currents
Spindle phosphoproteins
Herman Silljé
MS/MS, whereas other samples were enriched for phosphoWhen it divides, a cell undergoes an extreme makeover. Mipeptides by immobilized metal ion chromatography (IMAC), crotubules form an elaborate spindle structure, and replicatthen analyzed by MS/MS or LC/MS/MS. As expected, the ed chromosomes attach to it. researchers identified more When the time is finally right, dupeptide phosphorylation sites plicate chromatids are tugged in IMAC eluates than in samapart from each other, and ples not treated with IMAC resthe cell divides in two. Protein ins. An LC separation step also phosphorylation keeps many of increased the number of phosthese events on track. Although phopeptides detected. researchers know the identities Of the 736 phosphorylation of many of the proteins that are sites identified, 312 were presinvolved in spindle function, they ent in 72 known spindle prohaven’t pinned down all of the teins. Interestingly, ~90% of phosphorylation sites on these these sites had not been deproteins. So, Roman Körner and scribed previously. Other sites colleagues at the Max Planck were found in proteins not Institute of Biochemistry (Gerknown to be associated with many) performed a proteomics the spindle. The most frequentstudy of the proteins associatly observed type of phosphored with the human mitotic spinylation consensus site was for dle. A total of 736 phosphorylaproline-directed kinases, such tion sites were identified on 260 as the mitotic cyclin-dependent proteins. kinase Cdk1. Many of the sites Phosphoproteins abound. Researchers identified hundreds Körner and colleagues isowere evolutionarily conserved of phosphorylation sites on proteins that are associated with lated spindles from HeLa cells across three or more species. mitotic spindles. and separated the spindle proAccording to the researchers, teins on 1DE gels. Each lane was these findings must still be valcut into 18 pieces, and the proteins were digested in the gel idated using cell biology techniques. (Proc. Natl. Acad. Sci. with trypsin. Some samples were analyzed directly by LC/ U.S.A. 2006, 103, 5391–5396)
Predicting clinical outcomes Ureteropelvic junction (UPJ) obstruction is a common clinical problem among newborn children. In the condition, a blockage prevents the flow of urine from the kidney to the ureter; it can cause kidney damage in some cases. Surgery can relieve the blockage, but not all patients need such drastic measures; some will improve on their own. Doctors currently use invasive procedures to determine which newborns will need surgery, and the results of the tests are evaluated against arbitrary threshold values. Joost Schanstra and colleagues at l’Institut National de la Santé et de la Recherche Médicale (Inserm), Université Toulouse III Paul Sabatier, Centre Hospitalier Universitaire de Toulouse (all in France), and Mosaiques Diagnostics and Therapeutics AG (Germany) have taken a proteomics approach to UPJ prognosis. They found that they could correctly predict which patients © 2006 American Chemical Society
would require surgery with 94% precision by analyzing a collection of polypeptides in the urine. Using CE/MS, Schanstra and colleagues compared the peptide urinary patterns of adults and newborns. Interestingly, the newborn pattern was much more consistent among individuals than the adult pattern. Adults have a wide variety of eating and exercise habits that could explain this difference. Also, adults have had a greater chance to develop other kidney conditions that could affect the urinary proteome. The researchers classified 103 newborns according to current UPJ status: “No_OP” patients had UPJ and were not scheduled for an operation, “OP_Poss” patients had UPJ and were being monitored as possible candidates for surgery, and “OP” patients had severe UPJ and were scheduled for an operation. The researchers found 51 distinct polypeptides that distinguished patients in the
3 groups by using CE/MS. In a blind validation, independent groups of OP and No_OP newborns were evaluated with the candidate markers. The markers were 94% sensitive and were 80% specific for No_OP and 100% specific for OP patients. Of the 51 polypeptides, only 2 were identified in the study. One of the polypeptides is the 3 chain of type IX collagen, and the other is a fragment of the type V preprocollagen 2 chain. Next, the putative polypeptide markers were used to predict the clinical outcomes of OP_Poss patients. In a prospective blinded study of 36 OP_Poss newborns, the researchers predicted that 25 would need surgery and 11 would not. After waiting 9 months, the researchers followed up with the patients and found that their predictions were correct for 34 patients. Therefore, say the researchers, proteomics could be used for clinical prognostics. (Nat. Med. 2006, 12, 398–400)
Journal of Proteome Research • Vol. 5, No. 6, 2006 1293
currents Toolbox Delay-series correlation and convolution mapping Search engines such as Sequest or Mascot can help researchers identify proteins but don’t provide information about mutations, modifications, or trun cations. So, to obtain this kind of data, Juergen Kast and co-workers at the University of British Columbia (Canada) have applied a delay-series correlation technique to MS spectra as a supple ment to data generated by conventional search engines. In the method, spectra are mathematically correlated to each other. This process results in a trans formed view of the data that highlights mass shifts, which can indicate neutral losses or the presence of stable isotope labels, for example. Delay-series corre lation allows researchers to quickly and easily compare peaks within a single spectrum or between two spectra. By convolution mapping, researchers can discern whether a strong delay-series correlation is due to many small con tributions from several peaks or a few large contributions. Both mathemati cal methods were applied to LC/MS/MS stable isotope labeling by amino acids in cell culture (known as SILAC) data. (Anal. Chem. 2006, 78, 2600–2607)
Ontology Lookup Service Each biomedical community has its own lingo. Controlled vocabularies, or “on tologies”, exist to describe the anato my, development, pathology, gene func tions, and proteins for many species. To put all of these ontologies in one con venient place, Henning Hermjakob and colleagues at the European Bioinformat ics Institute (U.K.) have created the On tology Lookup Service (OLS). OLS is a database that contains 42 ontologies (~135,000 terms). Researchers can browse a complete ontology with a dy namically generated tree structure or search for information about one word. An auto-completion feature suggests terms as a user types a search term. To enhance the amount of information ob tained with each search, relationships between terms are color-coded. Mod ified ontologies are updated daily. The OLS source code is publicly available, and the database can be accessed at www.ebi.ac.uk/ontology-lookup. (BMC Bioinformatics 2005, 7, 97)
Apoptosis and the nuclear proteome During apoptosis, also called programmed cell death, chroNucleus Mitochondria Merge matin is condensed and DNA becomes fragClose association. Mitochondria are abundant in the nuclear invagi mented. To betnations (arrows) of apoptotic cells. (Adapted with permission. Copy ter understand right 2006 American Society for Biochemistry and Molecular Biology.) these events, David Han and co-workers at the University of Connectof the stable isotope labeling by amino icut School of Medicine and the Fred acids in cell culture (SILAC) method to Hutchinson Cancer Research Center incompare protein levels in control cells vestigated the nuclear proteome of huand cells in which apoptosis had been man leukemia T cells during apoptosis. induced. Instead of adding only one laThey identified several proteins that are beled amino acid to the cultures, the redifferentially regulated in apoptosis. searchers added two: leucine (Leu) and Han and co-workers used a variation lysine (Lys). This modified procedure
Activity assay for metallo proteases
Typically, proteomics researchers report that certain proteins are present in higher or lower levels when two or more groups are compared. However, some proteins that are present at the same levels in different groups could still be candidate biomarkers. For example, protein activities could be differentially regulated by posttranslational modifications (PTMs) that would not influence the abundance of a protein. But characterizing PTMs can be difficult with current proteomics methods. Therefore, Benjamin Cravatt and co-workers at the Scripps Research Institute applied their activity-based protein profiling (ABPP) technique to the study of metalloproteases, which are involved in many cellular processes. With a new set of ABPP probes, the researchers identified several metalloproteases that are activated in invasive cancer cells. Because metalloproteases display large variations in their active-site structures, Cravatt and co-workers developed three libraries of structurally diverse probes to target these en zymes. In each library, the probes (called HxBPyne probes) contain up to 10 amino acid side chains at a single position. After labeling a proteome sample with the probes, the research-
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ers add a small reporter tag at an alkyne group on the probe; this tag permits them to visualize or enrich for the probes that are bound to active enzymes. In an initial screen, the HxBPyne probes were added to cell and tissue proteomes and run on a 1DE gel. Some enzymes bound to several probes, whereas other enzymes were specific for probes with certain side chains. Commercially available human matrix metalloproteases also displayed probe-specific profiles. The researchers found that four of the probes labeled most of the enzymes, so they combined these probes in a mixture and added it to individual proteomes. With this procedure, they could simultaneously profile a broad range of metalloprotease activities. Invasive and noninvasive breast cancer and melanoma cell lines were then analyzed by ABPP and multidimensional protein identification technology (known as MudPIT). Several metalloproteases were selectively active in invasive cancer cells. For example, AlaAP and neprilysin activities were higher in invasive than in noninvasive melanoma cells. According to the researchers, additional investigations will be necessary to better understand what role these enzymes could play in cancer progression. (Nat. Chem. Biol. 2006, 2, 274–281)
currents improved the labeling coverage. The researchers passaged control cells with heavy amino acids (Leu-13C6 and Lys13C -15N ) and passaged apoptosis-in6 2 duced cells with light amino acids (Leu12C and Lys-12C -14N ). 6 6 2 The nuclei of control and apoptotic cells were combined and f ractionated with a sucrose gradient. The nuclear proteins were then sequentially extracted with three buffers. This extraction was performed to reduce the complexity of the nuclear proteome in subsequent analyses. As visualized on Coomassie Blue-stained 1DE gels, different proteins were extracted with each buffer. The first 2 buffers extracted ~43% and ~44% of the nuclear proteins. The final buffer extracted ~13% of the nuclear proteins. The nuclear proteins from control and apoptosis-induced cells were separated on a 1DE gel. Protein bands were excised, in-gel digested, and analyzed by LC/MS/MS. A total of 1174 unique proteins were identified from the 3 nuclear fractions. The levels of most of the quantified pro-
teins did not change drastically during apoptosis. However, 15 proteins were down-regulated and 50 proteins were up-regulated >2-fold in apoptosis-induced cells. The researchers validated the results for seven proteins by western blot and immunofluorescence analyses. Some proteins that were identified in the nuclear preparations are known mitochondrial proteins. Apoptotic nuclei have many invaginations that contain mitochondria, but these structures are not visible in control nuclei. When examining the SILAC data, the researchers found that a known mitochondrial protein called Bcl-2 homologous antagonist/killer (BAK) was up-regulated in apoptosis-induced nuclei. BAK was also up-regulated by western blotting and was visible in the nuclear invaginations of apoptotic cells by immunofluorescence. According to the researchers, this discovery suggests that nuclei and mitochondria are closely associated and that this association changes during apopto sis. (Mol. Cell. Proteomics 2006, doi 10.1074/mcp.M500162-MCP200)
Pencil lead as a MALDI matrix
A wide range of analytes, including small molecules, peptides, polymers, and actinides, can be analyzed by MALDI MS with pencil lead as a matrix, according to new results reported by Claudie Black and colleagues at AWE Aldermaston and the University of Southampton (both in the U.K.). Pencil lead requires less sample preparation than other matrices and is easily applied to the MALDI target plate. Although graphite rods and powders and amorphous nanocarbon previously had been used as MALDI matrices, those forms of graphite were more difficult to apply to a stainless-steel target plate than pencil lead. With pencil lead, one only needs to scribble on the target plate to obtain an even, reproducible coverage. Pencil lead is made of graphite with components such as clay and wax, which help it glide across paper. Thanks to those wax-based components, pencil lead also is easily applied to a MALDI target plate. Because pencil lead is a solventless matrix, one does not have to be concerned about solvent mixing, which often limits the kinds of samples that can
Toolbox SwePep peptide database In MS experiments, endogenous pep tides are difficult to identify because they are small (
