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Genome Technology s Lab Notebook: Reader Tips and Experiences: Jan 6, 2005

Following are scientists' responses to the question, "How would you fractionate plasma or serum prior to mass spec analysis to increase the number of identified proteins?"

 

For a complete list of scientists' responses, read Genome Technology, a GenomeWeb News sister publication.


"We have a new method that we call protein array pixelation that uses four separation dimensions - three at the protein level and one at the peptide level. These include depletion of the six most abundant proteins using an Agilent antibody column, microSol isoelectric focusing (a method we developed) and 1-D SDS PAGE followed by pixelation of the 1-D gels, digestion of gel slices with trypsin and nanocapillary HPLC followed by LC-MS/MS. When a high-sensitivity linear ion trap mass spectrometer is used, we can identify proteins spanning nine orders of magnitude including multiple proteins in the pg/ml range."


David W. Speicher, PhD

Director, Proteomics Laboratory

The Wistar Institute


"My research laboratory has been engaged in disease proteomics with a focus on plasma microparticles (MPs).

1) Preparation of plasma microparticles: On average, 30 ml of pooled plasma is required to prepare 2 mg MP protein for each 2D gel electrophoresis. First, pooled plasma will be spun at 3200g for 30 minutes to remove cell debris and insoluble materials. The supernatants will be collected and centrifuged at 250,000g for 1 hour to pellet MPs. The MP pellets will then be washed twice, once with 2X PBS and once with PBS. Finally, the MP pellets will be dissolved and sonicated in 2D gel rehydration buffer.

 

2) 2D gel electrophoresis: 2 mg of MP proteins will be dissolved in 500 µL rehydration buffer. The samples will be spun at 13,200g at 4°C for 10 minutes to remove insoluble particles. 450 µL of the supernatant will be subjected to Isoelectric Focusing Electrophoresis, pH 3¯10 nonlinear, at 20°C on an Amersham IPGphor using pre-made 24 cm IPG strips. Second dimension electrophoresis will be run on a 20x26 cm 12% SDS-PAGE on an Amersham Dalt II system.

 

Haifeng Mark Wu, MD

Director, Clinical Coagulation Laboratory

OhioStateUniversity

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