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Recent Research Papers of Note in Proteomics : Dec 4, 2008

Journal: Methods in Molecular Biology, 2009
Title: High-throughput protein expression using cell-free system
Authors: K Sitaraman; DK Chatterjee
According to the authors, one of the main challenges of the post-genomic era is developing efficient methods of protein synthesis. “The future of structural genomics and functional proteomics depends on the availability of abundantly expressing soluble proteins in a high-throughput manner,” according to the abstract. Presented is a protocol for expressing recombinant proteins with high yield in a standard 96-well plate format, using E. coli cell-free extract in a batch mode.

Journal: Rapid Communications in Mass Spectrometry: RCM, December
Title: ProteinQuant Suite: a bundle of automated software tools for label-free quantitative proteomics
Authors: B Mann; M Madera; Q Sheng; H Tang; Y Mechref; MV Novotny
The software suite comprises three stand-alone complementary computer utilities, ProtParser, ProteinQuant, and Turbo RAW2MGF. ProteinQuant Suite was validated initially using LC-MS/MS “generated for a mixture of standard proteins as well as standard proteins spiked in a complex biological matrix such as blood serum,” according to the abstract. It was applied to confirm the binding preference of standard glycoproteins to Con A lectin using a sample of both standard glycoproteins and proteins.

Journal: Nature Biotechnology, Nov. 30 [Epub ahead of print]
Title: MaxQuant enables high peptide identification rates, individualized ppb-range mass accuracies and proteome-wide protein quantifications
Authors: J Cox, M Mann
Described is MaxQuant, an integrated suite of algorithms developed for high-resolution, quantitative MS data. MaxQuant uses correlation analysis and graph theory to detect peaks, isotope clusters, and stable amino acid isotope-labeled peptide pairs as 3D objects in m/z, elution time, and signal intensity space. Using multiple mass measurements and correcting for linear and nonlinear mass offsets, the authors attained mass accuracy in the parts-per-billion range, a six-fold increase over standard methods.

Journal: Biomedical Chromatography: BMC, Nov. 27 [Epub ahead of print]
Title: Limitation of immunoaffinity column for the removal of abundant proteins from plasma in quantitative plasma proteomics
Authors: T Ichibangase; K Moriya; K Koike; K Imai
Authors tested the ability of immunoaffinity columns to remove abundant proteins from plasma. A plasma proteome analysis was performed using the fluorogenic derivatization-liquid chromatography-tandem mass spectrometry method combined with an IgY column.
They found that specific adsorption for albumin decreased as the number of uses of the column before its expiration date increased. They also found that hydrophobic high- molecular-weight compounds in plasma adsorbed onto the column materials surface “contributed to the functional changes from specific immunoaffinity adsorption into hydrophobic interaction,” they said in the abstract. “These results suggested that, in quantitative plasma proteomics studies, it is important to keep in mind the risk of not only the nonselective loss but also the changes in the adsorption ability of the immunoaffinity column,” they said.

Journal: Biotechnology Journal, Nov. 27, [Epub ahead of print]
Title: High-throughput protein production – Lessons from scaling up from 10 to 288 recombinant proteins per week
Authors: H Tegel; J Steen; A Konrad; H Nikdin; K Pettersson; M Stenvall; S Tourie; U Wrethagen; L Xu; L Yderland; M Uhlén; S Hober; J Ottosson
Described is how the protein expression and purification protocol has been optimized in the Human Protein Atlas project to produce recombinant proteins. Nearly 300 different proteins are handled weekly. The number of manual handling steps has been halved to 9 from 18 and the protein purification has been completely automated.

Journal: Electrophoresis, Nov. 26 [Epub ahead of print]
Title: Quantitative proteomics by fluorescent labeling of cysteine residues using a set of two cyanine-based or three rhodamine-based dyes
Authors: D Volke; R Hoffmann
In order to improve quantification by decreasing detection limits and increasing the dynamic range in gel-based proteomics, the authors report a multiplex analysis of protein samples using maleimide-activated cyanine-based and rhodamine-based dyes to permanently label all thiol-groups of cysteine-containing proteins. The detection limits in SDS-PAGE were about 10 nanograms per band and 2 nanograms for BSA, the authors report. Both cyanine- and rhodamine-based dyes also stained proteins that did not contain cysteines. “This side reactivity did not limit the method and might even extend its general use to proteins missing cysteine residues, but at a lower sensitivity,” according to the abstract.

Journal: Molecular & Cellular Proteomics, Nov. 25 [Epub ahead of print]
Title: Analysis of cell surface proteome changes via label-free, quantitative mass spectrometry
Authors: R Schiess; LN Mueller; A Schmidt; M Mueller; B Wollscheid; R Aebersold
Authors present a mass spectrometry-based strategy for the detection and quantification of cell surface proteome changes. The method is based on the label-free quantification of peptide patterns acquired by high mass accuracy mass spec using new software tools and the Cell Surface Capturing technology. The authors applied their strategy to monitor dynamic protein changes in the cell surface glycol-proteome of Drosophila melanogaster cells, leading to the construction of a cell surface glycoprotein atlas of 203 cell surface glycoproteins of D. melanogaster Kc167 cells. The results also indicated relative quantitative changes of cell surface glycoproteins in four different cellular states.

Journal: Genome Biology, Nov. 18 [Epub ahead of print]
Title: Proteomics studies confirm the presence of alternative protein isoforms on a large scale
Authors: ML Tress; B Bodenmiller; R Aebersold; A Valencia
The authors demonstrate that proteomics methods can detect the expression of stable alternative protein splice isoforms on a genome-wide scale. The alternative isoforms likely have regions that are disordered in solution, and specific methodologies may be necessary to identify the peptides.

Journal: Analytical Chemistry, Nov. 15
Title: Comparisons of mass spectrometry compatible surfactants for global analysis of the mammalian brain proteome.
Authors: EI Chen; D McClatchy; SK Park; JR Yates
Studies of protein expression in tissue such as brain require efficient and unbiased digestion of proteins that permit the identification of peptides by shotgun proteomic methods. To facilitate this, the authors have developed a digestion scheme using detergents compatible with mass spectrometry that improves membrane protein identification from brain tissue. The method yields almost 5,000 protein identifications from 1.8 mg of rat brain homogenate with an average of 25 percent protein-sequence coverage, and achieves a “remarkable reduction” in the amount of starting material needed to observe a broad spectrum of membrane proteins, the authors said in the abstract.

Journal: Analytical Chemistry, Nov. 15
Title: Characterization of strategies for obtaining confident identifications in bottom-up proteomics measurements using hybrid FTMS instruments.
Authors: AV Tolmachev; ME Monroe; SO Purvine; RJ Moore; N Jaitly; JN Adkins; GA Anderson; RD Smith
The authors explore three alternative strategies for high-throughput proteomics measurements using hybrid FTMS instruments: accurate mass and time tag; accurate precursor mass filter; and precursor mass and time filter. The APMF and PMTF methods are evaluated for coverage and confidence or peptide identifications and contrasted with the AMT tag strategy. The AMT approach, according to the authors, is preferred for studies requiring the highest number of identifications of peptides. The APMF approach does not require an AMT tag database and provides a “moderate level of peptide coverage combined with acceptable confidence values of approximately 99 percent,” according to the abstract. The PMTF approach resulted in “significantly” better peptide identification confidence of greater than 99.9 percent

Journal: Molecular & Cellular Proteomics, Nov. 15 [Epub ahead of print]
Title: MRMaid: the web-based tool for designing multiple reaction monitoring (MRM) transitions
Authors: JA Mead; L Bianco; V Ottone; C Barton; RG Kay; KS Lilley; NJ Bond: C Bessant
According to the authors, their tool is a novel alternative for the rapid design of MRM transitions for proteomics research. It uses a combination of knowledge of the properties of optimal MRM transitions with MS/MS evidence derived from interrogation of a database of peptide identifications and their associated mass spectra. MRMaid predicts retention time using a published model.

Journal: Molecular & Cellular Proteomics, Nov. 12 [Epub ahead of print]
Title: Bayesian nonparametric model for the validation of peptide identification in shotgun proteomics
Authors: J Zhang; J Ma; L Dou; S Wu; X Qian; H Xie; Y Zhu; F He
MS/MS with database searching allows for high-throughput identification of peptides in shotgun proteomics, but validating the database results is still a work in progress, the authors said in the abstract. In their study, they present a Bayesian nonparametric model for validating database results. Their model incorporates several techniques in statistical learning, including the compression of feature space with a linear discriminant function; the flexible nonparametric probability density function estimation for the variable probability structure in complex problem; and the Bayesian method to calculate the posterior probability. “Importantly, the BNP model is compatible with the popular target-decoy database search strategy,” according to the abstract.

Journal: Proteomics, Nov. 10 [Epub ahead of print]
Title:Peptide separation with immobilized pI strips is an attractive alternative to in-gel protein digestion for proteome analysis
Authors: NC Hubner; S Ren; M Mann
The authors investigate the recently introduced device for peptide separation with IPGs. The loading capacity for optimal peptide focusing is “well below 100 micrograms,” according to the abstract, and IEF is more efficient in the acidic than the basic pH region. The 24-well fractionation format resulted in about 40 percent more peptide identifications, but less than 20 percent additional protein identifications than the 12-well format. Peptide IEF identified a third more proteins with equal number of fractions compared to in-gel digestion, and low protein starting amounts still resulted in deep proteome coverage.

Journal: Proteomics, Nov. 10 [Epub ahead of print]
Title: IntelliMS: A platform to efficiently manage and visualize tandem mass spectral data
Authors: MS Kwon; HJ Lee; SK Jeong; EY Lee; SY Cho; YK Paik
The data management system incorporates a visualization function, IntelliMS, which can load data into a search engine. The platform, they said, filters out insignificant data, creates diagrams of the identification process from spectra to protein, and shares the resulting dataset.

Journal: Nature Medicine, November
Title: HIT: a versatile proteomics platform for multianalyte phenotyping of cytokines, intracellular proteins and surface molecules
Authors: MG Kattah; J Coller; RK Cheung; N Oshidary; PJ Utz
HIT, or high-throughput immunophenotyping using transcription, is a multi-analyte fluid-phase protein array technology. It employs a panel of monoclonal antibodies, each tagged with a unique oligonucleotide sequence serving as a molecular bar code. After a sample is tagged, T7 polymerase amplifies the tags, which are then hybridized to a DNA microarray for indirect measurement of each analyte. The technology has many applications, the authors said in the abstract, but in the study report its suitability for profiling cytokines, intracellular molecules, and cell surface markers.

Journal: Proteomics, November
Title: Quantitative protein microarrays for time-resolved measurements of protein phosphorylation
Authors: U Korf; S Derdak; A Tresch; F Henjes; S Schumacher; C Schmidt; B Hahn; WD Lehmann; A Poustka; T Beissbarth; U Klingmüller
Authors have developed a quantitative protein microarray “that monitors the activation of multiple signaling pathways in parallel and at high temporal resolution,” according to the abstract. A label-free sandwich approach was combined with near-infrared detection, enabling the quantitation of low-level phosphoproteins in limited biological samples corresponding to less than 50,000 cells, and with a standard deviation of about 5 percent. A customized software package, Quantpro, was used to help identify suitable antibody pairs by determining their accuracy and dynamic range.

Journal: Proteomics, November
Title: Enhanced detection and identification of multiply phosphorylated peptides using TiO2 enrichment in combination with MALDI TOF/TOF MS
Authors: A Schmidt; E Csaszar; G Ammerer; K Mechtler
Presented is a strategy for the analysis of complex phosphopeptides. The method combines peptide enrichment by titanium dioxide, separation by RP separation on monolithic columns, and mass spectrometry using high energy HE-CAD in a MALDI-TOF/TOF analyzer.

Journal: Proteomics, November
Title: Triplex protein quantification based on stable isotope labeling by peptide dimethylation applied to cell and tissue lysates
Authors: PJ Boersema; TT Aye; TA van Veen ; AJ Heck; S Mohammed
Authors described a “straightforward and cost-effective” triplex quantification method based on stable isotope dimethyl labeling at the peptide level. All proteolytic peptides are chemically labeled at their alpha- and epsilon-amino groups. Three isotopomers of formaldehyde to enable the parallel analysis of three different samples were used.

Journal: Nucleic Acids Research, Oct. 31 [Epub ahead of print]
Title: Sys-BodyFluid: a systematical database for human body fluid proteome research
Authors: SJ Li; M Peng; H Li; BS Liu; C Wang; JR Wu; YX Li; R Zeng
The database described contains 11 kinds of body fluid proteomes, including plasma/serum, urine, cerebrospinal fluid, saliva, human milk, and nipple aspirate fluid. Sys-BodyFluid contains more than 10,000 proteins and provides detailed protein annotations, including protein description, gene ontology, domain information, protein sequence, and involved pathways.

Journal: Analytical Chemistry, Oct. 28 [Epub ahead of print]
Title: On-line digestion system for protein characterization and proteome analysis
Authors: D Loìpez-Ferrer; K Petritis; NM Lourette; B Clowers; KK Hixson; T Heibeck; DC Prior; L Paša-Tolicì; DG Camp; ME Belov; RD Smith
The digestion system proposed reduces the number of sample manipulation steps for high-throughput proteomics, according to the authors. The system incorporates a pressurized sample loop into a LC-based separation system allowing for the simultaneous introduction of both sample and enzyme and producing a complete, rapid digestion. Using their platform coupled to an ion mobility TOF-MS, an ion trap MS or both, the authors digested both standard proteins and a complex Shewanella oneidensis global protein extract. The system denatured, digested, and separated product peptides in minutes, “making it amenable to online, high-throughput applications,” according to the abstract.

Journal: BMC Bioinformatics, Oct. 28 [Epub ahead of print]
Title: A new method for 2D gel spot alignment: application to the analysis of large sample sets in clinical proteomics
Authors: S Peres; L Molina; N Salvetat; C Granier; F Molina
Sili2DGel is presented to improve spot alignment in 2D gel-based work. The algorithm uses data from recursive gel matching and returns spot alignment positions for a given set of gels. The authors applied Sili2DGel to study the variability of the urinary proteome of 20 healthy individuals and concluded it performs “noiseless automatic spot alignment for variability studies (as well as classical differential expression studies) of biological samples,” according to the abstract.

Journal: Journal of Proteome Research, Oct. 28 [Epub ahead of print]
Title: Turnover of the human proteome: Determination of protein intracellular stability by dynamic SILAC
Authors: MK Doherty; DE Hammond; MJ Clague; SJ Gaskell; RJ Beynon
Authors analyzed time-dependent changes in the incorporation of a stable amino acid resolved precursor, a protocol they call “dynamic SILAC,” using 1D gel separation followed by in-gel digestion and LC-MS/MS analyses to profile the intracellular stability of almost 600 proteins from human A549 adenocarcinoma cells “requiring multiple measures of the extent of labeling with stable isotope labeled amino acids in a classic label-chase experiment,” according to the abstract. A profile can be built as turnover rates are acquired, allowing for the exploration of the “dynamic proteome” and of putative features that predispose a protein to a high or low rate of turnover. Measurement of the turnover rate can provide a unique insight into the processes of protein complex assembly and turnover, the authors said in the abstract.

Journal: Journal of Proteome Research, Oct. 28 [Epub ahead of print]
Title: Peptide sequence confidence in accurate mass and time analysis and its use in complex proteomics experiments
Authors: D May; Y Liu; W Law; M Fitzgibbon; H Wang; S Hanash; M McIntosh
Presented are new algorithms and a software implementation for “assigning confidence to peptide sequence assignments obtained through classic accurate mass and retention (ATM) matching techniques,” according to the abstract. Methods for integrating the algorithms and software implementation with standard proteomics workflows are also described. The algorithms are directed at increasing the number identifications of peptides and proteins in high-resolution mass spectrometry studies.

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