September 15, 2005 (Vol. 25, No. 16)

Technique of Choice for Protein Identification

The upcoming Mars mission in 2009 will carry mass spectrometry equipment, explained Firouz Naderi, Ph.D., of the NASA Jet Propulsion Laboratory, in a plenary address at the 53rd ASMS Mass Conference in San Antonio.

Using a combination of slides, live narration, and animated computer graphics, Dr. Naderi, the NASA MARS exploration program manager, held the audiences attention through the high-tension landing of Opportunity on the Martian surface. Noting that the lander came screaming down to the surface and bounced for a quarter of a mile after smashing into the planet, it is clear that miniaturization and durability will be highly desirable traits for the next generation of mass spect instruments.

Presentations at the conference also illustrated the reality that mass spectrometry is evolving into a user-friendly, affordable technology. More than half of the presentations dealt with proteomics, drug metabolism, and other bioanalytical applications.

Mass spec is now the technique of choice for the complex challenges of protein identification. In earlier years, the unraveling of protein amino acid sequences by Edman degradation was the preferred method; amino acids were sequenced by cleaving them one by one from the N-terminus of a protein.

Subsequently each amino acid was chromatographed using a high-performance liquid chromatography gradient. The resulting amino acids are identified based on their retention time, correlated to known standards. This powerful technique is exacting, but slow, and may require seven cycles of the sequencer to uniquely identify a protein in a sequence database.

On the contrary, mass spectrometry uses peptide masses to identify proteins, and with phenomenal speed can identify as many as 20 proteins in the space of a few minutes. The basis for the technology is the conversion of molecules into ions and their subsequent identification.

Various approaches are called into play. These include peptide mass fingerprinting by which a protein is digested with an enzyme and the peptide masses are then used to search a sequence database.

A second approach is the use of a sequence tag. In this instance a peptide is fragmented in a mass spectrometer and then a short stretch of amino acids is determined.

This tag, (peptide mass, sequence of the tag, starting and ending mass of the tag), is used to search a sequence database. Proteins can be correlated with the fragmentation of a single peptide using this technique.

Finally in MS/MS peptide identification, a peptide is fragmented in a mass spectrometer and the fragmented ion masses are then used to search a sequence database. Proteins can be correlated with the fragmentation of a single peptide using this technique. The identification of the protein becomes more reliable as the set of peptides identified increases.

A workhorse of mass spec technology is matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), in which a co-precipitate of a UV-light absorbing matrix and a biomolecule are irradiated by a laser pulse.

The ionized biomolecules are accelerated in an electric field, where they enter the flight tube and are separated according to their mass-to-charge ratio, causing them to reach the detector at different times. In this way each molecule yields a distinct signal. MALDI-TOF MS is sufficiently accurate to characterize proteins (after tryptic digestion) from completely sequenced genomes.

Needles in Haystacks

The power of mass spectrometry to analyze proteins has revolutionized proteomics with its speed, precision, and economy. However it has also posed new challenges resulting from the wealth of molecules that it reveals.

Given the overwhelming complexity of biological structures, there is great demand for techniques for separation or partitioning of cellular systems in order to limit the universe of host proteins to a manageable number, and to perform effective separation of complex mixtures, revealing the underlying information that the investigator seeks.

Moreover, according to studies on individual Drosophila heads using LC-ion mobility mass spectrometry by David Clemmer, Ph.D., and his colleagues at the University of Indiana, there is little variability in high-abundance proteins among individual specimens.

It appears that it is the lower abundance components of the proteome that play the most significant role in determining unique features of individuals. This need places a special challenge on the scientist to sort out these critical differences.

Zeroing in on Minor Proteins

One of the biggest challenges in the proteomics field is finding a way to isolate and purify low-abundance proteins for mass spectrometry analysis, stated Mike Malis, president of Alfa Wassermann Proteomic Technologies (www.

To overcome the difficulty of isolating low-abundance proteins, a nondenaturing continuous flow ultracentrifugation technology, referred to as Focus, is available through the company.

We are the leaders in this technology. Focus enables researchers to rapidly and efficiently separate, enrich, and accumulate organelles without damaging the subcellular information, said Elizabeth Arden, COO of Alfa Wassermann.

A typical tissue homogenate or cell lysate may contain thousands of spots representing different protein species when analyzed by 2-D electrophoresis. However, many of the proteins are present in such minute amounts that they will fall under the radar.

For this reason, Alfa Wassermann scientists have developed the Focus system, which takes advantage of the different buoyant densities of subcellular components using sucrose gradients to separate them.

The Focus technology facilitates the rapid fractionation of the organelles and organellar subtypes, permitting the enriched fractions to be analyzed by 2-D gel electrophoresis. Each 2-D gel will result in a manageable number of spots in the range of 200400, according to the company. These spots can be excised and readily subjected to mass spectrometry analysis.

Historically, sucrose gradients have been employed for separation of macromolecules and subcellular components. These analytical ultracentifuges employed swinging bucket rotors with large tubes holding the preparations. The problem with those systems, said Arden, is that the dimensions of the rotor contribute to a longer equilibration time of up to 48 hours.

With continuous flow centrifugation, a vertical tube rotor is employed with a radius of only a centimeter. This means that equilibration will occur in a few hours, and hundreds of milliliters can be processed. Since the system employs sucrose for building the gradients, the material is cheap and nontoxic.

High-purity preparations of subcellular organelles can be prepared in this fashion, digested into peptides with proteolytic enzymes, and immediately analyzed by mass spectrometry since the reagents are compatible with mass spectrometry instrumentation.

Differential Protein Expression

The molecular understanding of disease processes frequently hinges upon side-by-side comparison of normal and pathological states of cellular systems.

Invitrogen (www.invitrogen. com) has commercialized differential labeling of macromolecules in order to characterize unique proteins associated with disease conditions, including cancer, autoimmune dysfunction, and aging.

The companys product, SILAC (stable isotopic labeling by amino acids in cell culture), allows for the investigation of possible protein origins of cellular dysfunction. The kit was developed by R&D manager Marshall Pope, Ph.D., and his colleagues at Invitrogen.

The SILAC technologys history derives from the desire to reduce false positives in protein expression studies, Dr. Pope stated. The initial concept was originated by Xian Chin and his co-workers at the Los Alamos National Laboratory in New Mexico, he continued. The kit consists of a metabolic protein-labeling solution for identifying quantitative differences in protein abundance between two cell types.

The kits are designed to be compatible with mass spectrometry analysis so a variety of cellular differences can be unambiguously identified. The company cites one example in which cultured human breast cancer cells were compared with normal breast epithelial cells.

The cancer cells were grown in a medium containing heavylabeled forms of the amino acids lysine and arginine. The cells were mixed and lysed and then the membranes were isolated.

Without prefractionation more than 1,600 proteins were identified, of which 1,000 were membrane proteins and 250 were proteins of unknown function. When the proteins are subjected to analysis, the same protein will appear as a doublet of two peaks of equal height if there is no differential expression.

However, in cases in which the protein is differentially expressed, the doublet will consist of two peaks of unequal height. Following this procedure, 150 proteins were identified with increases in expression levels of twofold or more.

Another feature we built into the SILAC kit is a protein solubilizer solution, Pope said. With this proprietary formulation we avoid the use of Triton, CHAPS, and other solubilizers that are incompatible with mass spec.

Thus the SILAC kit is designed for use by biologists in a formulation that can be handed over to a mass spec core facility for processing. This resolves the problem of meshing biochemical and physical chemical technologies to build an optimal research protocol.

When we conceived the product we wanted it to fit a user-friendly format that required no detailed knowledge of the physics of mass spectrometry, but rather eliminated the barrier between the molecular biologist and the core facility, he continued.

Dr. Pope discussed another Invitrogen product that goes by the acronym ITRAQ. This technology achieves a nonmetabolic labeling of proteins allowing the identification and quantification of peptides by mass spectrometry without the necessity of growing cell in culture.

The platform uses amine-specific tagging reagents for chemical labeling for a side-by-side comparison of proteins in the same fashion as the SILAC kit. Free lysine residues in peptides can be tagged after a one-hour incubation.

Invitrogen partnered with Applied Biosystems (www. using their MS instrumentation and specific software to obtain an integrated solution to the challenges of quantitative molecular biology.

Peptide Modifications

Bruker Daltronics (www. introduced an improvement to its ion trap mass spectrometry (ITMS) system, according to Catherine Stacey, Ph.D, director of proteomics systems. The HCTultra PTM discovery system includes both convention CID (collision-induced dissociation) and ETD (electron transfer dissociation) fragmentation on a high-resolution ion trap instrument making this product unique.

The quadrupole ion trap mass spectrometer has been recognized for the last decade as a superior tool for biomolecular analysis, capable of identifying and describing a variety of molecules.

The fundamental difference of the ion trap is that all ions created over a given time period are trapped and then ejected into a conventional electron multiplier detector. This new method allows faster and more efficient ejection of the ion packet, thus greatly improving resolution.

Brukers Protein Post-Translational Modification (PTM) Discovery Tool represents a commercial application of electron transfer dissociation to the ion trap spectrophotometer. The module is a protein and peptide fragmentation technique that preserves glycosylation, phosphorylation, and other significant post-translational modifications. This provides an important step forward in deciphering the subtle modifications of proteins, which are known to have profound effects on phenotypic expression.

Peptide modifications are detected by various MS/MS approaches of which ETD has the advantage of preserving the modification on the amino acid during the fragmentation. The sequence, including the position of the modification, is easily confirmed by de novo sequencing or database searching, explained Dr. Stacey. Standard sequencing techniques will fail to identify many secondary modifications of proteins, so there is a strong demand for the Bruker technology.

Another platform, referred to as Clinprot and Clinprot micro, ties the companys mass spectrometry-based biomarker analysis technology to a collaborative in vitro diagnostics program with HealthLinx (Melbourne, Australia). This system is an integrated set of tools for biomarker discovery and clinical proteomics research, including software packages to advance clinical research and shorten timelines in biomarker discovery.

The Clinport workflow measures peptides and proteins that can be used to discover multimarker panels or profile patterns indicative of specific diseases. These multimarker panels or patterns have potentially better diagnostic specificity than single biomarkers.

The partnership is focused on discovery of potential biomarkers for malignancy in serum, plasma, urine, saliva, cerebral spinal fluid, or cell lysates. Clinprot supports a profiling workflow to detect patterns indicative of specific diseases in biological fluids. In a second workflow, individual biomarker candidates can be identified by means of the Bruker Daltonics TOF/TOF technology.

Sample preparation takes advantage of magnetic bead capture. These are microparticles with functional surfaces able to bind proteins and peptides. After elution the captured proteins and peptides are transferred to AnchorChips (a specialized sample support).

Profile spectra of the separated protein/peptide fractions are acquired using the FLEX-series MALDI-TOF instrument, taking advantage of bioinformatics for all major functionalities in biomarker detection and evaluation. This includes data on pretreatment, peak statistics, pattern recognition with direct feedback to the visualization, cross validation of the cluster analysis, and determination of sensitivity and specificity with independent test data.

Any future use of biomarker candidates in clinical environments requires identification of the respective peptides. The TOF/TOF functionality offered by Clinprot addresses this important task in clinical proteomics.

Biomarker candidates detected by ClinProTools can be subjected to TOF/TOF analysis. Metastable fragment ions of the respective precursor ion are analyzed after a second acceleration step, and a software package known as BioTools interprets the resulting fragment pattern and is used for peptide identification via database search.

The Clinprot micro solution is designed for cancer research and other clinical and diagnostics research laboratories engaged in biomarker discovery across a range of samples.

It represents a compact clinical system dedicated to peptide and protein profiling, biomarker discovery, and biomarker validation. It is derived from benchtop MALDI-TOF mass spectrometer from Bruker Daltonics FLEX series, providing biomarker detection sensitivity and resolution, as well as the reliability and reproducibility needed for validation studies of putative biomarkers for cancer and other diseases, according to the company.

The Clinprot micro system offers integrated sample-preparation tools for biological fluids and tissue extracts with comprehensive analysis, visualization, and model-building software.

Dr. Stacey considered the direction of technological developments in the companys product line. All mass spec vendors continue to innovate with technical enhancements to their existing products and introduce new products continuously.

The demand from customers tends to be for more performance, sensitivity, resolution, and better fragmentation, at a given price point, rather than for price or size reduction, she stated.

Diagnostic tests, via biomarker detection, using MALDI-TOF or ESI methods in combination with upfront sample preparation such as our ClinProt magnetic beads, is an emerging market for our company. We expect biomarker detection to grow in the clinical research world, although routine diagnostic testing is some where in the future.

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