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Human neurosphere under diff -CIII beta tubulin.  Image courtesy of Servio Ramirez, PhD, Department of Pathology and Laboratory Medicine, TUSM

RESOURCES FOR INVESTIGATORS

RESEARCH FACILITIES: PROTEOMICS RESEARCH FACILITY

Analysis Process

 

Some details of proteomic analysis are presented below to provide a better understanding of what happens to a sample once it is submitted.  Please be assured that we take extreme care of your samples to ensure the highest quality data possible.

 

Sample Preparation

Depending on the type of sample and the services recommended, sample preparation can take as short as a day or up to a maximum of two weeks.  Preparation processes may include ultracentrifugation, protein precipitation, buffer exchange, sample concentration, and total protein concentration estimation.

 

2D Gel Electrophoresis

Gel electrophoresis involves isoelectric focusing on IPG strips for first dimension separation followed by size separation by SDS-PAGE for the second dimension.  For first dimension separation, we run the sample in a group of two to three gels to ensure optimal separation for each experiment.  For the second dimension, the gels are run one at a time.  Please note that because this is a slow and exact process, there may be a queue to run the sample.  The average length of time is one to two weeks before stained 2DE gels can be obtained for a sample.

 

Gel Imaging

In order to assure the highest quality quantitative data, we optimize the image acquisition parameters for each sample.  It will take an average of two to three hours to image each gel.  Therefore, the time it takes to image the gels for your experiment depends on the number of gels and the queue for the imager.

 

Gel Image Analysis

Gel image analysis is time intensive.  Even though our sophisticated Z3 software aids analysis, manual intervention is required to assure accuracy and maximize information.  To provide a smooth transition to mass spectrometry analysis of excised protein spots from gels, we typically spend approximately one day summarizing and organizing the gel image data for individual spots to collect data on molecular weight, pl and staining intensity, as well as tracking information that is important for subsequent comparative studies.

 

Picking Protein Spots for Further Analysis

Because analysis of protein spots is time consuming and expensive, consultation will occur prior to choosing gel spots for analysis.  Gel picking entails staining the gel, imaging the gel, matching the gel to the experimental gels using Z3 analysis software, selecting the spots to be picked, picking and transferring the spots to a 96-well microtiter plate, and reimaging the gel after picking to ensure accurate picking.  Staining and imaging the gel takes one day, matching gels and creating a pick takes one day, and picking and reimaging the gel takes another day.  If the workload permits, this process will generally be completed within a week.

 

Trypsin Digestion

If gels are picked with the Xcise™ automated robotic system for 2DE gel, we can continue processing the gel plugs using the workstation, thus increasing throughput for the laboratory.  Depending on the number of spots to be digested, trypsin digestion will take one to two days.

 

Mass Spectrometry

Most gel samples are analyzed initially by MALDI TOF.  MALDI TOF is especially tolerant of the common contaminants extracted with peptides from an in-gel digestion.  If the protein is not confidently identified by peptide mass fingerprinting, the next approach is to acquire MS/MS data for selected peptides.  We will typically contact the investigator before proceeding with MS/MS to determine whether de novo sequencing may be necessary.  MALDI TOF analysis and database searching takes approximately two to three days for 20 protein spots.  This includes acquiring and processing the data, uploading a new database into MASCOT if necessary, scrutinizing the results, and preparing a report.  Similar to the other services, this process is done while multi-tasking and can take one week.  MALDI TOF/TOF acquisition and analysis can take an additional week.