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An unexpected DNA binding protein You never know what you’ll find when you perform a proteomics experiment. Using standard genomics and proteomics techniques, Michael Snyder and co-workers at Yale University identified a new DNA binding protein called Arg5,6, which is a yeast enzyme involved in the synthesis of arginine. This result is surprising because enzymes are not known to bind directly to DNA. Snyder and co-workers probed a yeast protein microarray with fluorescently labeled yeast genomic DNA to identify novel DNA binding proteins. The researchers detected >200 proteins with this assay, almost half of which were not previously known to bind to DNA. The rest of the positive proteins either bind directly to DNA, bind indirectly via a co-purifying protein on the microarray, or bind to DNA nonspecifically in vitro. The researchers tested eight of the proteins in ChIP (chromatin immunoprecipitation)/chip assays. Cells that expressed myctagged versions of the proteins were treated with formaldehyde to crosslink the proteins to DNA. The cells
Soft landings for proteins in liquids R. Graham Cooks and colleagues at Purdue University have demonstrated that protein ions can be deposited on liquid surfaces with their native structures intact via a technique called soft landing. The study builds on previous work in which the researchers used MS to mass-select protein ions and soft-land them in microarray formats on solid substrates. In the present work, the liquid surfaces were created by dissolving one of several MALDI matrixes in a glycerol solution, which was applied to a gold substrate. Protein solutions were ionized with an ESI source, and either a modified single-stage quadrupole or a home-built linear ion trap mass spectrometer was used to mass-select and deposit the ions. The material was then analyzed—without any additional treatment—by ESI, electroson© 2005 American Chemical Society
Proteomics and genomics. A schematic of the protein chip and ChIP/chip assays. (Adapted with permission. Copyright 2004 American Association for the Advancement of Science.)
ic spray ionization, or MALDI MS. If a liquid surface is used instead of a solid one, it does a better job of preserving the activity of the proteins, and the proteins can be purified from the liquid, the researchers say. In addition, one can either perform direct MS analysis of the material or add proteolytic enzymes to the liquid surfaces and analyze the fragments using standard MS methods. ( J. Am. Soc. Mass. Spectrom. 2004, 15, 1874–1884)
Multiplexed studies of cellular signaling To help tease out complex cellular signaling pathways, Paul J. Utz and colleagues at Stanford University School of Medicine have applied reversed-phase protein (RPP) microarrays to the study of site-specific phosphorylation of proteins. This multiplexed approach uses just nanoliter volumes of whole-cell lysate per microarray
were then lysed, and the proteins were immunoprecipitated with antibodies against the myc tag. DNA that was recovered from this step was fluorescently labeled and used to probe a yeast genomic microarray. Three proteins bound to DNA in the ChIP/chip assay, and one of these proteins, Arg5,6, was studied further. Snyder and co-workers performed PCR on the DNA fragments that co-purified with Arg5,6. DNA from seven different mitochondrial and nuclear genes bound to the protein. In a gel-shift assay, purified Arg5,6 reduced the mobility of one of these genes in a native polyacrylamide gel; this indicates that the protein bound to the gene. The researchers used real-time PCR to measure transcript levels in a yeast strain in which the gene for Arg5,6 was deleted, and they observed that certain transcript levels were reduced. These data suggest that Arg5,6 regulates gene expression in addition to its function as an enzyme. (Science 2004, 306, 482–484)
spot; hence, it is suitable for analyzing rare cells. Utz and colleagues used RPP microarrays to profile 62 signaling components in Jurkat T lymphocytes. They identified a previously unknown correlation between cross-linking of the CD3 receptor and dephosphorylation of the kinase Raf-1 at the Ser259 site. To demonstrate the method’s potential for working with rare cells, the researchers looked at phosphorylation of proteins known as STAT factors, which are thought to be involved in the interleukin-2 receptor signaling cascade, in CD4+CD25+ regulatory T cells. Such cells exhibit regulatory activity both in vitro and in vivo. The researchers suggest that the RPP approach can be used to study other types of modifications in cellular signaling, including glycosylation. (Nat. Med. 2004, 10, 1390–1396)
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TOOLbox Differential proteomics without isotope labeling Jacques Colinge and colleagues at GeneProt (Switzerland) have developed a new semiquantitative method, called peptide match score summation (PMSS), to compare protein samples without isotope labeling. PMSS sums peptide identification scores and gives results similar to those from spectrum sampling (SpS), which is a method that takes into account the number of spectra that were used to identify each protein. To validate that certain proteins are present at different levels, the researchers combined PMSS and SpS with statistical tests, such as the local pooled error test (LPET) and the t-test. LPET allows researchers to perform fewer experimental repetitions than the t-test to arrive at the same confidence level. The researchers analyzed mixtures of standard proteins, plasma samples, and spiked plasma samples with the new method. Threefold changes in plasma proteins were detected with ~90% confidence. (Anal. Chem. 2005, 77, 596–606)
Deconvolution of ESI mass spectra ESI produces multiply charged ions with more than one charge-carrying species. To assign the proper charge states to these ions, Simin Maleknia and Kevin Downard at Griffith University, Sciformatics, and the University of Sydney (all in Australia) have developed the charge ratio analysis method (CRAM). Unlike other ESI deconvolution programs, CRAM does not assume that the charge-carrying species is uniform for all ions. Instead, CRAM calculates the charge states of the ions to determine the masses of the chargecarrying species. These data are then used to calculate the molecular weight of the analyzed molecule. The researchers applied CRAM to the study of lysozyme, ubiquitin, and calmodulin. The new method can be used with low-, medium-, or high-resolution mass spectrometers. (Anal. Chem. 2005, 77, 111–119)
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Sensing sugars Zeev Rosenzweig and colleagues at the University of New Orleans have developed fluorescent sensors for detecting carbohydrates and glycoproteins. The sensors could potentially be used in arrays for high-throughput screening. Rosenzweig and colleagues attached ConA, a carbohydrate binding protein, to 1.6-µm polystyrene particles. The ConA molecules were tagged with the fluorescent marker FITC. The FITC–ConA particles maintained the carbohydrate binding ability for up to 5 days in solution. The investigators incubated Texas Redlabeled dextran molecules with the ConAmodified particles. The interactions between the dextran and the ConA, which took 15 min to reach equilibrium, could be observed by fluorescence resonance energy transfer (FRET). The FITC fluorescence decreased by 50%, while the Texas Red fluorescence increased by the same amount. When unlabeled carbohydrates or glycoproteins were introduced, they competed with the dextran molecules to bind to the
ConA molecules on the particles. The FRET efficiency between the FITC on ConA and the Texas Red on dextran decreased with increasing concentrations of carbohydrates or glycoproteins. The particles were able to distinguish between monomeric carbohydrates like mannose and galactose. But millimolar levels of the monomeric carbohydrates were needed for effective inhibition of FRET between FITC and Texas Red. With glycoproteins, the investigators found that the FRET inhibition was dependent on the number of sugar moieties present on the proteins. Glucose oxidase was more efficient at inhibiting FRET than avidin because it carried more sugar groups. The different binding capabilities of carbohydrates and glycoproteins to the sensors could be useful for discriminating between the two types of molecules. Rosenzweig and colleagues suggest that the capabilities of the FRET-based sensors can be expanded by substituting ConA with other types of carbohydrate binding proteins. (Anal. Chem. 2005, 77, 393–399)
Miniaturized lectin affinity chromatography Bingcheng Lin and colleagues at the Dalian Institute of Chemical Physics, the Shanghai Academy of Life Sciences, and the Graduate School of the Chinese Academy of Sciences (all in China) have miniaturized lectin affinity chromatography for the separation of glycoproteins on a microfluidic chip. The integrated system reduces the analysis time from 4 h to 400 s compared with conventional lectin affinity chromatography, and only 300 pg of glycoprotein is required. The researchers prepared a lectin affinity monolith column in the microchannel of a microfluidic chip using a two-step procedure. First, they synthesized a porous monolith matrix by UVinitiated polymerization. Then they immobilized pisum sativum agglutinin (PSA) on the monolith matrix. To demonstrate the miniaturized system, Lin and colleagues performed Microchip layout. (1) Running buffer reserseparations on three different glycoprovoir, (2) eluent buffer reservoir, (3) sample teins—turkey ovalbumin, chicken ovalreservoir, (4) sample waste reservoir, (5, 6) bumin, and ovomucoid. All of the glycowashing reservoirs, and (7) waste reservoir. proteins were readily separated into various fractions with different affinities toward the immobilized PSA. The results could be used to deduce information on the structure of glycans. (Anal. Chem. 2004, 76, 6941–6947)
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currents Chip-based glycopeptide analysis
Inkjet printing damages proteins
Alina Zamfir and colleagues at the University of Münster (Germany) and Advion BioSciences (U.K.) have developed an off-line CE/chip-based negative-ion nano-ESI/quadrupole TOFMS platform for characterizing glycopeptides in complex biological mixtures. The introduction of fully automated chip-based nano-ESI-MS greatly increases the throughput and sensitivity of off-line CE/MS. Although such an approach has been successfully implemented for peptides and proteins, this is the first time it has been applied to carbohydrate analysis. The researchers used the approach to analyze a complex mixture of O-glycosylated peptides and amino acids from the urine of patients with Schindler’s disease. Such patients have an inherited deficiency of the lysosomal enzyme -N-acetylhexosaminidase, which causes a 100 higher concentration of O-glycans in urine compared with healthy controls. The characterization of O-glycosylated amino acids and peptides in urine thus serves as an important diagnostic of the disease. ( J. Mass Spectrom. 2004, 39, 1190–1201)
A caveat now exists for using inkjet printers to produce protein microarrays. Gary Nishioka and colleagues at H&N Instruments have discovered that a model enzyme can be damaged by rapid compression during the printing process. Inkjet printing has several advantages in producing microarrays—it is quick, contactless, and inexpensive. But some concern has emerged about shear damage to the molecules and nonspecific adsorption of the molecules to the tubes in the printer. Nishioka and colleagues have now found a third source of problems. The investigators studied the enzyme peroxidase by fluorescence as they printed the enzyme into 96-microwell plates. The enzyme’s activity significantly decreased, even when the solution was compressed slowly. However, the addition of a solution of trehalose and glucose to the peroxide solution helped protect the enzyme against damage by compression. ( J. Am. Chem. Soc. 2004, 126, 16,320– 16,321)
Microwaving proteins
C-terminal ladder
N-terminal ladder
Liang Li and colleagues at the University of Alberta (Canada) have developed a microwave-based method for sequencing proteins and identifying posttranslational modifications (PTMs). Unlike traditional methods of generating polypeptide ladders, the new technique specifically produces N- and C-terminal peptides and appears to cleave many different peptides and proteins. The researchers digest proteins and peptides by controlled acid hydrolysis. HCl is added to the sample protein or peptide, which is then microwaved for 1 min. Acid hydrolysis alone can generate peptides that are solely N- or C-terminal, but exposure of the reaction to microwave irradiation speeds up the process. Longer periods of irradiation, however, produce many internal peptides, and this can make sequencing difficult. The peptides are analyzed by MALDI-TOFMS. γ α β N C Peaks differ by one amino acid A 1 A 2 A 3 A 4 • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • A n- 1 A n or by one amino acid and a HCI and microwave irradiation PTM, so protein identification and determination of PTMs are easy and fast. Impurities of 20. They observed little or no bias against standard, low-abundance proteins that were spiked into the sample. (Rapid Commun. Mass Spectrom. 2004, 19, 9–14)
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To reduce the number of time-consuming manual validations needed for processing MS/MS data, Youhe Gao, Wei Sun, and colleagues at the Chinese Academy of Medical Sciences/Peking Union Medical College have developed a computer program that automatically filters MS/MS spectra searched by Sequest. Called the Advanced Mass Spectrum Screener (AMASS), the new program discriminates between positive and negative matches by using the same spectral properties as manual validations. It measures the match percentage of high-abundant fragment ions and the continuity of b or y ion series. AMASS increases the number of correct peptide identifications by effectively removing noisy spectra, false interpretations, and poor fragmentation spectra. The program can be used along with the Rscore filter for even better results. (Mol. Cell. Proteomics 2004, 3, 1194–1199)
Alexander Schmidt and colleagues at the Max Planck Institute of Biochemistry (Germany) have developed a new isotope labeling technique that is capable of quantitative proteome profiling on a global scale. The new method, called isotope-coded protein label (ICPL), tags all free amino groups in isolated intact proteins and therefore is applicable to any protein sample. In ICPL, proteins derived from two different cells or tissues are extracted, alkylated, and differentially labeled at the free amino groups with either isotopeencoded (heavy) or isotope-free (light) ICPL tags. After the protein mixtures are combined, any separation method
Cell state A
Cell lysis, alkylation, and ICPL labeling
All amino groups labeled with light ICPL tags
Cell state B
All amino groups labeled with heavy ICPL tags
Combine
Fractionation on the protein level
Digest Fractionation on the peptide level
Mass spectrometry Relative abundance
Automatic validation of MS/MS spectra
New isotope labeling technique
Quantification (MS)
m/z Relative abundance
TOOLbox
Identification (MS/MS)
m/z
Follow the flow. Overview of how ICPL works. (Adapted with permission. Copyright 2004 John Wiley & Sons.)
can be used to reduce the sample complexity at the protein level—and after digestion, at the peptide level. Identical peptides derived from each of the two labeled protein samples differ in mass because they are modified with either the heavy or light isotope. Consequently, they appear as doublets in the MS spectra. The ratios of the ion intensities of these peptide pairs provide quantitative information about the relative abundance of the parent proteins in the original sample. The proteins are then identified via peptide mass fingerprinting or collision-induced dissociation followed by a database search using sophisticated algorithms. (Proteomics 2004, 4, doi 10.1002/pmic.200400873)
De novo algorithm A key part of de novo peptide sequencing algorithms is the scoring function, which is used to evaluate how well a candidate peptide matches a given experimental spectrum. Ari Frank and Pavel Pevzner have developed a new scoring method for de novo interpretation of peptides that outperforms other de novo algorithms. Called PepNovo, the new algorithm relies on a hypothesis test to determine whether peaks in a mass spectrum are more likely to have been produced under a fragmentation model or under a probabilistic model that treats peaks as random events. The researchers tested PepNovo on ion trap MS/MS data and achieved superior results compared with those from popular de novo algorithms. In the future, they plan to expand the score models to include additional charge states and to create models for data from other types of mass spectrometers such as Q-TOF. PepNovo is available at www-cse.ucsd. edu/groups/bioinformatics/software. html. (Anal. Chem. 2005, 77, doi 10.1021/ac048788h)
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Producing proteins for structural proteomics Producing stably folded, soluble proteins is the main challenge for high-throughput structure determination. To meet this challenge, John Markley and colleagues at the Center of Eukaryotic Structural Genomics (CESG), the University of Wisconsin–Madison, and the Medical College of Wisconsin have developed a cell-free method for producing folded, soluble proteins that are ready for NMR structure determination studies. Not every protein can be expressed in E. coli. Cell-free alternative methods, such as the wheat germ approach, bypass some of the problems with protein expression in E. coli. Another advantage of cell-free methods is that the protein labeling strategies are easier to achieve. Markley and colleagues demonstrated the wheat germ cell-free approach with a particular target from the model plant Arabidopsis thaliana. The approach involved four steps. First, the investigators cloned the gene for the target into a special plasmid for in vitro transcription. During the second step, a small-scale
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screen was carried out to assay the plasmid for protein expression and solubility. Next, a larger-scale production of 15N-labeled protein was done to test its applicability for NMR structure determination. In the final step, sufficient quantities of 13C- and 15N-labeled protein were produced for multidimensional, multinuclear NMR structure determination. The investigators say that the approach has been used to screen potential open reading frames (ORFs) in different organisms. CESG researchers have used it to screen 153 ORFs in A. thaliana and 85 in the human genome. Half of the ORFs produced proteins that were >50% soluble. Incorporation of stable isotopes into the proteins was 95% efficient. Markley and colleagues say that the most significant advantage of their wheat germ cell-free approach is the production of more folded, soluble proteins than is possible with the E. coli approach. The investigators also suggest that certain steps in their approach could be automated. (Nat. Methods 2004, 1, 149–153)
