A new method, MAP (mixing after purification)-SILAC, to quantitatively investigate the interactions of protein complexes by mass spectrometry is described. In combination with the traditional SILAC approach, stable and dynamic components are distinguished by the differences in their relative abundance ration changes.
Recent Research Papers of Note in Proteomics: Nov 8, 2007
Journal: Journal of Molecular Graphics & Modelling, Nov.26
Title: Protein radial distribution function (P-RDF) and Bayesian-regularized genetic neural networks for modeling protein conformation stability: Chymotrypsin inhibitor 2 mutants
Authors: M Fernandez; J Caballero; L Fernández; JI Abreu; M Garriga
Authors report the extension of the radial distribution function scores formalism to proteins for encoding 3D structural information with modeling purposes.
Journal: Biochemical and Biophysical Research Communications, Nov. 23
Title: 2Dbase: 2D-PAGE database of Escherichia coli
Authors: C Vijayendran; S Burgemeister; K Friehs; K Niehaus; E Flaschel
The authors present a web-based integrated proteome database to store, compare, analyze, and retrieve information generated by 2D polyacrylamide gel electrophoresis and mass spectrometry. Features of the database include the ability to query and data-mine applications to access the stored proteomics data; efficient comparison of specific protein spots in comparable proteome maps; and analysis of data with integrated classification for cellular functions of gene products of E. coli.
Journal: Analytical Biochemistry, Nov. 15
Title: Identification of protein-binding peptides by direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of peptide beads selected from the screening of one bead-one peptide combinatorial libraries
Authors: MM Marani; E Oliveira; S Côte; SA Camperi; F Albericio; O Cascone
The strategy incorporated streptavidin as the model protein and a combinatorial library of 6,561 peptides synthesized on ChemMatrix resin by the divide-couple-recombine method. The authors used 4-hydroxymethylbenzoic acid as the linker and incorporated five residues of Gly at the C termini to increase the final peptide molecular weight.
“Positive control peptides with the HPQ motif and negative control peptides without the HPQ motif evidenced that the linker and the five residues of Gly have neither impaired the specific binding nor facilitated unspecific binding,” according to the abstract. Positive beads were isolated and washed after the library was screened. The beads were sliced into two and four pieces, deposited onto the stainless steel MALDI sample plate and treated with ammonia vapor to release the peptides. Also, 26 beads chosen randomly from the library were treated in the same manner. The samples were analyzed by MALDI-TOF MS. The peptides, according to the abstract, were unambiguously identified with very good reproducibility between the bead pieces, “thus evidencing the good homogeneity of the bead.”
Journal: Proteomics, Nov. 7
Title: Multidimensional liquid phase protein separations in conjunction with stable isotope labeling for quantitative proteomics
Authors: BF Assiddiq, JC Williamson; AP Snijders; K Cook; MJ Dickman
Authors describe an approach combining the advantages of high-throughput, automated, reproducible protein separations with accurate protein quantitation done in a mass spectrometer. With the approach, they were able to identify a number of differentially expressed proteins, they say. The approach also overcomes caveats associated with multidimensional liquid phase protein separations “including the presence of multiple proteins present in a single chromatographic peak.”
Journal: Proteomics, Nov. 7
Title: HUPO BPP pilot study: A proteomics analysis of the mouse brain of different developmental stages
Authors: J Wang; Y Gu; L Wang; X Hang; Y Gao; H Wang; C Zhang
C57/B16 mouse brains of three developmental stages at embryonic day 16, postnatal day 7, and 8 weeks were provided by the HUPO Brain Proteome Project executive committee. The whole brain proteins of each animal were individually prepared using 2-DE coupled with PDQuest software analysis. The protein spots representing developmentally related or stably expressed proteins were prepared with in-gel digestion then MALDI-TOF/TOF MS/MS. Analysis was done with the Mascot search engine searching SwissProt and NCBInr database. The 2-DE gel maps of the mouse brains of all of the developmental stages were obtained and submitted to the Data Collection Center. The authors identified alpha-enolase, stathmin, actin, C14orf166 homolog, 28 000 kDa heat- and acid-stable phosphoprotein, 3-mercaptopyruvate sulfurtransferase and 40 S ribosomal protein S3a. Western blotting analysis further demonstrated that enolase is a protein up-regulated in the mouse brain from embryonic stage to adult stage.
Journal: Nature Methods, Nov. 4
Title: Semi-supervised learning for peptide identification from shotgun proteomics datasets
Authors: L Käll; JD Canterbury; J Weston; WS Noble; MJ MacCoss
Authors describe an algorithm called Percolator which they say improves the rate of confident peptide identifications from tandem mass spectra. Percolator uses semi-supervised machine learning “to discriminate between correct and decoy spectrum identifications,” they say in the abstract. It correctly assigns peptides to 17 percent more spectra from a Saccharomyces cerevisiae dataset and up to 77 percent more spectra from non-tryptic digests, relative to a fully supervised approach, they say.
Journal: Nucleic Acids Research, Nov. 2 [Epub ahead of print]
Title: NetworKIN: a resource for exploring cellular phosphorylation networks
Authors: R Linding; L Juhl Jensen; A Pasculescu; M Olhovsky; K Colwill; P Bork; MB Yaffe; T Pawson
Authors describe how the NetworKIN database can be used for both global and targeted molecular studies. Users can query the database of precomputed kinase-substrate relations or obtain predictions on novel phosphoproteins. The database contains a predicted phosphorylation network with 20,224 site-specific interactions involving 3,978 phosphoproteins and 73 human kinases from 20 families.
Journal: Analytical Chemistry, Nov. 1
Title: Top-down proteomics on a chromatographic time scale using linear ion trap Fourier transform hybrid mass spectrometers
Authors: BA Parks; L Jiang; PM Thomas; CD Wenger; MJ Roth; MT Li; PV Burke; KE Kwast; NL Kelleher
A method is described for high-resolution tandem mass spectrometry of intact proteins on a chromatographic time scale. Authors were able to identify 22 yeast proteins with molecular weights from 14 to 35 kiloDaltons using LC-MS/MS. Using anion exchange chromatography to fractionate a whole cell lysate before online LC-MS/MS, they detected 231 metabolically labeled protein pairs from Saccharomyces cerevisiae. An additional 39 proteins were identified and characterized from LC-MS/MS of selected anion exchange fractions and automated localization of multiple acetylations on histone H4 was accomplished “on an LC time scale from a complex protein mixture,” according to the abstract. “To our knowledge, this is the first demonstration of top-down proteomics … on linear ion trap Fourier transform (LTQ FT) systems using high-resolution MS/MS data obtained on a chromatographic time scale,” the authors say. An additional 39 proteins were identified and characterized from LC-MS/MS of selected anion exchange fractions and automated localization of multiple acetylations on histone H4 was accomplished “on an LC time scale from a complex protein mixture,” according to the abstract. “To our knowledge, this is the first demonstration of top-down proteomics … on linear ion trap Fourier transform (LTQ FT) systems using high-resolution MS/MS data obtained on a chromatographic time scale,” the authors say.
Journal: Analytical Biochemistry, Nov. 1
Title: Protein phosphorylation analysis by site-specific arginine-mimic labeling in gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
Authors: YH Ahn; ES Ji; KH Kwon; JY Lee; K Cho; JY Kim; HJ Kang; HG Kim; JS Yoo
Authors introduce a method for analyzing protein phosphorylation in which phosphorylation sites are labeled with guanidinoethanethiol by beta-elimination/Michael addition prior to proteolysis and mass spectrometry analysis. The technique, they say, is particularly useful in conjunction with gel-based technology because all processes can be carried out in excised gel slices, minimizing sample loss and contamination.
Journal: Journal of Proteome Research, November
Title: Two-dimensional reversed-phase x ion-pair reversed-phase HPLC: An alternative approach to high resolution peptide separation for shotgun proteome analysis
Authors: N Delmotte; M Lasaosa; A Tholey; E Heinzle; CG Huber
Authors say their 2D separation scheme has a lower degree of orthogonality but higher separation efficiency, more homogeneous distribution of peptide elution and easier experimental handling, compared to the classical strong cation exchange x ion-pair reversed phase approach. About 13 percent more peptides and 7 percent more proteins could be identified with the approach described in the paper in 30 percent less analysis time. Combining the classical approach with the alternative approach, 871 proteins were identified in a cytosolic protein separation, representing 29.1 percent of all proteins annotated in the genome of C glutamicum.
Journal: Proteomics, Oct. 31 [Epub ahead of print]
Title: Two-dimensional separation of human plasma proteins using iterative free-flow electrophoresis
Authors: M Nissum; S Kuhfuss; M Hauptmann; C Obermaier; U Sukop; R Wildgruber; G Weber; C Eckerskorn; J Malmström
Authors propose a strategy to mine for low abundance proteins in human plasma, using 2D free flow electrophoresis to separate proteins and peptides in solution based on their isoelectric point prior to LC-MS/MS. Plasma proteins were first separated into specific FFE fractions. Tryptic digests of the separated proteins were generated and then separated using FFE. The approach, they say, revealed low-abundant protein and several tissue leakage products.
Journal: Protein Science: A Publication of the Protein Society, Oct. 26 [Epub ahead of print]
Title: A rapid and universal tandem-purification strategy for recombinant proteins
Authors: AJ McCluskey; GM Poon; J Gariépy
Authors report the design and use of a novel tandem (His)(6)-calmodulin (HiCaM) fusion tag, combining two purification strategies, immobilized metal affinity, and hydrophobic interaction chromatography, in a simple two-step procedure.
Journal: Biology Direct, Oct. 25 [Epub ahead of print]
Title: RAld DbS: Peptide identification using database searches with realistic statistics
Authors: G Alves; AY Ogurtsov; YK Yu
A database search method is proposed using a simple scoring scheme for the identification of peptides in MS-based proteomics. They collected the scores of peptides in the database for every experimental spectrum examined and found “good agreement” between the collected score statistics and their theoretical distribution. They used student’s T-tests to quantify the degree of agreement between the theoretical distribution and the score statistics collected. Combined with reported P-value for a peptide hit using a score distribution model, the new measure prevents “exaggerated” statistics.
Journal: Nature, Oct. 25
Title: Clathrate nanostructures for mass spectrometry
Authors: TR Northen; O Yanes; MT Northen; D Marrinucci; W Uritboonthai; J Apon; SL Golledge; A Nordström; G Siuzdak
Authors introduce nanostructure-initiator mass spectrometry, which they say is a tool for spatially defined mass analysis. The method uses “initiator” molecules trapped in nanostructure surfaces or “clathrates” to release and ionize intact molecules adsorbed on the surface, which responds to ion and laser irradiation. “The lateral resolution (ion-NIMS about 150 nm), sensitivity, matrix-free and reduced fragmentation of NIMS allows direct characterization of peptide microarrays, direct mass analysis of single cells, tissue imaging, and direct characterization of blood and urine,” according to the abstract.
Journal: Nucleic Acids Research, Oct. 25 [Epub ahead of print]
Title: CEBS chemical effects in biological systems: A public data repository integrating study design and toxicity data with microarray and proteomics data
Authors: M Waters; S Stasiewicz; BA Merrick; K Tomer; P Bushel; R Paules; N Stegman; G Nehls; KJ Yost; CH Johnson; SF Gustafson; S Xirasagar; N Xiao; CC Huang; P Boyer; DD Chan; Q Pan; H Gong; J Taylor; D Choi; A Rashid; A Ahmed; R Howle; J Selkirk; R Tennant; J Fostel
Chemical Effects in Biological Systems is an integrated public repository for toxicogenomics data and includes the study design and timeline, clinical chemistry and histopathology findings in microarray and proteomics data. It is designed to allow queries using data conditions and subject responses. After identifying an appropriate set of subjects, a researcher can then move to the microarray module of CEBS to carry out gene signature and pathway analysis. It currently holds 22 studies of rats, four of mice and one of C. elegans. CEBS can also accommodate data from studies of human subjects.
Journal: Chemistry, Oct. 24 [Epub ahead of print]
Title: A nanoporous reactor for efficient proteolysis
Authors: L Qiao; Y Liu; SP Hudson; P Yang; E Magner; B Liu
Authors describe a nanoreactor based on mesoporous silicates to be used for tryptic digestion of proteins within the mesochannels. “Cyano-functionalized mesoporous silicate (CNS), with an average pore diameter of 18 nm, is a good support for trypsin, with rapid in situ digestion of the model proteins, cytochrome c and myoglobin,” they say. Peptides were analyzed with MALDI-TOF MS.
Journal: PLoS ONE, Oct. 24
Title: Optimized protein extraction for quantitative proteomics of yeasts
Authors: T von der Haar
Authors have developed a novel technique for extraction proteins from S. cerevisiae based on chemical lysis and simultaneous solubilization in SDS and urea. The method can be used for different Saccharomycetes yeast species and varying growth conditions, they say. It is also suitable for high-throughput extraction in a 96-well format. The resulting extracts can be post-processed for use in non-SDS compatible procedures such as 2D gel electrophoresis.
Journal: Proceedings of the National Academy of Sciences of the United States of America, Oct. 23 [Epub ahead of print]
Title: Identifying the subproteome of kinetically stable proteins via diagnonal 2D SDS/PAGE
Authors: K Xia; M Manning; H Hesham; Q Lin; C Bystroff; W Colón
Authors show the application of a diagonal 2D SDS/PAGE assay for the identification of kinetically stable proteins in complex mixtures. The method was applied to the lysate of E. coli. Upon proteomics analysis, they identified 50 unredundant proteins that were SDS resistant.
Journal: Physiological Genomics, Oct. 22
Title: Relative quantification of peptide phosphorylation in a complex mixture using 18O labeling
Authors: JR Smith; M Olivier; AS Greene
Authors describe a method they developed to determine the degree of phosphorylation of a peptide in a complex mixture without enrichment or operation of the mass spectrometer in negative ion mode. Yeast lysate with known amounts of synthetic peptides was labeled with (16)O and (18)O during hydrolysis. One sample was treated with phosphatases, and then both samples were pooled. The degree of phosphorylation present before treatment was inferred by using the intensity of the dephosphorylated peptide peaks.
Journal: Analytical Chemistry, Oct. 15
Title: Dual electrospray ion source for electron transfer dissociation on a hybrid linear ion trap-Orbitrap mass spectrometer
Authors: DK Williams Jr.; GC McAlister; DM Good; JJ Coon; DC Muddiman
Authors altered a dual electrospray ionization source to simultaneously produce cations and anions to do rapid electron transfer dissociation ion/ion reactions on a hybrid linear ion trap Orbitrap MS. Unlike the pulsed dual ESI sources, this source separates the emitters in space instead of time, by physically switching which one is in front of the atmospheric inlet, the authors say. The new configuration allows for “substantially enhanced spray stability and decreased switching time, allowing for more tandem-MS spectra per unit time.”
Journal: Analytical Chemistry, Oct. 12 [Epub ahead of print]
Title: Pseudo Internal Standard Approach for Label-Free Quantitative Proteomics
Authors: T Tabata; T Sato; J Kuromitsu; Y Oda
A novel label-free quantitative method of proteome analysis using pseudo internal standards was developed, derived from northern blotting analysis, “in which housekeeping genes are used as standards to normalize and compare target gene expression levels in different samples,” according to the abstract. In many proteomics studies, the authors say, the expression levels of most proteins are not changed under different conditions. As a result, they can be used as pseudo internal standards.
Journal: Molecular & Cellular Proteomics, Oct. 12 [Epub ahead of print]
Title: Identifying dynamic interactors of protein complexes by quantitative mass spectrometry
Authors: X Wang; L Huang