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Readers’ Tips, How-To’s, Experiences: Jan 1, 2005

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Q: How do you fractionate plasma or serum prior to mass spec analysis to increase the number of identified proteins?

A. 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

A. 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
Ohio State University

A. The most successful approach in our hands has been one that combines complementary techniques both at the level of protein fractionation and peptide/protein identification by mass spectrometry. We commonly separate intact proteins by non-denaturing techniques followed by standard denaturing separation on one (1DE) or two (2DE) dimensional electrophoresis. 2DE is interfaced with MALDI-TOF peptide mass fingerprinting and LC-ESI/MS/MS, and 1D gels sequentially fractionated and analyzed by LC-ESI/MS/MS. Albumin and other abundant protein depletion by commercially available kits can be helpful to increase coverage of the proteome, and the most successful approaches will likely analyze the same sample both with and without depletion of abundant proteins.

Thomas M. Vondriska, PhD
Proteomics Laboratory
David Geffen School of Medicine at UCLA

A. Our approach is based on the use of specific chemical probes that can selectively tag and facilitate subsequent isolation of a target peptide of protein. Since most serum proteins are glycosylated, selective isolation of glycopeptides provides several advantages for serum protein analysis. First, the most abundant serum protein, albumin, does not contain N-linked glycosylation motifs and therefore is effectively transparent to the analysis. Second, many serum proteins are post-translationally altered by phosphorylation, glycosylation, acetylation, methionine oxidation, protease processing, and other mechanisms, resulting in multiple forms for each protein.

Hui Zhang, PhD
Institute for Systems Biology

Reference for method of using specific chemical probes to selectively tag and facilitate subsequent isolation of a target peptide of protein:
Submitted by Hui Zhang, PhD
Institute for Systems Biology

Reference:
14. Zhang H, Li XJ, Martin DB, Aebersold R: Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry. Nat Biotechnol 2003, 21(6):660-666.

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If an RNAi experiment results in insufficient gene knockdown, do you try the experiment again? If so, what approach do you use to improve the knockdown?

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