Proteomics and Metabolomics in Transfusion Medicine.pptx

Proteomics and metabolomics in transfusion medicine

1. Proteomics and Metabolomics in Transfusion M edicine Dr Atif Irfan Khan

2. Outline Introduction Proteomics Definition Techniques Role in transfusion medicine Limitations and future prospects Metabolomics Definition Techniques Role in transfusion medicine Limitations and future prospects

3. I ntroduction Genomics Transcriptomics Proteomics Metabolomics DNA RNA PROTIEN METABOLITE Transcription Translation Enzymatic action

4. Proteomics The name protein is derived from the Greek term proteios meaning the first rank was used for the first time by Berzelius in 1838 The term “proteome” originates from the words protein and genome , it was coined by Wilkins and colleagues in 1996

5. Proteomics Definition Proteomics is the total protein content of a cell or that of an organism . It represents the entire collection of proteins encoded by the genome in an organism . Proteomics is the understanding of the structure, function, and interactions of the entire protein content of an organism, tissue, cell or fluid.

6. Proteomics

7. Methods Separation of proteins Staining and digestion of proteins Identification of protein

8. Seperation Methods Proteomic technologies Conventional techniques Chromatographic based techniques Ion exchange chromatography Size exclusion chromatography Affinity chromatography ELISA Western blotting Advance techniques Protien microarray Analytical protein microarray Functional protein microarray Reverse phase protein microarray Gel based approaches Mass spectrometry Edman sequencing Quantitative techniques ICAT- Isotope coded affinity tag based protein profiling SILAC-Stable Isotope Labeling by/with Amino acids in Cell culture iTRAC - Isobaric tags for relative and absolute quantitation High throughput techniques X-ray chromatography NMR spectroscopy

9. Gel based separation technique 2 dimensional gel electrophoresis 2D-GE Available since 1975 Separates complex protein mixtures based on isoelectric point and molecular weight The separated proteins are visualized by staining with colloidal Coomassie brilliant blue (CBB), or silver nitrate Higher resolution separation can be achieved by using several 2D-GE with overlapping narrow pH gradients After staining gel can be scanned and subject to computer based analysis of protein

10. Gel based separation technique 2D-GE The two dimension (2-D) First dimension is based on isoelectric point known as isoelectric focusing(IEF) Second dimension  molecules are then separated at 90 degrees from the first electropherogram according to molecular mass

11. Gel based separation technique 2D-GE Advantages Relatively low cost Easy to use Automation possible Disadvantage N ot a high-throughput technique N ot suitable for the separation of membrane proteins I ts sensitivity is not satisfactory for low-abundance proteins

12. Gel based separation technique 2D-GE Other improved modification Quantitative proteomic techniques such as difference gel electrophoresis (DIGE) Uses fluorescent cyanine series of dyes and allows relative quantitative analyses of up to three samples sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE )

13. Non gel based separation technique U sed in detecting protein/structure not accessible using 2D-GE, such as the detection of low-abundant or hydrophobic (membrane) proteins Relies on separation 2D peptides rather than protein then characterization using MS Liquid chromatographic methods are commonly used Protein sample are degraded to smaller units by enzyme like trypsin B ased on two or more biophysical characteristics such as surface charge (ion exchange chromatography), hydrophobicity (hydrophobic interaction chromatography), or affinity to particular compounds; affinity , dye ligand, reversed-phase liquid chromatography (RPLC )

14. Non gel based separation technique 2D-LC Protein sample is reduced, alkylated, and digested in solution Peptide mixture is fractionated through 2D-LC using a strong cation exchange column T he derived fractions are further separated, on the base of hydrophobicity M ultidimensional protein identification technology (MUDPIT ) is an application of it

15. Non gel based separation technique Another modification of this test is combined fractional diagonal chromatography (COFRADIC ) Advantages of non gel based methods H igh-throughput capabilities P ossibility of full automation D irect integration with MS H igher sensitivity S maller amount of starting material needed

16. Mass spectrometry Used for analysis of proteins and peptides since 1989 Main components of mass spectrometer Ion source (the device that brings the analytes into gas phase and ionises them ) One or more mass analysers - measures the mass-to-charge ratio (m/z) of the ionised analytes A detector , which registers the number of ions at each m/z value

17. Mass spectrometry In ESI (electron spray ionization) ions are formed from a liquid solution, while in MALDI a laser pulse induces the sample to sublimate out of a dry crystalline matrix. Gaseous ions formed by MALDI and ESI are accelerated into the mass analyser by an electric potential the m/z ratios are determined by the motion of the ions through the mass analyser . The detector converts the stream of ions into a voltage that is interpreted by a computer and converted to a mass spectrum

18. Mass spectrometry Types of MS Time-of-flight (TOF), Quadrupole (Q), triple quadrupole or linear ion trap (LIT), ion trap (IT), Fourier transform ion cyclotron resonance (FTICR) Advantages H ighly sensitive, requiring only 10-15 femtomoles of peptides as starting materials, and extremely accurate

19. Protein Microarrays Basic principle is capture of specific proteins at specific sites The chips consist of a glass or plastic surface spotted with a checkerboard-like grid of molecules that captures the protein Micro-volumes of reagents and samples is delivered to the chip, where bound molecules are detected through a secondary antibody tagged with a fluorescent marker or directly Scanners are used to read the chips D edicated software is used to analyse and interpret the data

20. Protein Microarrays

21. Applications in TM RBC Application in RBC membrane study dates back to 1980s 2D-GE was applied to study the membrane in diseased individuals ESI-MS/MS, SDS-PAGE, MS were used in profiling RBC proteins The structure & function relationship in the erythropoietin receptor signaling complex, and the protein involved in signaling The surface proteins of malaria infected RBCs T he influence of type 2 diabetes mellitus on RBC membranes

22. Applications in TM RBC storage lesions have been studied Length of storage and proteins that accumulates has been done using LC-ESI-MS/MS which also compared pre storage leukoreduced and non leukoreduced RBC C hanges of the RBC cytoskeleton during storage of SAGM preserved non leukoreduced RBC unit using 2D-GE, with staining by CBB stain

23. Applications in TM Platelets In 1979, Clemetson used 2D-GE to analyze platelets Various systems of MS has been used to study platelet structures at various stages of its activation 2D-GE has also been employed in detecting various platelet proteins P hosphorylated proteins and the impact of protein phosphorylation on signal transduction pathways/cascades The platelet releasate of activated platelets The platelet microparticle proteome

24. Applications in TM Platelet storage lesions DIGE and MS has been used in identifying important protein markers that can be used as markers of storage lesions Affects of Various preparation methods of platelets on storage lesions Characterizing platelet storage lesion with approach to monitoring the in vitro quality of stored PC and predicting their in vivo performance

25. Applications in TM Plasma In 1977 human plasma proteins were first profiled through 2D-GE, closely associated with the history of proteomics itself it is the most complex human-derived proteome large proportion of it is albumin (55 %) 2D-GE and LC-ESI-MS/MS has been employed Proteomics has been used to evaluate the effect of pathogen inactivation techniques

26. Applications in TM Plasma Other plasma derivative products have been studied using 2D-GE coupled with MALDI-MS/MS or LC-ESI-MS/MS PCC- study processing and to improve quality IVIG

27. Applications in TM Granulocyte Not a lot has been applied on this Studies have addressed regulation of neutrophil function, pathways induced by proinflammatory mediators

28. Applications in TM CD34+ haematopoietic stem cell Functional genomics approaches have been used to study myeloid differentiation , applying different proteomic techniques, such as oligonucleotide microarrays and 2D-GE MS, and DNA microarrays D ifferentiation is a critical step in stem cell development , during which proteomic changes are observed as a cell undergoes successive development

29. Proteomic – work flow example

30. Method - RBCs stored for 7 and 42 days were used. After extraction of membrane proteins with a deoxycholate containing buffer, band 3 complexes were co-immunoprecipitated on magnetic beads coated with two anti-band 3 antibodies. Both total membrane protein extracts and eluates (containing band 3 complexes) were separated on SDS-PAGE, proteins were ingel digested and analyzed by LC-MS/MS , adenylosuccinate lyase (ADSL ), a-adducin and flotillin-2 were further quantified using western blots. Results- ADSL abundance tended to increase during storage in both total membrane protein and band 3 complexes, whereas a-adducin mainly tended to stay onto the membrane extract. Flotillin-2 was equivalently present in total membrane proteins whereas it clearly co-immunoprecipitated with band 3 complexes during storage

31. Limitation and future prospects of proteomics limitation Cost Highly technical and time consuming Difficult for transfusion services to employ cutting edge expertise for such processes Future prospects Rapidly developing science which offers potential for improvement in transfusion services Complexity of blood and its component is potential area that could be exploited with these technologies

32. Metabolomics Metabolomics is a large scale study of small molecules called metabolites within cells bio fluids tissue or organisms Collectively these small molecule and their interaction within biological system are known as metabolome It involves comprehensive and simultaneous systematic profiling of multiple metabolite levels and their systematic and temporal changes to different factors

33. Metabolomics- G eneral application

34. Methods/Techniques Nuclear magnetic resonance (NMR) Mass spectrometry (MS) Gas chromatography- Mass spectrometry (GS-MS) Liquid Chromatography- Mass spectrometry (LC-MS) Capillary electrophoresis- Mass spectrometry (CE-MS) Fourier Transform infrared spectroscopy (FTIR)

35. Nuclear magnetic resonance T he molecular structure of a material is analyzed by observing and measuring the interaction of nuclear spins when placed in a powerful magnetic field Resonance  frequency of a substance is usually directly proportional to the strength of the applied magnetic field. T his feature is exploited in imaging techniques

36. Nuclear magnetic resonance

37. Nuclear magnetic resonance Application Analysis of molecular structure of unknown substances Analysis of mixture Study of dynamics Chemical reaction speed, structure binding site, and interaction

38. Metabolomic workflow steps STEP 1: Extraction Extraction of and analysis of hydrophilic and lipophilic small molecule fraction Seperated by protein precipitation e,g ice cold methanol STEP 2: analysis NMR, MS, LC Example- 13 C isotope labelled glucose to study glycolysis/ pentose phosphate pathway

39. Metabolomic workflow steps

40. Metabolomic workflow steps STEP 3: bioinformatics and post data collection elaboration Post data acquisition of data either through NMR or MS, it is integrated into a software that categorizes it/analyses it STEP4: statistical analysis

41. Application in Transfusion Medicine Storage lesion RBC metabolic storage lesions- info on energy metabolism ATP, DPG, lactate, G6PD Corresponding accumulation of oxidative stress markers such as oxidized lipids To Compare these affects in different preservation and additive solution methods Donor variability of storage lesions Considering genomic back ground and gene expression Platelet storage lesion

42. Materials and Methods Red cell concentrates were stored in SAGM, AS-1, AS-3 or PAGGSM, and sampled fourteen times spanning Day 1–46 . These were analysed with ultra-high-performance liquid chromatography (UHPLC ) coupled to mass spectrometry( MS ) analysis affording quantitative metabolic profiles of both intra- and extracellular red cell metabolites . Results Differences were observed in glycolysis, purine salvage, glutathione synthesis and citrate metabolism on account of the storage solutions

43. Application in Transfusion Medicine Cell processing Comparing apheresis and buffycoat methods of platelet preparation to see effects on glycolysis and TCA cycle

44. Methods PCs either untreated ( uPCs ) or INTERCEPT treated ( iPCs ) were sampled along the 7-day storage period. First, metabolites were extracted and analyzed using ultra-high pressure liquid chromatography (UHPLC )— high resolution mass spectrometry( MS ) followed by statistical analysis, urate, a major plasma antioxidant , in the platelet function using flow cytometry based assays Results oxidative damages in stored iPCs compared to uPCs , in particular alteration of the purine and the glutathione metabolism

45. Future aspects of metabolomics C ould potentially become surrogate for markers of blood cell storage quality Could help in developing alternate storage solutions A dvances in understanding of donor and recipient safety as well as effectiveness of transfusion therapy

46. References Liumbruno GM. Proteomics: applications in transfusion medicine. Blood Transfusion. 2008 Apr;6(2):70 . Bryk AH, Wiśniewski JR. Quantitative analysis of human red blood cell proteome. Journal of proteome research. 2017 Jul 24;16(8): 2752-61 . Introduction to proteomics Principles and Applications. C. Mishra Aslam B, Basit M, Nisar MA, Khurshid M, Rasool MH. Proteomics: technologies and their applications. Journal of chromatographic science. 2017 Feb 1;55(2): 182-96 .