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Preparing samples for mass spectrometry analysis is not as simple as merely isolating total protein from its biological source. They also need to be in a chemical environment that is MS friendly and allows their interrogation. Often enzymatic digestion and/or depletion/partitioning/enrichment are required to obtain a usable sample; but what are the best sample preparation methods, and how might they impact experiments? By Gautam Thor Inclusion of companies in this article does not indicate endorsement by either AAAS or Science, nor is it meant to imply that their products or services are superior to those of other companies. Until the early 1990s, mass spectrometry (MS) researchers were generally restricted to the study of small, thermostable molecules that could be easily ionized. Two breakthroughs occurred in quick succession in the late 1980s when John Fenn (who received part of the 2002 Nobel Prize in Chemistry) developed electrospray ionization (ESI) and Koichi Tanaka introduced matrix associated laser desorption ionization (MALDI) for large molecules, based on the work of Franz Hillenkamp and Michael Karas. These methodologies enabled MS analysis of a much broader range of biological molecules, without any need for chemical derivatization. Finding the Needle in a HaystackNow, the way in which samples are prepared for MS experiments is frequently determined by, and can have significant impact on, the subsequent steps in the procedure. Sample preparation procedures prior to MS need to serve dual functions in proteomic studies. First, they must provide adequate isolation of the protein(s) of interest, and second, the buffers used should provide a suitable environment for subsequent MS analysis. One of the biggest challenges, particularly in clinical, diagnostic, and prognostic applications such as those associated with biomarker discovery, is dealing with both the sheer complexity and the large dynamic range of the samples used. Jerald Feitelson, business development, intellectual property, and alliance manager at Beckman Coulter, points out that "although any single protein preparation technology is unlikely to be ideal for all samples, the more one can simplify the sample, reduce the dynamic range of protein concentrations, enrich for medium and low abundance proteins, and remove interfering peptide mass fingerprints the better the mass spectrometry results will be and the deeper one can dig into the proteome." Fluids like blood, saliva, and urine can contain many thousands of proteins and protein fragments that cover a dynamic range of 10 or more orders of magnitude, whereas most MS workflows are restricted to three or four logs of concentration, at best. Roughly 85 percent by mass of the human serum proteome is comprised of only six highly abundant proteins, including albumin, immunoglobulins of the G and A subtypes, transferrin, haptoglobin, and α1-antitrypsin. But this handful of components can mask the mass spectra of the low abundance (and often more interesting) proteins. In fact, early research in biomarker discovery using low resolution, but rapid, MS technologies such as surface enhanced laser desorption ionization (SELDI) was unable to establish any reproducible or clinically valid biomarkers (most of the signature differences turned out to be nonspecific components of the proteome). In order to overcome this hurdle, several approaches to sample preparation were developed that rely on depleting samples of highly abundant proteins. Depletion procedures often involve immunoaffinity-based methods using polyclonal or monoclonal antibodies—immobilized on a solid phase—to capture and retain the most abundant proteins. "The Multiple Affinity Removal System was designed to be highly selective for the removal of 14 highly abundant proteins from human serum or plasma," states Scott O'Brien, product line manager at Stratagene, an Agilent Technologies division. The kit makes use of a specifically formulated buffer "to minimize protein-protein interactions and ensure efficient capture and release of targeted proteins," says O'Brien. Immunoaffinity capture using avian antibodies may provide higher selectivity and lower cross-reactivity than other comparable products, since chicken antibodies display less cross-reactivity with mammalian proteins. GenWay Biotech (recently acquired by Sigma-Aldrich) provides Seppro affinity depletion technology built from a library of 700 chicken-derived IgY antibodies, while Beckman Coulter has been offering, for research use only, ProteomeLab IgY-12 spin and liquid chromatography column kits that are able to selectively partition 12 highly abundant proteins found in plasma or serum, enriching the sample up to 25-fold. Calbiochem (part of EMD Biosciences) makes use of a different methodology: the company's ProteoExtract kit includes an albumin/IgG removal component which uses a combination of the albumin-specific resin and an immobilized protein A polymeric resin. After removal of the high abundance proteins, the kit allows for the selection of partial or full proteome extraction, a precipitation step to concentrate the sample, and an enzymatic digestion if required. Novagen's (also a part of EMD Biosciences) ProteoEnrich CAT-X kit separates proteins under nondenaturing conditions based on their binding to the strong cation exchange resin. Almost the entire proteome should bind to the matrix under slightly acidic conditions, and partial proteomes can be eluted using a salt gradient. One profound drawback of depletion strategies results from the tendency for abundant proteins, particularly albumin, to bind other serum proteins and protein fragments. Thus it is possible that depletion of highly abundant proteins might also remove desirable ones. Bio-Rad addresses this problem with its ProteoMiner technology, which provides a large and highly diverse library of hexapeptides bound to a chromatographic support, allowing each unique hexapeptide to bind to a unique protein sequence. When the sample is added, the limiting binding capacity of the beads enables high abundance proteins to rapidly saturate their ligands, and excess protein is washed out. In contrast, the low abundance proteins are concentrated on their specific ligands. Following elution from the beads, the dynamic range of proteins in the sample is greatly reduced. Kate Smith, Bio-Rad's product manager for expression proteomics, explains that "since our depletion strategy does not entirely discard all the high abundant proteins, we get a quantitative analysis of the low abundance proteins and limit the potential codepletion of proteins associated with the highly abundant proteins." The procedure described above not only depletes abundant proteins but also provides a means to segment the sample, demonstrating an alternative strategy for protein sample preparation: enrichment. Separating the Wheat from the ChaffIn contrast to depletion strategies for sample preparations, enrichment processes allow for the preparation of specific subsets of proteins. These can be achieved either through affinity purification using posttranslational chemical modifications (for example, glycosylation or phosphorylation) or by the isolation of cellular subpopulations (such as organelles or plasma membrane fractions). "Most biofluid samples for clinical discovery experiments need to be reduced, alkylated and trypsin digested, and then subjected to a sample cleanup by reverse phase HPLC [high performance liquid chromatography]," says Mary F. Lopez, director, biomarker research initiatives in mass spectrometry at Thermo Fisher Scientific. In her opinion, the most successful approaches seem to be enrichment strategies for target pathways rather than depletion of interfering proteins. However, she also says that "the choice of targeted-enrichment strategy depends on how much is known about the protein or family of proteins of interest." Lopez indicates that proteomics-based identification of relevant biomarkers is a very active area of research. "The objective is biomarker candidates or signatures that can be used in a targeted, clinically relevant quantitative assay. However, the necessary sensitivity level in blood, urine, or even tissue extracts is often achievable only through an enrichment strategy." Enrichment strategies frequently use solid-phase extraction (SPE), a standard biochemical technique that uses the affinity of solutes within a liquid (mobile) phase for the solid (stationary) phase over which that sample is passed. Either the desired analytes of interest are collected in the flow through or, if they are retained on the stationary phase, they can then be removed with an appropriate eluent. Jeff Zonderman, director of clinical and toxicology LC/MC from Thermo Fisher Scientific, points out that "the most widely used pre-MS clinical sample preparation method is probably SPE due to its large selectivity." However, he acknowledges that researchers frequently use more than one selective technology to adequately reduce the complexity of a sample. One example is Thermo's Transcend system with TurboFlow technology, which puts a unique spin on pre-MS chromatographic separation. As Zonderman explains, introducing turbulence into the system—by running cartridges at predetermined high speeds—creates a more uniform velocity profile across the column, and "provides a means to obtain high volume, high demand separation." Varian's OMIX product line uses SPE in a pipette tip to provide a high throughput application for microextraction of proteins and peptides. The company claims that the small sorbent bed makes for a cleaner elution into a smaller volume. A similar product from Millipore, the ZipTip, is made to be compatible with a number of automated liquid handling systems and, according to the company literature, "provides a reproducible, high recovery method for concentrating and purifying femtomoles to picomoles of peptides, proteins, and oligonucleotides." "Selective enrichment is an area we are pursuing vigorously," states Andrew Emili, professor at the Donnelly Centre for Cellular and Biomolecular Research, at the University of Toronto, Canada. "For example, we are using both HPLC and aptamer phage display to physically isolate targets of interest from complex mixtures prior to MS detection." Emili claims that their approach, which is still in the research phase, is faster and cheaper for isolating proteins or peptides than raising antibodies for immunoenrichment. Teasing Apart the CellIf depletion strategies are too blunt an instrument and enrichment procedures too specific, the limitations introduced by the complexity of the MS samples could be addressed, at least in part, by the use of strategies targeting information-rich subsets of the proteome. This includes using standard subcellular fractionation technologies such as Poppers kits from Pierce (now a part of Thermo Fisher Scientific) and EMD Bioscience's ProteoExtract Subcellular Proteome Extraction kit, both of which are capable of enriching for nuclear, cytoplasmic, and mitochondrial proteins. The Qproteome kits from Qiagen, in contrast to straightforward differential centrifugation methods, promise to provide high purity of isolated subcellular fractions and soluble proteins, with concomitant depletion of high-abundant proteins such as IgG and albumin. "At the same time," says Ute Boronowsky, global product manager, "the procedure is very gentle, using only a benchtop centrifuge, leaving subcellular organelles such as mitochondria and plasma membranes intact, and retaining proteins in a native conformation with full biological activity." Although it is predicted that nearly 30 percent of all proteins are phosphorylated, phosphoproteins tend to be of low abundance and often with multiple phosphorylation sites of varying stoichiometries, which presents several challenges for MS analysis. Enrichment of phosphoproteins from complex mixtures can be performed by affinity chromatography using immobilized antibodies specific for phosphoserine, phosphothreonine, and phosphotyrosine residues, obtainable as commercial kits. Another technique more amenable to MS analysis is the use of immobilized metal affinity chromatography (IMAC) as it relies on the affinity of phosphate groups for certain metal ions (e.g., Fe3+ or Ga3+) bound to tethered chelating reagents presented on solid phase supports. Pierce's Phosphopeptide Isolation kit and Sigma-Aldrich's PhosphoProfile I Phosphopeptide Enrichment kit are both designed to simplify the isolation and identification of phosphopeptides from protein digests through the specific interaction of phosphate groups with immobilized gallium. Using a different process involving magnetic beads as the carrier and zirconium ions for capture, Calbiochem's ProteoExtract Phosphopeptide Capture kit enables isolation of phosphorylated peptides for downstream identification by MS. The company also produces the ProteoEnrich ATP-Binders kit which uses a novel resin with covalently bound ATP on a flexible linker to obtain samples enriched for protein kinases and other ATP-binding proteins. Other subproteomes often targeted are those of the intracellular and plasma membranes, of particular interest because they are the location of many proteins involved in cell signaling. However, these proteins have proved difficult to analyse, as their hydrophilic regions are preferentially detected by commonly used MS preparative methods. This necessitates high sequence coverage in order to accurately map sites of posttranslational modification and agonist/antagonist binding, elucidate stoichiometry, and identify sites of protein-protein interaction. Reagents that can increase sequence coverage of hydrophobic proteins are therefore in great demand. Novagen's ProteoExtract Native Membrane Protein Extraction kit is designed for the isolation of membrane proteins from mammalian cells and tissues. The extremely mild procedure yields a solution of integral membrane and membrane-associated proteins in their nondenatured state. Life Technology's (previously Invitrogen) Invitrosol MALDI Protein Solubilizer includes the ability to prepare both hydrophobic and hydrophilic proteins and peptides directly for MALDI time-of-flight MS analysis without the need for subsequent acid hydrolysis. Pushing the Limits of TechnologySample preparation methods are highly dependent on the proteomic query of interest. Generally, protein purification procedures require nonvolatile buffers, solubilizing agents, EDTA, and detergents to ensure that the proteins retain their native conformation. Some MS applications might require relatively harsh treatments to expose certain structures or hydrophobic components, while others, such as ESI-MS, might require more gentle buffers that retain macromolecular associations, but could require meticulous cleanup of interfering reagents prior to MS analysis. Although approaches relying on the depletion of potentially interfering, highly abundant proteins in clinical samples are often used, the approach runs the risk of throwing out the proverbial baby with the bathwater, since some of these high abundance proteins might have proteins of interest associated with them. These issues can be overcome by partitioning procedures that not only separate out the unwanted proteins, but also allow for the analysis of the bound sets of fragments, too. The combination of proteome partitioning and fractionation allows for the enrichment of low abundant proteins, as well as abrogation of the masking effect created by peptides derived from highly abundant proteins. Complementary biochemical manipulations for sample preparative methods include enrichment strategies that target subsets of the proteome such as cellular organelles or functional groups—the choice being determined by the specific target in mind. Although preparative technologies for MS have improved dramatically in the past decade, ironically the extraordinary ability and power of MS instruments is still being held back by the seemingly mundane biochemical limitations imposed by the procedures for sample preparation.
Gautam Thor has a Ph.D. in Neurobiology and is currently a freelance writer based in San Diego, California DOI: 10.1126/science.opms.p0900032
SFC ColumnsA new line of supercritical fluid chromatography (SFC) columns brings new phases and selectivities to SFC separations, expanding the use of this technology to analytical and preparative applications. SFC offers several advantages over high-performance liquid chromatography, including improved resolution, faster separations, and higher throughput. SFC also reduces the use of toxic solvents. SFC columns have applications in pharmaceutical and academic research as well as medicinal chemistry. Phenomenex’s new SFC offerings include the Luna HILIC (hydrophilic interaction liquid chromatography) columns with unique, cross-linked diol chemistry for analysis of polar metabolites; Synergi Polar-RP (ether-linked phenyl phase) for polar and aromatic analysis; and Lux chiral phases.Phenomenex Liquid Chromatography SystemThe AKTAready liquid chromatography system is designed for process scale-up and production for drug development and full-scale production following good laboratory practice and good manu-facturing practice standards. The system can improve cost efficiency and productivity by saving time and expenditures for startup, labor, and consumables. AKTAready operates with ready-to-use, disposable flow paths, eliminating the risk of cross-contamination and the need for cleaning and validation of cleaning procedures. The system includes the chromatography unit, Unicorn software, and a disposable ReadyToProcess Flow Kit including sensors and detection flow cells. The software includes an installation wizard with instructions and reports for column installation. The system is supported by extensive regulatory product documentation and services, including validation documentation, product documentation, and more.GE Healthcare Stain-Free ImagingThe Criterion Stain Free imaging system for detection and quantitation of proteins consists of a new formulation of Bio-Rad’s Criterion precast gels, a Criterion Stain Free imager, and Image-Lab software. The new system enables scientists to bypass the staining and destaining steps of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualize protein samples in 2.5 minutes after electrophoresis. In addition, the system captures digital images of gels to make record-keeping easy, with no need for a gel drying step. Benefits of the system include one-touch instrument operation, equal or improved sensitivity compared with a Coomassie stain, compatibility with protein immunoblotting and mass spectrometry, and digital images and data that are easy to share, print, and store. There is no organic waste.Bio-Rad Laboratories Antibody Purification ToolsThe PureProteome Protein A and Protein G Magnetic Beads enable researchers to perform small-scale purifications faster and more easily. The beads provide high binding capacity for high recovery of protein. The beads also ensure reproducible separation of immunoglobulins (IgG) by allowing for total removal of buffers and complete recovery of beads. Magnetic beads were developed to ensure rapid and reproducible purifications by immobilizing the beads to the side of the tube allowing for total removal of buffers with no loss of sample. In the past, magnetic beads have had significantly lower binding capacities than other bead-based purification media, but PureProteome beads overcome this by binding higher amounts of IgG. PureProteome beads are available with an optional magnetic rack that immobilizes the beads against the side of the tube in seconds, allowing for reproducible purifications to be performed quickly and easily.Millipore Fluorescent Detection KitThe DetectX Glutathione Fluorescent Detection Kit is designed to quantitatively measure free and oxidized glutathione in a single sample. The kit makes use of a proprietary molecule, ThioStar, that covalently binds to the free thiol group on glutathione to yield a highly fluorescent product. After mixing the sample with ThioStar for 15 minutes, the user reads the glutathione-generated signal at 510 nm in a fluorescent plate reader. Addition of the provided reaction mixture to the wells converts all the glutathione disulfide into free glutathione, which then reacts with the excess ThioStar during a second 15-minute incubation to detect total glutathione.Luminos
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Science. ISSN 0036-8075 (print), 1095-9203 (online)
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