PROTEOMICS OF REDIVAC FLUID: COMPARATIVE ANALYSIS OF PROTEIN CLUSTERS IN PERITONEAL LAVAGE FLUID AND GYNAECOLOGICAL CANCER

EUGENE LEONG WENG KONG1, RAMARAO SERIRAMALU2, PUTERI SHAFINAZ ABDUL RAHMAN2,3, NOOR AZMI MOHAMAD ADENAN1 AND ONN HAJI HASHIM2,3.

1Department of Obstetrics and Gynaecology, 2Department of Molecular Medicine, Faculty of Medicine, 3University of Malaya Centre for Proteomics Research, 50603 Kuala Lumpur.

Objectives Results

Conclusions

References

The method may be used to analyse the protein compositions of post-operative drain fluids from various cohorts of surgical patients.Materials & Methods

Gynaecological cancer is a cancer that develops in a woman’s reproductive organs. The signs and symptoms for gynaecological cancer remain to be vague and thus hinder early detection. Therefore search for biomarkers using body fluids to provide early detection is urgently needed. Peritoneal lavage fluid is obtained at laparotomy pre-definitive surgery when saline is introduced into the peritoneal cavity, and then removed by clean sterile syringe. The fluid is then examined for malignant (cancer) cells under a procedure called peritoneal washing cytology [1]. Interestingly, the fluid and its protein constituents have not been studied previously. In the present study, we have subjected proteins of redivac fluids from patients (with consent) with gynaecological cancer and the peritoneal lavage fluid as control to 2-dimensional electrophoresis (2-DE) and mass spectrometry.

1. http://hepatitis.about.com/od/pqr/g/PeritonealFluid.htm2. Abdul-Rahman, P. S., Lim, B. K., & Hashim, O. H. (2007).

Expression of high-abundance proteins in sera of patients with endometrial and cervical cancers: Analysis using 2-DE with silver staining and lectin detection methods. Electrophoresis, 28(12), 1989-1996.

3. Gruys, E., Toussaint, M. J., Upragarin, N., Van, E. A., Adewuyi, A. A., Candiani, D., et al. (2005). Acute phase reactants, challenge in the near future of animal production and veterinary medicine. J Zhejiang Univ Sci B, 6(10), 941-947.

Acknowledgement• This work was funded by research grant UMRG

RG278/10HTM from the University of Malaya, Kuala Lumpur.• Mass spectrometry analysis was performed with the access to the Proteomic facility at the Medical Biotechnology Laboratory, Faculty of Medicine, UM. • Special thanks to Ms. Nazirah Abrahim for her assistance in

sample collection and poster design.

Fig 1. Representative 2-DE profile of peritoneal lavage fluid (left) and redivac fluid from a patient with gynaecological cancer (right).

Table 1. Maldi ToF/ToF results obtained for proteins spots detected in Fig. 1.

Discussions• The 2DE protein profile obtained from peritoneal

lavage fluid is comparable with the redivac fluid of the gynaecological patient. However, a significantly higher number of low molecular proteins were resolved in the redivac fluid.

• The proteins identified in Table 1 mainly falls under the category of acute phase proteins. Acute phase proteins are proteins whose concentrations are altered in response to trauma, infection and inflammation[3].

• Visual comparison between 2DE profile shows over-expression of haptoglobin (11), Ig alpha-1 chain C region (15), vitamin D-binding protein (16) and serum albumin fragments (21, 22, 23).

• At present, image analysis of the expression of proteins clusters from both group of samples are performed by using Image Master Platinum version 7.0.

Collection of peritoneal lavage fluid (n=3) & redivac fluid from patients with gynaecological

cancer (n = 3)

Separation by 2-dimensional electrophoresis (2-DE) and silver staining [2]

Image analysis using Image Master Platinum 7

Mass spectrometry analysis

Spot No Spot/ Cluster ID Matched Protein IdentityAccession number

(Swiss-Prot)

Theoretical Mass (Da) Theoretical pI Mascot Score No. of peptides

matchedSequence

Coverage (%)

1 TRF Serotransferrin P02787 77000 6.81 603 12 182 ALB Serum albumin P02768 69321 5.92 669 13 233 HPX Hemopexin P02790 51643 6.55 380 8 184 ABG Alpha-1B-glycoprotein P04217 54239 5.58 359 8 18

5 DBP Vitamin D-binding protein P02774 52929 5.40 1151 16 47

6 AAT Alpha-1-antitrypsin P01009 46707 5.37 900 15 397 KNG Kininogen-1 P01042 71912 6.34 114 4 68 AHS Alpha-2-HS-glycoprotein P02765 39300 5.43 220 4 10

9 LRG Leucine-rich alpha-2-glycoprotein P02750 38154 6.45 118 4 12

10 APOA 4 Apolipoprotein A-IV P06727 45371 5.28 405 12 3111 HAP Haptoglobin P00738 45177 6.13 446 9 19

12,13 AATf Alpha-1-antitrypsin fragment P01009 46707 5.37 374/114 19/8 27/16

14 ZAG Zinc-alpha-2-glycoprotein P25311 33851 5.57 51 2 715 Ig Ig alpha -1 chain C region P01876 37631 6.08 93 4 12

16 DBP Vitamin D-binding protein P02774 22995 5.76 554 6 45

17 HAP Haptoglobin P00738 45177 6.13 264 4 1318 TTY Transthyretin P02766 15877 5.52 137 2 17

19, 21,22,23 ALBf Serum albumin fragment P02768 69321 5.92 229/445/214/146 12/9/6/6 8/13/8/420 SAA Serum amyloid A protein P02735 13524 6.28 88 2 27