Establishment of a near-standard two-dimensional human urine proteomic map

1. Establishment of a near-standard two-dimensional human urine proteomic map J. Oh, J-H Pyo, R-H. Jo, S-II Hwang, S-C. Kang, J-H. Jung, E-K. Park, S-Y. Kim, J-Y. Choi, J. Lim Proteomics 2004, 4:3485-3497 Erica

2. 2-dimensional gel electrophoresis • Powerful tool for characterizing complex protein mixtures. • Up to 10,000 protein spots can be analyzed simultaneously • 2-step process that separates proteins according to isoelectric focusing (IEF) and SDS-polyacrylamide gel electrophoresis.

3. 2-dimensional gel electrophoresis • Powerful tool for characterizing complex protein mixtures. • Up to 10,000 protein spots can be analyzed simultaneously • 2-step process that separates proteins according to isoelectric focusing (IEF) and SDS-polyacrylamide gel electrophoresis. According to what protein property does each technique separate the proteins from the initial mixture? - IEF - SDS-PAGE

4. Extraction of protein Isoelectric focusing (1D gel) Ettan™ IPGphor™ MALDI-TOF SDS-PAGE Spot excision; Trypsin digestion staining

5. Mass Spec Principles Sample + _ Detector Ionizer Mass Analyzer

6. Typical Mass Spectrum Relative Abundance aspirin m/z ratio: Molecular weight divided by the charge on this protein 120 m/z-for singly charged ion this is the mass

7. Artificially trypsinated Fragmented using trypsin Artificial spectra built Spot removed from gel Peptide Mass Identification Spectrum of fragments generated MATCH Library Database of sequences (i.e. SwissProt)

8. Extraction of protein Isoelectric focusing (1D gel) Ettan™ IPGphor™ MALDI-TOF Where can things go wrong amongst these steps? SDS-PAGE Spot excision; Trypsin digestion staining

9. Possible applications of 2-D electrophoresis: - proteome analysis - cell differentiation - detection of disease markers - monitoring therapies - drug discovery - cancer research - purity checks - microscale protein purification.

10. Establishment of a near-standard two-dimensional human urine proteomic map Objectives: - Establish optimal techniques for purifying urinary proteins prior to 2-DE. - Create a proteomic map of normal healthy human urinary proteins.

11. Methods Fig.2. Urinary protein preparation for 2-D gel analysis. - Human clean catch first urine samples from 20 healthy males and 20 healthy females, in 20’s.

12. Methods Fig.2. Urinary protein preparation for 2-D gel analysis. - Human clean catch first urine samples from 20 healthy males and 20 healthy females, in 20’s. Sampling prepping: - “healthy” individuals? - age range?

13. Human: NH4SO4 & TCA precipitation. Moomoo: NH4SO4 & TCA precipitation. Human: dialysis Fig. 1. Comparison of dialyzed and nondialyzed urine proteins on 2-D gels.

14. So what have we lost here?

15. Fig.3. Comparison of urinary protein profiles on 2-D gels. Fig.4. 2-DE profile of the proteins from the pooled urine.

16. Female urine Male urine Fig.5. Comparison of female and male urinary proteins.

17. Fig.6. Classification of identified proteins according to cellular location.

18. Fig.6. Classification of identified proteins according to cellular location. What do you think about the biases involved in constructing this pie chart?

19. Table 2

20. Affi-Gel Blue treated urine Untreated urine Fig.7. What were they trying to show here? Any problems encountered?

21. Summary • Elimination of albumin, salts, and interfering small molecules from urine enhances low-abundant protein profiles and improves the resolution and reproducibility of the 2-D gel. • Gel protein patterns varied between individuals as well as within the same individual whose urine was sampled on two different days. • The 2-D gel proteomic map obtained from the pooled urine of 20 healthy individuals helps to establish a near-standard urine protein profile on a 2-D gel.

22. Summary • Elimination of albumin, salts, and interfering small molecules from urine enhances low-abundant protein profiles and improves the resolution and reproducibility of the 2-D gel. • Gel protein patterns varied between individuals as well as within the same individual whose urine was sampled on two different days. • The 2-D gel proteomic map obtained from the pooled urine of 20 healthy individuals helps to establish a near-standard urine protein profile on a 2-D gel. What should their future prospects be? i.e. What other questions/biases should they be addressing?

23. Would you trust your doctor who ran a 2-D gel of you and told you that you were inflicted with a disease?