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Sample Preparation Solutions for Cellular Protein Fractionation Poster, Rev A

T Slyker, B Gillece-Castro, M Nguyen, L Sapp, MG Brubacher, and WB Strong


Presented by the Authors at The Association of Biomolecular Resource Facilities (ABRF)

February 1013, 2003
Denver, Colorado

Abstract
2-D gel electrophoresis (2DE) is a widely used, proven method for proteome analysis. The quality and value of the information obtained from a 2DE experiment is highly dependent upon the initial sample preparation. In order to identify the most complete array of cellular proteins it is often necessary to reduce the complexity of the protein sample. A strategy for reduction in sample complexity is especially important when analysis of low-abundance and membrane proteins is the goal. Ideally the method(s) employed should be simple, reproducible, general to a wide variety of cell types, and result in a low conductivity protein sample that is free of substances that interfere with 2DE. With these aims in mind, we will present several solutions for convenient and efficient extraction of cellular proteins into discrete, more easily manageable fractions that are enriched in certain classes of proteins such as cytosolic, nuclear, membrane, and signaling. The majority of these procedures are intended to provide tools that simplify the preparation of membrane proteins that are generally considered difficult to isolate. These proteins are of considerable interest due to their roles in signal transduction and cell-to-cell interactions among other functions. 2DE and MALDI peptide mass fingerprinting data will be presented to illustrate the effective application of these techniques to improved sample prefractionation and protein identification.


Introduction
High-resolution 2DE is the most widely used separation method for global proteome analysis. Continuous improvements in the technique are expanding the capabilities of gel separations and removing limits on the types of proteins that can be resolved. Here, we describe new sample preparation kits that enhance the detection capabilities of 2-D gels.

These protocols address two key issues in applying 2-D gels to proteomics analysis of complex samples. One issue is how to handle both the enormous chemical diversity and the wide dynamic range of proteins in a biological sample so that lowabundance and housekeeping proteins can be monitored and identified. We have developed a number of protocols to quickly prepare highly enriched protein fractions based on protein solubility. One protocol separates a complex sample into enriched fractions of nuclear and cytoplasmic proteins. A second protocol enriches for membrane-associated signaling proteins. Other protocols enrich for membrane proteins. All protocols enhance the capability to monitor and identify proteins of biological interest.

The other issue is how to solubilize these hydrophobic proteins so they can be separated in 2-D gels. A number of new detergents and more stringent chaotropic agents have been incorporated into a complete protein solubilization buffer (PSB), which efficiently solubilizes hydrophobic proteins for immediate application to a 2-D gel. Using both the simplified enrichment and enhanced solubilization protocols, we significantly expanded the capabilities of applying 2-D gels for proteome analysis of complex cellular extracts.


Methods
Sample Preparation
Mouse liver tissue samples were processed with three different sample preparation kits to extract and enrich specific subclasses of proteins. Instructions for each kit were followed exactly. Protease inhibitors were added to the starting buffers immediately prior to use to prevent proteolysis during extraction. Following extraction, if required, protein samples were processed with the ReadyPrep 2-D cleanup kit to remove salts and detergents and to create a low-conductivity sample suitable for isoelectric focusing (IEF). This cleanup kit was also employed, if required, to concentrate proteins from dilute samples. Prior to IEF all samples were solubilized in PSB, to which 50 mM DTT, 2 mM TBP, and 0.2% Bio-Lyte ampholytes (310) were added, and applied to 17 cm, pH 310 NL, ReadyStrip IPG strips. Approximately 455 g of protein was loaded to each strip. E. coli samples were processed with the ReadyPrep membrane II kit protocol. Extracted membrane proteins were applied to 24 cm, pH 47 ReadyStrip IPG strips.


2-D Electrophoresis
The IPG strips were focused using a PROTEAN IEF cell for ~60,000 V-hr at a final focusing voltage of 10,000 V. The cell was set for rapid voltage ramping. Focused IPG strips were loaded onto 816% SDS-PAGE gels following equilibration for 10 min with DTT containing buffer followed by 10 min with iodoacetamide containing buffer. Following electrophoresis, gels were fixed for 30 min, stained with Bio-Safe Coomassie stain for 1 hr, and then destained for at least 2 hr before scanning with a GS-800 densitometer.


Protein Identification
2-D gel images were processed for analysis with PDQuest 2-D analysis soft ware and spots of interest were cut from the gels using the ProteomeWorks spot cutter. Peptide mass fingerprint data was obtained from excised 2-D gel spots using the MassPREP station robotics system for protein digestion and MALDI target spotting. Gel spots were destained, reduced, alkylated, dried, digested with modified trypsin (Promega) while heated at 37C for 5 hr, and extracted with an acidic solution. Peptides were then automatically spotted onto MALDI target plates mixed with a matrix solution of 2 mg/ml -cyano-4-hydroxycinnamic acid. MALDI mass spectra were acquired on a M@LDI-LR instrument in reflector mode. A nitrogen laser (= 337 nm, Laser Science) was pulsed at 20 Hz. Ions were accelerated to 15 kV after a timelag focusing pulse of 2,750 V at 500 ns. Data were collected from the sample well and a near point lock mass (ACTH clip 1839, Sigma). The MALDI mass spectra were processed to identify monoisotopic peaks using the Micromass algorithm MaxEntLite. The search engine used to identify peptide mass fingerprint data was ProteinLynx Global SERVER 2.0 software.

Results
Three separate sample preparation protocols were used to fractionate and enrich mouse liver protein samples prior to 2DE. Although the proteins for each gel were prepared from the same starting material, the final gel results following fractionation provide completely different views of the mouse liver proteome (Figure 1). Proteins that were unique to the fractionated sample were selected for identification. Table 1 shows selected proteins from these gels that are associated with the cellular structures enriched for in the sample separation protocols. Many of these identified proteins are membrane associated.

E. coli samples were treated using a carbonate extraction procedure to extract and enrich for membrane proteins (Figure 2). Table 2 shows that many of the proteins that were extracted using this procedure are membrane-associated E. coli proteins.


Conclusions
Applying specialized sample preparation solutions for the fractionation of cellular proteins from complex protein mixtures provides a simple and rapid method for enrichment of both lowabundance and membrane-associated proteins prior to sample analysis by 2-D electrophoresis. Understanding the cell biology of membrane proteins is especially important in the search for new drug targets, as ~50% of the known drug targets are membrane proteins. We have shown that application of four new kits for protein fractionation can provide a simple way to differentiate the proteome of a complex sample into a number of manageable fractions. Key features of these kits include:

Rapid separations Extraction from a crude sample to an enriched, gel-ready sample takes <3 hr

Reproducible results Each kit is based on proven chemistries and provides detailed instructions for application

Increased protein solubilization The PSB solution has been optimized to enhance solubilization of membrane-associated proteins for 2-D separation

Improved 2-D results The ReadyPrep 2-D cleanup kit provides quantitative recovery of samples with elimination of detergent and salt contaminants

Applying these sample prep kits to enrich proteins from complex biological samples should enhance the quality of the data that is obtained with any 2-D proteomics program.


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Bio-Safe, GS-800, PDQuest, PROTEAN, ProteomeWorks, ReadyPrep, and ReadyStrip are trademarks of Bio-Rad Laboratories, Inc. Global SERVER, M@LDI-LR, MassPREP, and ProteinLynx are trademarks of Waters Corp. Coomassie is a trademark of Imperial Chemical Industries, PLC. Triton is a trademark of Union Carbide Chemicals and Plastics Technology Corp.


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