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 Celera Genomics Inc. cDNA microarray Tandem mass spectrometer (MS/MS) MALDI-TOF MS

Proteomics solution IEF SDS-PAGE

2-Dimension Electrophoresis (2-DE) for Protein Separation 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. Speaker: C. C. Wu Date: 31/10/2001

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. NH3+ COOH pH < pI Positive charge NH3+ COO- pH = pI NH2 COO- pH > pI Negative charge Net charge pH Isoelectric point

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

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

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

Immobilized pH Gradient (IPG) Acrylamide monomer Acidic buffering group: COO- CH2 = CH-C-NH-R O Basic buffering group: NH3+ Polyacrylamide gel

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

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

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

IPG strip rehydration and sample loading Rehydration solution 2-DE sample 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 Anode (+) electrode Cathode (-) electrode 30 voltage 12hr

First dimension: Isoelectric focusing 1. Place electrode pads (?) 2. 200 V step-n-hold 1.5hr 3. 500 V step-n-hold 1.5hr 4. 1000 V gradient 1500vhr 5. 8000 V gradient (?) 36000vhr Holder cover IPG strip Electrode pads Time Voltage

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

Protocol of silver stain: 50% methanol 25% acetic acid 4hr ddH2O 30 sec ddH2O x 3 times 30min/time 3% Na2CO3 0.0185% formaldehyde 0.004% DTT solution 30min 2.3M citric acid 0.1% AgNO3 30min 5% acetic acid 25% methanol

2-DE separation of soluble E. coli proteins