SELDI-TOF

Question 1

Describe Surface-Enhance Laser Desorption Ionizaton-Time of Flight (SELDI-TOF) mass spectometry as an approach for the rapid detection and quantification of proteins.

Answer 1

1. SELDI-TOF info
2. Array Preparation
3. Applications
4. Case study: Ovarian Cancer
5. Advantages
6. Disadvantages

Question 2

1. SELDI-TOF info

Answer 2

• surface enhanced laser desorption and ionization-time of flight
• ProteinChip technology
• mass spectometry based technology used to identify biomarkers/proteins that are changing in expression between clinical samples
• does not rely on digesting proteins with trypsin, analyses intact proteins
• components;
  
  1. proteinChip Arrays = seperation
  2. proteinChip Reader = detection
  3. proteinChip Software = analysis
• can be used to analyse a variety of sample types e.g tumor tissue and cultures, nuclear extracts and serum

Question 3

2. Array Preparation

Answer 3

• each chip has 8 spots for 8 samples and a specific chromatographic surface for pulling down particular proteins of interest, can be chemical or biological by nature, complex protein sample is placed on proteinChip Array by using a funnel for each spot and allowed to incubate for 30 mins to 1 hr
• unbound proteins are removed by washing the ProteinChip with an appropriate stringency buffer to refine the types of bound proteins retained
• energy absorbing matrix (EAM) is applied to each spot (excess at 100:1) and is left to dry, heat from the instrument will help it dry quicker, laser hits chip surface, matrix absorbs that energy and starts to vibrate, ablates off chip surface with protein embedded within it and is ionized, ionized proteins are detected and their mass accurately determined by time-of-flight mass spectometry, light hits faster than heavier
• map of proteins (trace view) is generated showing their accurate mass and relative ion intensity

Question 4

3. Applications

Answer 4

1. research proteomics (protein-protein interactions)
   - receptor-ligand assays e.g antibody on ProteinChip that binds to a particular protein
   - SELDI-TOF more beneficial for analysis as opposed to ELISA as SELDI-TOF can recognises the 2 different isoforms of IL-8 whereas ELISA cannot. this is advantageous as these 2 isoforms have very different biological functions within cells, bindinig onto different receptors driving tumorigenesis
   - these protocols can also be carried out by robots which are much more efficient as results are reproducible due to no petty human error
2. process proteomics (process proteomics)
   - pharmaceutical companies can utilise large scale columns with IMAC-Ni bound to its surface that wil bind onto their desired product and use imidazole to elute a very pure form of their product

Question 5

4. Case study: ovarian cancer

Answer 5

• 184 serum samples taken from patients with ovarian cancer, patients with benign tumours and healthy donors and were analysed on strong anion-exchange surfaces using SELDI-TOF
• a series of protein biomarkers were found that were significantly chanfing in abundance in blood samples of patients wit ovarian cancer compared to the control group
• SELDI-TOF not able to identify which protein was increased in abundance as the sample protein was not broken into peptides that could be searched against a database, however as its mw and the surface it binds to is known the protein is able to be isolated and enriched

Question 6

5. advantages of SELDI-TOF

Answer 6

• high-throughput technique
• minimal sample volume required
• ability to analyze proteins from a variety of crude biological samples (can directly test native proteins/peptides)
• prefractionation of samples with ProteinChips which allows seperation of proteins based on their chemical characteristics
• allows sample binding to the surface of a ProteinChip array (anionic, cationic, metallic, normal phase and hydrophobic)

Question 7

6. Disadvantages of SELDI-TOF

Answer 7

• lack of tandem mass spectometry: doesn’t provide peptide or protein identification
• low mass resolution
• low mass accuracy
• cost of the ProteinChip array and fractionation kit
• the software (CiphergenExpressTM) will reveal many dozens of ‘statistically significant’ candidate biomarkers when small numbers of samples (e.g 5 to 10) are used for ‘discovery’ studies