Proteomics Facility

2D Gel Electrophoresis and Image Analysis

Two-dimensional gel electrophoresis is used to separate complex protein samples on the basis of isoelectric point (pI) and molecular weight. The first-dimension separation is performed using the PROTEAN IEF cell and ReadyStrip precast immobilized pH gradient (IPG) strips, providing stable and consistent separations. Standard broad-range pH gradients are used for separation of total protein on a single gel over a full pH 3-10 range. Narrow-range gradients provide greater resolution. The Criterion cell system utilizes 11cm IPG strips for first-dimension separation along with 13.3 x 8.7cm Criterion precast SDS-PAGE gels for second-dimension separation.

A variety of protein detection reagents are available, including silver stain, Coomassie Brilliant Blue, and SYPRO Ruby. Both silver and Coomassie stains are compatible with Criterion precast gels and require a minimum of 10-100 ng of protein per spot for detection. SYPRO Ruby is sensitive to as little as 1-10ng of protein per spot. It is compatible with high throughput protocols and downstream analysis, including mass spectrometry and Edman sequencing. SYPRO Ruby also allows for detection of glycoproteins, lipoproteins, low molecular weight proteins, and metalloproteins.

Image analysis is performed using the Investigator ProPic Robotic Workstation from Genomic Solutions. Spot picking is carried out using the Investigator HT Analyzer from Genomic Solutions. The two systems are linked together providing high reproducibility and eliminating human error when selecting desired protein spots.

Enzymatic Digestion of Proteins

Proteins are typically processed for MS analysis by in-gel reduction/alkylation and trypsin digestion, but other enzymes can be used if necessary. Digestion of proteins can also be performed in solution. In-gel digestion can be carried out robotically in order to minimize sample contamination by keratin.

MALDI-MS and MS/MS Analysis

A peptide solution is applied to a DHB or cinnamic acid derivative matrix, ionized by laser desorption, and analysed by a Thermo Finnigan MALDI LTQ mass spectrometer. Both peptide mass fingerprinting (MS analysis) and peptide sequencing (MS/MS analysis) can be performed.

LC-ESI-MS and MS/MS Analysis

A peptide solution, usually in a volume of approximately 10 uL, is loaded onto a C18 trap column. Peptides and then eluted with an acetonitrile/water gradient onto a 75 um x 10 cm reverse-phase (C18) analytical column at approximately 500 nL/min. The eluant from the second column is directly analyzed by a Micromass Q-TOF Micro mass spectrometer following electrospray ionization. A survey scan is performed in MS mode. The instrument switches automatically into MS-MS mode when peptides are detected.

Protein Identification

Data acquired by MS analysis are processed using Protein Lynx Global Server 2.2.5. The resulting output list is then submitted for database searching using Mascot (http://www.matrixscience.com). Based on analysis of the database search results, assignments are made for proteins in the mixture. Protein assignments are made based on several factors. Using MS/MS data, a protein assignment can be made with a single peptide - assuming that the peptide contains six or more amino acids and there is no ambiguity in the mass spectrometry data.

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