Life Science Quarterly Summer 2002

Summer 02 Proteomics

 Life Science
Quarterly
 A TECHNICAL APPLICATION NEWSLETTER VOL 3   ISSUE 2   Summer 2002

 Proteomics

Proteomics: New Products for Protein Analysis

By Staci Rohn
Sigma-Aldrich Corporation, St. Louis, MO, USA

The Human Genome Project, one of the most widely heralded projects in science, has provided a great deal of data about the location of genes and their sequence. However, the true biological function of these genes lies in the expression of their encoded proteins. The full range of proteins found in an organism is known as the “proteome” and the study of the proteome is known as “proteomics”. Whereas DNA is composed of four nucleotides of similar structure, proteins are built from 20 different amino acids that differ greatly in size, charge, and structure. To complicate analysis, proteins are often modified following translation. These post-translational modifications include phosphorylation and glycosylation, though a number of other modifications exist as well. Thus, the complex nature of proteins makes proteomics a task of enormous proportions. Analysis of proteins starts with sample preparation, followed by protein separation, then characterization, and finally image analysis. Scientists who want to study proteins from start to finish must use multiple suppliers to obtain the necessary materials.

One of the companies quickly becoming a leading supplier in this field is Sigma-Aldrich. Sigma-Aldrich has taken enormous initiative and is making huge strides in the proteomics market thanks to years of experience in protein preparation and analysis, strategic alliances, and gritty determination. Working with companies like Proteome Systems™ (the company that coined the term “proteomics”) in Australia and Shimadzu Biotech™ (formerly Kratos Analytical™) in the UK has allowed Sigma-Aldrich to offer products that simplify proteomics. Calling themselves “The Proteomics Alliance™,” Sigma-Aldrich, Proteome Systems, and Shimadzu Biotech combined their talents in late 2000. The opportunity was enormous – a virtually unrivaled combination of reputation, resources, and expertise that could offer everything needed to run a comprehensive high-throughput proteomics facility. Reformulating and repackaging existing chemical products was the most obvious approach for Sigma-Aldrich. Working with the Alliance, they quickly released two lines of kits: one for sample preparation and one for the calibration of MALDI mass spectrometers. A multitude of other products has followed, each created to solve known problems in proteomics research.

Sample Preparation

The first step of proteomics research involves the isolation of proteins from a biological sample. Many proteins of interest are low abundance or membrane-associated, and therefore difficult to solubilize. Detergents, protease inhibitors, and reducing and alkylating reagents facilitate the protein solubilization and stabilization. However, standard detergents leave behind approximately 10% of the proteins, which poses an obvious problem for researchers studying low abundance proteins.

Higher protein loads, improved solubility, and increased visibility of low abundance proteins are the key benefits of Sigma’s ProteoPrep™ sample preparation products. In addition, the ProteoPrep kit format provides pre-measured and pre-weighed products in individual vials, which allows researchers to obtain all the needed reagents at one time, with one product. It also significantly reduces the waste and time typically associated with reagent preparation. These kits are for specific or targeted protein extraction, and include new, innovative detergent formulations that significantly refine results on 2-D electrophoresis gels.

There are three ProteoPrep kits from which to choose, depending on the needs of the researcher. The Universal Extraction Kit (Product Code PROT-TWO) sequentially separates soluble cytoplasmic and membrane fractions. The Membrane Extraction Kit (Product Code PROT-MEM) allows total extraction of membrane-associated proteins, while the Sample Extraction Kit (Product Code PROT-TOT) provides multiple reagents for testing or optimizing extraction conditions for total protein extracts. The data below demonstrate the superior results achieved with the ProteoPrep Universal Extraction Kit in an E. coli extraction (Figure 1).

Isolation of specific organelles often aids in the study of targeted proteins and cellular processes. Intact mitochondria are often the target of protein sample preparation, particularly for researchers studying proteomics, or apoptosis and its relationship to diseases. An intact mitochondrial fraction can be obtained with the Mitochondria Isolation Kit (Product Code MITO-ISO1). This kit allows small-scale isolation of an enriched, functional mitochondrial fraction from both “hard” or “soft” animal tissue samples. Easy-to-follow protocol diagrams are included with an additional stain to determine the integrity of the inner mitochondrial membrane. The integrity of the outer mitochondrial membrane can be determined using the Cytochrome c Oxidase Kit (Product Code CYTOC-OX1). The Chloroplast Isolation Kit (Product Code CP-ISO) provides plant researchers a convenient procedure for the isolation of intact chloroplasts from plant leaves.

Protein Separation

The analytical separation of proteins is one of the most challenging steps of proteomics research. The key is finding the most efficient way to achieve accurate results. Sigma-Aldrich recognizes 2-D electrophoresis as the most reliable technique currently available to obtain rapid results, in large scale, and in parallel.

Reproducibility, accuracy, and comprehensiveness are the hallmarks of the Sigma-Aldrich electrophoresis system. The ProteoSilver™ Silver Stain Kit (Product Code PROT-SIL1) contains prepared solutions of silver staining reagents along with detailed instructions to achieve optimal results. The kit has been proven to have an extremely low background, as shown in (Figure 2) . ProteoSilver’s detection limit of 0.1 ng/mm2 of protein (BSA) allows for superior detection of low abundance proteins and increased stability of the bands or spots. The MALDI-MS compatible ProteoSilver Plus™ Kit (Product Code PROT-SIL2) contains the same reagents found in ProteoSilver, with two additional reagents that help simplify the destaining required prior to MS characterization.

Protein Characterization

Multiple tools for protein characterization are available, but mass spectrometry is the most common method used by researchers today. Mass spectrometers are incredibly sensitive instruments that use molecular ionization to generate the mass-to-charge ratio of proteins and peptide fragments from protein digests, which leads to molecular weight determination and protein identification. Researchers may use the results to deduce theoretical information about the amino acid sequence of a protein. Over the past decade MALDI-TOF mass spectrometry has become easier to use, less expensive and more accurate. However, using poorly packaged, impure calibration reagents can reduce ionization efficiency and spectra. Again, Sigma-Aldrich offers assistance.

ProteoMass™ kits save significant time when calibrating MALDI instruments. Sigma’s stringent quality control ensures an absence of impurities and contaminant interference by eliminating the possibility of Na+ and K+ leaching from packaging. Working with Shimadzu Biotech, the manufacturer of the Kratos Analytical AXIMA-CFR mass spectrometry system, Sigma-Aldrich has designed and tested the kits to ensure researchers using ProteoMass achieve accurate spectra and increased ionization efficiency. In addition, the kits offer multiple matrices, solvents, and standards. The standards can be mixed and matched to target a specific mass range. Each standard is packaged in multiple vials, which ensures fresh product is available, and eliminates the possibility of using old, opened products that may have degraded on the shelf. Three kits are available, each targeted to a specific mass range, and suitable for either external or internal calibration.

The spectra in (Figure 3) and (Figure 4) were obtained using the ProteoMass Peptide MALDI-MS Calibration Kit (Product Code MS-CAL2), for a mass range of standards between 757 Da and 3494 Da.

Image Analysis

This final step of proteomics involves analyzing and cataloging the identified protein structures. Bioinformatics has become increasingly important in this area. Proteome Systems rounds out the proteomics process with bioinformatic systems that efficiently generate data needed in protein analysis. In particular, the systems are geared to high-throughput proteomics in which the amount of data generated is far too extensive to be managed on an individual level.

Summary

While companies supplying the proteomics market have boundless opportunity to stake their claim, few have taken the initiative of Sigma-Aldrich. The optimized products now available provide proteomics researchers a comprehensive means to perform their research quickly, with products that have already been tested in leading high-throughput proteomics facilities worldwide. Using the full platform from the Proteomics Alliance, including equipment such as the Xcise™ or ElectrophoretIQ™ from Proteome Systems, and the Axima™ mass spectrometer from Shimadzu, means using systems and products that work together, backed by the expertise and support from pioneers in the field. The race to map the human proteome is underway, and Sigma-Aldrich has proven its commitment.


ORDERING INFORMATION
Product Code Product Description Units
MS-CAL1 ProteoMass Peptide and Protein MALDI-MS Calibratiion Kit; for the mass range of standards between 757 Da - 66,430 Da 1 kit
MS-CAL2 ProteoMass Peptide MALDI-MS Calibration Kit; for the mass range of standards between 757 Da - 3,494 Da 1 kit
MS-CAL3 ProteoMass Protein MALDI-MS Calibration Kit; for the mass range of standards between 5,730 Da - 66,430 Da 1 kit
PROT-TWO ProteoPrep Universal Extraction Kit 1 kit
PROT-MEM ProteoPrep Membrane Extraction Kit 1 kit
PROT-TOT ProteoPrep Sample Extraction Kit 1 kit
C 0356 Cellular and Organelle Solubilizing Reagent 25 ml
T 7567 Tributylphosphine Stock Solution 5 x 0.5 ml vials
A 3221 Alkylating Reagent, Iodoacetamide 5 x 56 mg vials
RELATED PRODUCTS
PP0100 ProteoProfile™ Trypsin In-Gel Digest Kit 1 kit
CP-ISO Chloroplast Isolation Kit; for the isolation of intact chloroplasts from plant leaves 1 kit
MITO-ISO1 Mitochondria Isolation Kit; for the isolation of an enriched, functionalmitochondrial fraction from animal tissue 1 kit
CYTOC-OX1 Cytochrome C Oxidase Assay Kit; for the determination of cytochrome c oxidase activity in soluble and membrane bound mitochondrial samples 1 kit
T 6567 Trypsin, Proteomics Grade 5 x 20 mg vials
G 1041 EZBlue™ Gel Staining Reagent 500 ml
I 7281 ProteoGel™ IPG Equilibration Buffer 1 bottle
C 4105 ColorBurst™ Electrophoresis Markers 1 vial (500 ml)
C 4236 Chemichrome™ Western Control 1 vial
SUPPORTING LITERATURE
Literature on products mentioned can be ordered online or by calling 1-800-325-3010.
ProteoPrep Sample Preparation and Organelle Isolation Kits - ESP
ProteoMass MALDI-MS Calibration Kits - DZH
Crystallography Kits - ETW
Trypsin In-Gel Digest Kits - EVI
Post-Translational Modification - EVH
Electrophoresis for Proteomics - EGR

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