currents Ambient thermal dissociation generates fragments outside the mass spectrometer
through a heated coiled tube to induce dissociation and from there, depending on the experiment, one of two things can In the world of fragmentation methods for protein and pep- happen. If ionic fragments are of interest, they can be passed tide MS, collision-induced dissociation (CID) has long been through a transfer capillary directly into the instrument. To isolate neutral fragments, king. But recently, several alternative interfering ionic species methods, including are first sorted and direct- electron capture and ed out of the gas flow, and Transfer N2 electron transfer dis- then the neutral fragments Coiled tube capillary + sociation, have been are reionized before pass- Mass spectrometer – developed and may ing into the mass spec- ESSI force CID to share its trometer. Hot (Reduced throne. These com- Neutral species are no- pressure) Ambient plementary methods toriously difficult to detect provide valuable data because the traditional about ionic species reionization process un- Thinking outside the box. Experimental setup for ambient thermal dissociation by giving different der vacuum can be tricky and subsequent reionization of neutral fragments from peptide or protein ions. patterns of fragment and requires specialized ion intensities when instrumentation. But with compared with CID. Now, R. Graham Cooks and colleagues ambient thermal dissociation, ions are generated using sim- at Purdue University have developed a new fragmentation ple on-line corona discharge ionization. method called ambient thermal dissociation that reports on Because this method provides some unique dissociation unique ionic species and, for the first time, allows researchers information and is only a slightly modified form of ESSI, the to isolate and reionize neutral fragments at ambient pressure researchers hope that this technique will be smoothly inte- outside of the mass spectrometer. grated into the arsenal available to proteomics researchers as Ambient thermal dissociation starts with electroson- a supplement to other dissociation methods. (J. Am. Chem. ic spray ionization (ESSI). Peptide or protein ions then pass Soc. 2007, 129, 5880–5886)
Tag for improved limits of detection with ESI MS High hydrophobicity boosts ESI performance. So, to improve the limits of detection for peptides in proteomics studies, David Muddiman and colleagues at North Carolina State University designed hydrophobic tags that can be added to cysteines. The new strategy, called augmented limits of detection for peptides with hydrophobic alkyl tags (ALiPHAT), allows researchers to detect peptides that are present at low levels. Although other hydrophobic tagging methods have been described recently, they are not ideal. Side reactions can occur, and extensive sample cleanup may be required with these techniques. Some labeling reactions do not go to completion or the modification is lost during collision-induced dissociation (CID). With ALiPHAT, however, the tagging step is simple and goes to completion. The tag, an octylcarboxyamidomethyl (OCAM) group, is added to cysteines during the alkylation and reduction step that typically is performed on peptides in shotgun proteomics studies. In addition, the modification is not lost during © 2007 American Chemical Society
CID fragmentation, so it does not interfere with the interpretation of MS/MS spectra. As a proof of principle, Muddiman and colleagues alkylated laminin nonapeptide and b-type natriuretic peptide-32 (BNP) with 2-iodo-N-octylacetamide, which leaves an OCAM group on the peptide, or with iodoacetamide, the typical alkylation reagent. Compared with the conventional modification, the OCAM tag improved the chromatographic performance and ESI response for laminin. When it was attached to BNP, which is a larger peptide, only the ESI response was improved. When the investigators performed MS/MS on OCAM–BNP, however, they obtained a better sequence coverage compared with BNP alkylated with the typical reagent. The researchers say that the tagging method will be particularly useful for targeted proteomics studies. (Anal. Chem. 2007, 79, 3989–3995)
Mixed-bed ion-exchange resin for enhanced MudPIT Strong-cation exchange (SCX) LC typically is used as the first separation dimension in the shotgun proteomics
method called multidimensional protein identification technology (MudPIT) because it is orthogonal to reversedphase (RP) chromatography, and the buffer conditions are usually suitable for downstream RPLC and MS analyses. To achieve the best SCX performance when separating peptides, researchers must add an organic modifier to the mobile phase; however, this modifier is incompatible with RPLC and the high-concentration salt buffers required for SCX elution. To optimize the first separation dimension of MudPIT while retaining compatibility with RPLC and MS, John Yates and co-workers at the Scripps Research Institute developed a mixedbed ion-exchange resin composed of a blend of anion and cation exchange (ACE) materials. ACE separations greatly improved the recovery of peptides. In addition, an increase in orthogonality with RPLC was observed. The performance of the ACE mixedbed system was assessed with a tryptic digest of five standard proteins and a yeast cell lysate. Various combinations of weak-anion exchange (WAX) and SCX resins were tested as well as tandem
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currents SherLoc for subcellular localization To get a quick hint at where a protein resides, Hagit Shatkay and colleagues at Queen’s University (Canada) and the University of Tübingen (Germany) developed a computational system, called SherLoc. Sequence features, including N-terminal targeting peptides, internal signal anchors, sorting motifs, and the overall amino acid composition of a protein, are integrated with text information drawn from PubMed abstracts associated with the protein’s SwissProt entry. Unlike other methods that include text information to make predictions, SherLoc only selects a subset of distinguishing terms that are likely to be associated with a particular subcellular location. If a protein does not have a SwissProt entry or its entry does not include PubMed identifiers, the researchers use the text of close homologues in the analysis. These text terms are weighted and analyzed in combination with the outputs of the sequence classifiers. SherLoc is available at www-bs. informatik.uni-tuebingen.de/Services/ SherLoc. (Bioinformatics 2007, doi 10.1093/bioinformatics/btm115)
Panoramics Protein homology and redundancy in database records can confound current search engines, so Bret Cooper and co-workers at Johns Hopkins University and the U.S. Department of Agriculture Agricultural Research Service developed a probability model to assess whether peptides are correctly assigned to proteins. Peptide scores from Mascot, the size of the queried database, the number of peptides in the database with molecular weights similar to that of the precursor ion, and the frequency with which the sequence appears in the database are factors that the model uses to derive probability estimates. On the basis of this model, an algorithm groups proteins that have the same sets of matched peptides and calculates the probabilities that the identified proteins are correct with respect to the searched database. The algorithm is included in the Panoramics software platform, which is freely available from the researchers. (Anal. Chem. 2007, 79, 3901–3911)
combinations of WAX and SCX columns. The best results were obtained with a 2:1 weight ratio of WAX and SCX resin packed in the same column. For the yeast cell lysate, the number of peptide identifications was increased by 100% with ACE materials as compared with SCX resin alone. Phosphopeptides enriched from a HeLa nuclear extract also were analyzed with the ACE mixed bed. The material enriched phosphopeptides, particularly those with a low pI value. Overall, the new system resulted in a 94% increase in the number of identified phosphopeptides compared with SCX. Therefore, the researchers say that the ACE mixed-bed system is a simple and effective alternative to SCX for Mud PIT analyses. (Anal. Chem. 2007, 79, 3623–3634)
A feedback strategy leads to high proteome coverage
Quantitative proteomics method for fast events Signaling proteins can be modified within a few seconds, but most MSbased sample preparation protocols only enable researchers to take time points within 30 seconds of stimulation. To analyze processes that occur on a much faster timescale, Blagoy Blagoev, Jens Andersen, and colleagues at the University of Southern Denmark and the Max Planck Institute for Biochemistry (Germany) developed a new experimental design called quantitative proteomic assessment of very early cellular signaling events (qPACE). With qPACE, isotopically labeled cells and a growth factor are pumped into a mixing chamber. To quench the reaction, a fixing solution of 70% ethanol
PHOTODISC
Toolbox
Eventually, shotgun proteomics experiments hit a wall. Beyond a certain number of experi- ments, researchers typically only identify a few new proteins. So, instead of wasting effort on replicates that would yield just a handful of novel protein identi- fications, Ruedi Aebersold and co-workers at the University of Zurich, ETH Hönggerberg Fruit fly proteome. Researchers developed and ap(Switzerland), the Institute for plied a new experimental design to increase proSystems Biology, the Univer- teome coverage of D. melanogaster. sity of Denmark, LEO Pharma (Denmark), and Utrecht Univer- sity (The Netherlands) developed a dif- proteomics. By performing statistical ferent approach, called analysis-driven analyses, the researchers conclud- experimentation (ADE). ed that short proteins and basic ones With ADE, researchers perform it- were underrepresented in these ex- erative cycles of experimentation and periments. They performed gel filtra- analysis. The knowledge they gain at tion to identify additional short proteins and free-flow electrophoresis at pH 4– each point is used to better direct their efforts in subsequent experiments. 7 to target acidic proteins. Several pro- Aebersold and co-workers compiled teins, including those that are secret- a high-quality Drosophila melanogased or span membranes, were identified ter proteome list of 9124 proteins for with ADE. The proteins identified with various cell types and developmental this strategy provided proof that >60% stages with the new experimental de- of all fruit fly gene models are correct. sign. They also applied these data to Novel genes and genes that were in- the validation or correction of fruit fly correctly annotated also were dis- gene models. covered with the method. Finally, the Several parameters, such as length, scientists compiled an extensive pro- pI, and number of transmembrane do- teotypic peptide (PTP) library of 37,279 mains, were determined for the D. melaPTPs for D. melanogaster. (Nat. Bionogaster proteins identified by shotgun technol. 2007, 25, 576–583)
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currents at –20 °C was pumped into the system. Samples were taken at 0, 1, 5, 10, and 60 seconds. Phosphorylations were detected as soon as 5 seconds after stimulation. The researchers say that, in principle, even shorter time points (
