.3470 ± 2.8741	2.2750 ± 0.8691^a	3.3500 ± 1.4021	5.0803 ± 2.5679	15.1212 ± 2.8105^a
3500	61.7487 ± 14.6609	30.5922 ± 5.7805^a	31.5265 ± 10.0698^a	40.5131 ± 9.1222^a	52.9948 ± 16.3305
4176	6.2371 ± 1.4741	13.1376 ± 2.6474^a	6.2852 ± 2.1996	5.2620 ± 1.1088	9.9945 ± 2.4824^a
4375	26.4306 ± 14.9442	5.9400 ± 0.8360^a	3.9134 ± 2.8521^a	14.0446 ± 10.938	7.9466 ± 1.9342
4593	14.0845 ± 4.7759	5.6422 ± 1.5177^a	4.5188 ± 2.4300^a	9.6446 ± 5.1590	7.2117 ± 1.0144
4967	4.9403 ± 1.0946	10.6572 ± 0.5707^a	2.1225 ± 0.6708^a	3.2305 ± 1.0138	4.8149 ± 0.3702
5188	3.4296 ± 0.4815	5.7278 ± 1.3448^a	2.4621 ± 1.3072	1.7072 ± 0.2569^a	2.9478 ± 1.5139
5293	6.9409 ± 1.8886	0.3202 ± 0.4205^a	3.0108 ± 1.9187^a	4.9164 ± 6.3486	1.7028 ± 0.6838^a
5844	5.9792 ± 0.6388	17.5006 ± 5.1685^a	6.7709 ± 2.6764	7.7226 ± 1.7386	10.7055 ± 5.2839^a
6182	4.4867 ± 0.2486	2.7742 ± 0.4657^a	3.0496 ± 0.3867^a	4.6527 ± 1.7981	5.0486 ± 1.0129
6834	10.1435 ± 2.4405	3.7008 ± 1.2856^a	4.9548 ± 0.7583^a	7.1938 ± 1.3778	6.6784 ± 1.5826
7059	95.5744 ± 12.6410	51.3755 ± 4.5712^a	56.3343 ± 14.521^a	69.6360 ± 7.4089^a	66.2583 ± 31.1963
7123	19.2276 ± 3.7736	9.9541 ± 1.7377^a	10.4156 ± 1.0938^a	16.2178 ± 1.5618	14.8913 ± 2.2784
7269	32.2728 ± 5.1274	13.8246 ± 2.4549^a	18.0912 ± 1.5334^a	27.5697 ± 2.8288	25.2043 ± 5.7100
7941	2.3275 ± 1.4976	7.4351 ± 1.7531^a	4.4476 ± 1.7824	4.4009 ± 1.1501	6.2284 ± 0.9935^a
7993	2.8034 ± 1.1242	7.2934 ± 1.7074^a	5.2011 ± 1.2473^a	5.4604 ± 1.1784^a	7.8795 ± 6.2284
8049	2.2933 ± 0.9355	12.1950 ± 2.9269^a	4.9726 ± 2.4829	5.3588 ± 2.9818	5.1762 ± 0.7322^a
8280	6.1233 ± 1.5604	29.3106 ± 3.2833^a	17.6307 ± 8.5842	20.8140 ± 9.0664^a	25.1188 ± 3.4754^a
8375	12.3169 ± 4.4709	30.9582 ± 5.0366^a	29.5462 ± 13.9287^a	31.5016 ± 11.922^a	38.1846 ± 8.3291^a
8577	2.1622 ± 1.2673	6.8483 ± 1.3505^a	5.8367 ± 2.8003	5.0572 ± 2.0863	6.7491 ± 1.3291^a
9338	9.1229 ± 2.6825	22.3046 ± 5.7089^a	29.2782 ± 9.9409^a	23.1440 ± 6.4920^a	12.6337 ± 3.4252
12807	0.5029 ± 0.1219	1.8068 ± 0.8740^a	1.2366 ± 0.6019^a	0.7553 ± 0.4362	1.4422 ± 1.1389
13750	22.1437 ± 8.4222	7.8945 ± 0.4529^a	11.4521 ± 2.9112^a	12.3117 ± 3.0116^a	13.4735 ± 7.4224
13969	5.6126 ± 2.1838	2.1891 ± 0.3789^a	3.5527 ± 0.9604	3.5131 ± 0.5755^a	3.4203 ± 1.7100
14866	0.1590 ± 0.0728	0.9651 ± 0.3580^a	1.1524 ± 0.9373^a	0.3975 ± 0.3095	1.0006 ± 1.1270^a
26145	0.2555 ± 0.0806	0.1349 ± 0.0327^a	0.2908 ± 0.2866	0.1777 ± 0.0504	0.0805 ± 0.0299^a

Data presented as mean ± SD.

P < 0.05 versus allograft control; Student’s t-test.

m/z, mass-to-charge ratio; MTX, methotrexate.

A search of the UniProtKB/Swiss-prot database (using pI and mass information for the 28 proteins presented in Table 2) revealed that two of the candidate proteins were likely to be complement component C3f fragment and complement component 4A (C4A anaphylatoxin) (Table 3).

Table 3.

UniProtKB/Swiss-prot database search results, showing matching protein isoelectric point (pI) and mass information (obtained using the TagIdent protein identification tool; CM-10 array mass and pI range, 4.0–14.0), for two protein peaks identified as potential rejection-related proteins from surface-enhanced laser desorption/ionization time-of-flight mass spectrometry analysis of serum from LEW(RT-1¹) rats, which had received either heart allografts from DA(RT-1^a) rats or heart syngrafts from LEW(RT-1¹) rats.

m/z	Matching information from UniProtKB/Swiss-prot database
m/z	Name of protein	Mw	pI	Comment
1950	Complement C3f fragment	1948	9.32	Derived from the proteolytic degradation of complement C3
8577	C4a anaphylatoxin	8594	9.50	Derived from proteolytic degradation of complement C4; a mediator of local inflammation

m/z, mass-to-charge ratio; Mw, molecular weight; pI, isoelectric point.

Discussion

Proteomic profiles obtained using SELDI-TOF MS can be valuable for disease diagnosis.^8,9 The emergence of new peaks, and alterations of peak intensities, can reflect the critical protein release and concentration change in response to tissue damage or disease.¹⁰

The present study identified 28 transplant rejection-related proteins as potential biomarkers for rejection. A search of the UniProtKB/Swiss-prot database revealed likely matches: complement component C3f fragment (1950 Da) and C4a anaphylatoxin (8577 Da). This suggested that complement system activity was elevated following organ transplantation, concurring with a report that peripheral synthesis of complement is required for the donor organ response to tissue stress, and for priming alloreactive T cells in mediating transplant rejection.¹¹ Published information on the remaining 26 proteins was limited: in some cases even the protein name was unclear, and as such they are not discussed further.

Research has indicated that a complement component 3 (C3) polymorphism in donor kidneys could be used as a marker to diagnose rejection.^12,13 Another study failed to confirm an association between C3 polymorphisms and transplant outcome.¹⁴ C3 processing by C3 convertase has been reported to be the central reaction in both classical and alternative complement pathways.¹⁵ C3 convertase catalyses the cleavage of C3 into C3a and C3b by C3, indicating that the concentration of C3 could fluctuate when the complement system is activated. C3b is rapidly split in two, to form inactivated C3b (iC3b) and C3f, which is then released.¹⁶ The complement C3f fragment may, therefore, be a more reliable marker than C3 in assessing immunological status following transplantation. In the present study, the protein with m/z 1950 Da (which is likely to be complement C3f fragment) displayed a peak intensity that was significantly higher in the allograft control group compared with the syngraft control group, allograft methotrexate group and allograft rapamycin group, suggesting this may be a risk factor for rejection.

Complement component 4a (C4a anaphylatoxin) is derived from proteolytic degradation of complement component 4 (C4).¹⁷ It is reported that C4 is of critical importance in the clearance of immune complexes¹⁸ and low levels of C4a have been associated with diabetes-related microangiopathy.¹⁹ Low level C4a-induced endothelial dysfunction has been described as resulting from defective clearance of immune complexes, which may deposit in nearby tissues and cause local damage.²⁰ For this reason, C4a has been suggested as a regulatory factor for endothelial protection. In the area of transplantation, immune complexes are considered to contribute to graft-vessel damage, leading to graft loss. The present study demonstrated that the m/z 8577 Da protein (likely to be C4a anaphylatoxin) had a peak intensity significantly lower in the allograft control group compared with the syngraft control and allograft methotrexate and rapamycin groups, which may suggest that low C4a could be a risk factor for tissue rejection.

Both methotrexate and rapamycin exhibited the capacity to reduce allograft rejection, in the present study. Moreover, the combination of methotrexate and rapamycin presented additive effects in prolonging cardiac allograft survival. In the allograft methotrexate and rapamycin group, 11 of the 28 rejection-related proteins had significantly different peak intensities compared with the allograft control group. Most of the peak intensities of these 11 proteins were closer to those of the syngraft control group, compared with the allograft methotrexate-alone or allograft rapamycin-alone groups. As a relatively longer mean survival time can be achieved by cotreatment with methotrexate and rapamycin, the concentration of these 11 proteins may be associated with additive immunosuppression; thus, they may provide potential drug targets for the development of immunosuppressive agents. There was, however, one exception. Compared with the allograft monotherapy groups, the peak intensities of the protein with an m/z of 3376 Da in the allograft methotrexate and rapamycin group was far removed from that of the syngraft control group. It could be speculated that as-yet unknown metabolic or drug–drug interactions between the different agents may explain this result.

There were some limitations to the present study. First, this was an animal study and a relatively small number of animals were used. Thus, large-scale animal experiments and clinical trails are needed to clarify in more detail the diagnostic value and clinical significance of these proteins.

In conclusion, SELDI-TOF MS proteomic analysis identified 28 transplant rejection-related proteins that may be potential diagnostic biomarkers for rejection. Two protein peaks – m/z 1950 Da and 8577 Da – were identified from the published literature as complement component C3f fragment and C4a anaphylatoxin, respectively, and may have potential for post-transplant rejection diagnosis. In the allograft methotrexate and rapamycin group, 11 of the 28 proteins may be potential cotreatment targets for the combined effects of these agents. The rejection-related proteins identified may be subject to post-translational modifications, cleavage and degradation. Thus, to identify more biomarkers of rejection and therapeutic targets (in order to reduce the use of immunosuppressive agents), further investigations are required. Such investigations should help to identify accurately, and purify, the corresponding proteins of these specific peaks.

Footnotes

Declaration of conflicting interest

The authors declare that there are no conflicts of interest.

Funding

This research received funding from the National Nature Science Fund of China, grant no. ZR09-6-02.

References

Orlando

. Finding the right time for weaning off immunosuppression in solid organ transplant recipients. Expert Rev Clin Immunol 2010; 6: 879–892.

Xiu

Uchida

. Simplified method of heterotopic rat heart transplantation using the cuff technique: application to sublethal dose protocol of methotrexate on allograft survival. Microsurgery 2001; 21: 16–21.

Sakuma

Xiu

Uchida

. Short-course methotrexate and long-term acceptance of fully allogeneic rat cardiac grafts: a possible mechanism of tolerance. Transpl Immunol 2002; 10: 49–54.

Tang

Tornatore

Weinberger

. Current developments in SELDI affinity technology. Mass Spectrom Rev 2004; 23: 34–44.

Issaq

Veenstra

Conrads

. The SELDI-TOF MS approach to proteomics: protein profiling and biomarker identification. Biochem Biophys Res Commun 2002; 292: 587–592.

Grus

Joachim

Pfeiffer

. Analysis of complex autoantibody repertoires by surface-enhanced laser desorption/ionization-time of flight mass spectrometry. Proteomics 2003; 3: 957–961.

Ono

Lindsey

. Improved technique of heart transplantation in rats. J Thorac Cardiovasc Surg 1969; 57: 225–229.

Petricoin

Liotta

. SELDI-TOF-based serum proteomic pattern diagnostics for early detection of cancer. Curr Opin Biotechnol 2004; 15: 24–30.

Clarke

. Proteomic research in renal transplantation. Ther Drug Monit 2006; 28: 19–22.

10.

Elased

Cool

Morris

. Novel mass spectrometric methods for evaluation of plasma angiotensin converting enzyme 1 and renin activity. Hypertension 2005; 46: 953–959.

11.

Sacks

Zhou

. The role of complement in the early immune response to transplantation. Nat Rev Immunol 2012; 12: 431–442.

12.

Brown

Kondeatis

Vaughan

. Influence of donor C3 allotype on late renal-transplantation outcome. N Engl J Med 2006; 354: 2014–2023.

13.

Damman

Daha

H Leuvenink

. Association of complement C3 gene variants with renal transplant outcome of deceased cardiac dead donor kidneys. Am J Transplant 2012; 12: 660–668.

14.

Varagunam

Yaqoob

Döhler

. C3 polymorphisms and allograft outcome in renal transplantation. New Engl J Med 2009; 360: 874–880.

15.

Nakagawa

Komorita

. Complement component C3-derived neutrophil chemotactic factors purified from exudate of rat carrageenin-induced inflammation. Biochem Biophys Res Commun 1993; 194: 1181–1187.

16.

Ganu

Müller-Eberhard

Hugli

. Factor C3f is a spasmogenic fragment released from C3b by factors I and H: the heptadeca-peptide C3f was synthesized and characterized. Mol Immunol 1989; 26: 939–948.

17.

Cui

Ferreri

Hugli

. Structural characterization of the C4a anaphylatoxin from rat. Mol Immunol 1988; 25: 663–671.

18.

Schifferli

Peters

. The role of complement and its receptor in the elimination of immune complexes. N Engl J Med 1986; 315: 488–495.

19.

Barnett

Mijovic

Fletcher

. Low plasma C4 concentrations: association with microangiopathy in insulin dependent diabetes. Br Med J (Clin Res Ed) 1984; 289: 943–945.

20.

Lappas

. Lower circulating levels of complement split proteins C3a and C4a in maternal plasma of women with gestational diabetes mellitus. Diabet Med 2011; 28: 906–911.