j gen intern med. 2008 january 23(suppl 1): 78–84.
Analytical Tools in Proteomics Method Principle Advantages Disadvantages Electrophoresis Electrophoresis When an electric field is applied to a solution containing a protein that has a net positive or negative charge, the protein migrates at a rate that depends on its net charge, size, and shape. Gels must be stained before proteins can be visualized. Rarely useful by itself as proteins cannot be accurately identified without the use of another detection technique such as immunoblotting or mass spectroscopy. SDS-PAGE Proteins migrate through inert matrix gel of polyacrylamide. Pore size is adjustable to retard protein of interest. SDS is a negatively charged detergent that unfolds proteins and frees them from other molecules. Proteins migrate at different rates toward positive electrode. Separates all types of proteins, even those insoluble in water. One-dimensional separation method has limited resolution. Closely spaced bands or peaks tend to overlap. Can only resolve a small number of proteins. Two-dimensional gel electrophoresis Combines 2 separation procedures. First dimension: the solubilized, denatured proteins are separated by their isoelectric point (pH where net charge is 0) in a polyacrylamide gel. Second dimension: the narrow gel containing proteins separated by isoelectric focusing undergoes electrophoresis at a right angle in SDS-PAGE to separate by size. Good resolution of mixture. Comparison of multiple gels facilitated by image analysis software. Posttranslational modifications can be discerned. Resolution of protein approximately 1ng/mL. Presence of high abundance proteins (i.e., albumin, immunoglobulins) may obscure low abundance proteins. Low throughput. Final identification of protein requires spot removal from gel, digestion, and analysis of peptides by mass spectrometry. Unable to resolve low molecular weight proteins (<10,000Da). Not easily amenable to multivariate analysis. Two-dimensional fluorescence difference gel electrophoresis Labels complex mixtures with fluorescent dyes before conventional two-dimensional electrophoresis. Different cyanine dyes are used to label protein from different samples and will be excited and emit at different light wavelengths. Up to three different samples can be labeled and mixed together (test, control, reference). Analysis of differences between mixtures is simplified. Ratio of protein expression can be obtained in a single gel, and an internal standard can be used in each gel to reduce gel-to-gel variation. Very sensitive. Presence of high abundance proteins (i.e., albumin, immunoglobulins) may obscure low abundance proteins. Low throughput. Final identification of protein requires spot removal from gel, digestion, and analysis of peptides by mass spectrometry. Many spots cannot be identified because of lack of material. Unable to resolve low molecular weight proteins (<10,000Da). Protein array Protein arrays Multiplex protein arrays, cytokine arrays, tissue microarrays In most common form, antibodies to known proteins are tethered to a surface (beads, nitrocellulose, etc.) and then detected using principles of immunoassays. High sensitivity and throughput. Multiple analytes can be measured simultaneously. Identification of potential targets already known. Limited antibody availability and specificity. Required some prior knowledge of expressed proteins. May not detect isoforms of analyte. Cost per sample may be prohibitive. Mass spectroscopy (MS) MS Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) Determines the precise mass of protein or peptide fragment from protein. Protein/peptide samples are mixed with organic acid matrix, dried on metal slide, and blasted by laser ionizing the peptide, which is accelerated in an electric field toward a detector. The time it takes to reach the detector is determined by the charge and mass. Peptide sequence information can be obtained with tandem mass spectrometers (MS–MS). Highest resolution is for molecules <3,000Da in size. Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) Comparable to MALDI, the difference being that SELDI uses chromatographic chip arrays to selectively bind subsets of proteins from complex samples. The surfaces can be washed to remove nonspecifically bound proteins and substances that can interfere with the ionization process (salt, detergents, etc.). High throughput via automation. Requires minimal sample preparation. Can be combined with prefractionation of material to enhance the detection of lower abundant proteins. No direct identification of proteins. Less sensitive to high molecular weight protein (>20kDa). May have instrument-to-instrument variation. Stable isotope labeling Biological samples are labeled with different stable isotopes using modifying agents targeting a specific amino acid (e.g., ICAT). After separation and mass spectrometry, peptides from the 2 samples differing in mass units specific for the isotope used (e.g., 8-Da mass shift for ICAT) can then be used to provide relative quantification. Wider proteome coverage than other methods. Can obtain quantitative information on a large number of proteins; Usually yields IDs of relevant proteins. Technically demanding; very low throughput capability; samples need to be trypsinized before analysis; reliable quantitative measurements likely on most abundant proteins. Adapted from Hoehn and Suffredini.12
mass spectrom rev. 2006 25(3): 450–482.
Since its introduction 30 years ago (Comisarow & Marshall, 1974a, Comisarow & Marshall, 1974b), FTICR-MS has gradually emerged as a powerful technology for the analysis of biological samples, but has only recently begun to move away from its niche role. Approaches for both “top-down” and “bottom-up” proteomics have used the high resolution and mass measurement accuracy (MMA) afforded by FTICR-MS to determine peptide/protein identities from parent masses, along with a powerful array of methods to identify peptide/protein sequences and modifications based upon fragmentation patterns. Significant advances in FTICR-MS technology have been reported over the last decade (Belov, et al., 2000b, Marshall, et al., 1998, Marshall, 2000, Paša-Tolić, et al., 2002, Zubarev, et al., 1998). In addition, there are a number of excellent reviews available on FTICR-MS (Holliman, et al., 1994, Marshall, 1996, Marshall & Guan, 1996, Marshall, et al., 1998, McLafferty, 1994) and its use in proteomics (Bergquist, 2003, Bogdanov & Smith, 2005, Page, et al., 2004). Interested readers are directed to these reviews for additional information. FTICR-MS is attractive for proteomics because it simultaneously provides high sensitivity, high MMA, and a wide dynamic range (Belov, et al., 2000b, Bruce, et al., 1999, Marshall, et al., 1998). In some regards, the high sensitivity analyses afforded by FTICR-MS are surprising since 30 to 50 charges typically must be trapped in an FTICR cell to provide a S/N >3, in contrast to conventional ion trap and time of flight mass spectrometers that can obtain a measurable signal from only a single ion. However, when considered from the viewpoint of overall ion utilization efficiency, the advantage of FTICR-MS compared to ion trap MS is the ability to trap 103 to 104 larger ion populations, and thus provide measurements of a much larger fraction of the ions produced by an electrospray ionization (ESI) source at any given point in a chromatographic separation. The data quality at low signal levels is also better in that relative isotopic peak intensities based upon hundreds of ions do not suffer from the stochastic issues associated with smaller ion populations, while the high resolution of measurements greatly reduces background due to “chemical noise”. These qualities provide the basis for studying either small cell populations or biofluid volumes, and enable measurement of changes in relative protein abundances for low level species with improved fidelity relative to other technologies. The ability of FTICR-MS to measure masses with a high level of mass accuracy (e.g., ppb to ppm levels) could be considered the most important feature of FTICR-MS for proteomics analysis. This level of routinely achievable MMA allows peptides to be identified without the need for one at a time peptide selection and MS/MS analysis for identification. Obtaining broad proteome coverage in bottom-up analyses often involves dealing with extremely complex mixtures of peptides. Despite the high resolution and high mass measurement accuracy afforded by FTICR-MS, only a limited view of the proteome can be obtained unless the mixture has been separated by some method prior to mass spectral analysis. Without such separations or fractionation, one is both limited by the dynamic range achievable in a single spectrum and, more importantly, by the fact that even very high levels of resolution are insufficient for extremely complex peptide mixtures. Thus, even with the use of extended signal averaging, spectral congestion becomes a limiting issue since the more abundant species within a sample prevent the detection of many of the less abundant species.
mass spectrom rev. 2006 25(3): 450–482.
Figure 2 Plot of observed elution time versus predicted elution time for 2925 peptides from a S. oneidensis global tryptic digest.
mass spectrom rev. 2006 25(3): 450–482.
Figure 3 Typical mass spectrum and isotopic distributions for a global soluble yeast tryptic digest analyzed by LC-FTICR-MS. Reprinted in part with permission from Shen, et al., 2001a. Copyright 2001 American Chemical Society.
mass spectrom rev. 2006 25(3): 450–482.
Figure 4 Isotopic distribution of a peptide from a proteome analysis of an organism grown on normal (14N) media (left distribution) and a distribution for the same peptide grown on 15N isotopically labeled media (right).
mass spectrom rev. 2006 25(3): 450–482.
Figure 5 Two-dimensional plot of filtered data from a single LC-FTICR-MS analysis. The region within the blue box is shown in Figure 6.
mass spectrom rev. 2006 25(3): 450–482.
Figure 6 Zoomed region of the two-dimensional mass and time plot in Figure 5.
mass spectrom rev. 2006 25(3): 450–482.
Figure 8 2D plot of the UMCs from an LC-MS analysis used for NET alignment.
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Figure 11 Comparison of peptide absolute abundance values for two S. oneidensis samples from two biological conditions. Logarithmic scales have been used to better illustrate the data at lower peptide abundances.
mass spectrom rev. 2006 25(3): 450–482.
Figure 12 (A) A sulfhydryl-reactive solid phase isotope-coded reagent. Aminopropyl-beads were covalently linked with a photosensitive linker, a stable isotope-coded leucine residue, and a sulfhydryl reactive group. For the heavy isotope version, the leucine residue contains six 13C and one 15N as indicated by the bold letters in the structures. The photosensitive linker can be cleaved by UV (365 nm) illumination (the line indicates the cleavage site). (B) The isotope-coded label attached to a sulfhydryl group in cysteinylpeptides after photocleavage. The light and heavy isotope-coded labels have a mass difference of 7.017 Da.
mass spectrom rev. 2006 25(3): 450–482.
Figure 14 LC-FTICR-MS analysis of a tryptically digested SPICAT-labeled cell lysate from 10,000 mammary epithelial MCF7 cells: (A) Total ion chromatogram reconstructed from the DREAMS set of spectra. (B) Corresponding 2D plot. (C) An illustrative peptide pair.
mass spectrom rev. 2006 25(3): 450–482.
Figure 16 (A) A partial 2D plot showing 16O/18O labeled Cys-peptide pairs enriched by QCET. (B) Three examples of peptide pairs with their sequences, corresponding proteins, and the 16O/18O ratios.
