Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) Mass spectrometry for protein identification 2-Dimensional Gel Electrophoresis MALDI-TOF Mass Spectrometry Speaker: Dr. J. S. Yu Date: 3/21/2002

The age of X-omics and biotechnology: Genomics: Human genome project Transcriptomics: cDNA microarray Proteomics: Development and involvement of mass spectrometry MALDI-TOF MSTandem mass spectrometer (MS/MS)cDNA microarray Celera Genomics Inc.

Proteomics solution IEF SDS-PAGE

2-Dimension Electrophoresis (2-DE) for Protein Separation Speaker: C. C. Wu Date: 31/10/2001 The core technology of proteomics is 2- DE: At present, there is no other technique which is capable of resolving thousands of proteins in one separation procedure.

Isoelectric point (pI): Isoelectric point is the pH of a solution at which the net charge of protein is zero. In electrophoresis there is no motion of the particles in an electric field at the isoelectric point. Net charge pH Isoelectric point NH 3 + COOH NH 3 + COOH pH < pI Positive charge NH 3 + COO - NH 3 + COO - pH = pI NH 2 COO - NH 2 COO - pH > pI Negative charge

sample pH 9 - pH 3 + Isoelectric focusing (1 st dimension) General principle and protocol of 2-Dimension Electrophoresis MW pH gradient SDS-PAGE Ampholytes polyacrylamide 2nd dimension

Traditional Equipment for Isoelectric focusing (IEF): Ampholytes polyacrylamide Cathode (-) electrode solution Anode (+) electrode solution

Traditional 2-Dimensional Electrophoresis Anode (+) electrode solution Cathode (-) electrode solution Disadvantage: cathodic drift Ampholyte polyacrylamide pH 3 pH 3 pH 3 pH 9 pH 7 pH 5 Time

Immobilized pH Gradient (IPG) Polyacrylamide gel Acidic buffering group: Basic buffering group: CH2 = CH-C-NH-R O COO - NH 3 + Acrylamide monomer

Gradient maker plastic support film Production of Immobilized pH Gradient (IPG) strip A C B F E D acidic basic pH 3 pH 10

IPGphor (IEF System) Amersham Pharmacia Biotech Inc. Protein IEF Cell Bio-Rad Laboratories Equipment for Isoelectric focusing (IEF):

Lysis solution: 8M Urea 4% NP-40 or CHAPS 40mM Tris base Sample preparation Cell line Lysis solution Sonication vacuum Lysis solution Centrifugation Measurement of [protein] 2-DE sample

IPG strip rehydration and sample loading 2-DE sample Rehydration solution Rehydration solution: 8M Urea 2% NP-40 or CHAPS 2% IPG buffer (Ampholyte) 0.28% DTT Trace Bromophenol blue IPG strip holder Position the IPG strip

IPG strip rehydration and sample loading Strip holder Cathode (-) electrode Anode (+) electrode 30 voltage 12hr

First dimension: Isoelectric focusing 1. Place electrode pads (?) V step-n-hold 1.5hr V step-n-hold 1.5hr V gradient 1500vhr V gradient (?) 36000vhr Time Voltage Holder cover IPG strip Electrode pads

Second dimension: SDS-PAGE SDS equilibration SDS-PAGE SDS equilibration buffer 50 mM Tris-HCl 6 M Urea 30% Glycerol 2% SDS Trace Bromophenol SDS SDS-PAGE 0.5% agarose in running buffer SDS-PAGE Marker in paper IPG strip

Protocol of silver stain: 50% methanol 25% acetic acid 4hr ddH 2 O x 3 times 30min/time 0.004% DTT solution 30min 0.1% AgNO 3 30min ddH 2 O 30 sec 3% Na 2 CO % formaldehyde 2.3M citric acid 5% acetic acid 25% methanol

2-DE separation of soluble E. coli proteins