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Recent Research Papers of Note: Nov 20, 2009

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Journal:Journal of Proteome Research , Nov. 11 [Epub ahead of print]
Title: Liquid chromatography-tandem and MALDI imaging mass spectrometry analyses of RCL2/CS100-fixed, paraffin-embedded tissues: Proteomics evaluation of an alternative fixative for biomarker discovery
Authors: A Mangé; P Chaurand; H Perrochia; P Roger; RM Caprioli; J Solassol

Described is an investigation of the compatibility of RCL2/CS100, a non-cross-linking, non-toxic, and non-volatile organic fixative, with shotgun proteomic analyses. Protein extraction protocols that are compatible with mass spectrometry and which were investigated include RCL2/CS100-fixed and fresh-frozen colonic mucosa, breast, and prostate tissues. Peptides and proteins identified from RCL2/CS100 were "comprehensively" compared with peptides and proteins identified from matched fresh-frozen tissues using a bottom-up approach based on nano-RPLC-MS/MS. "Results showed that similar peptides could be identified in both archival conditions and the proteome coverage was not obviously compromised by the RCL2/CS100 fixation process," according to the abstract. Performing MALDI tissue profiling and imaging mass spectrometry, the authors observed a "high level of agreement" in protein expression "as well as excellent agreement between the images obtained from RCL2/CS100-fixed and fresh-frozen tissue samples," they said.


Journal:Nucleic Acids Research , Nov. 11 [Epub ahead of print]
Title: The proteomics identifications database: 2010 update
Authors: JA Vizcaíno, F Reisinger; H Barsnes; JM Foster; J Rameseder; H Hermjakob; L Martens

According to the abstract, during the past two years, the PRIDE database has grown substantially and now comprises 60 different species, more than 2.5 million protein identifications, 11.5 million peptides, and more than 50 million spectra as of September. Described are new and improved features in the database, including a revised submission process, which now includes direct submission of fragment ion annotations. Visualization of spectrum fragmentation annotations of tandem MS spectra are also now possible. The authors also describe recent developments in the PRIDE BioMart interface "which now allows integrative queries that can join PRIDE data to a growing number of biological resources such as Reactome, Ensembl, InterPro, and UniProt," according to the abstract.


Journal:Journal of Proteome Research , Nov. 10 [Epub ahead of print]
Title: Combination of FASP and StageTip-based fractionation allows in-depth analysis of the hippocampal membrane proteome
Authors: JR Wiśniewski; A Zougman; M Mann

According to the abstract, membrane proteomics presents challenges due the incompatibility of "desirable strong detergents" with downstream analysis. The authors recently demonstrated the efficient removal of SDS by the filter-aided sample preparation method. Here, they combine FASP with a previously described small-scale membrane enrichment protocol, in which they identified more than 1,000 membrane proteins in a single mouse hippocampus in a single LC-MS/MS run without protein or peptide prefractionation. To extend proteome coverage, they developed a simple anion exchange fractionation method in a StageTip format. Separating peptides into six fractions, a duplicate analysis resulted in the identification of 4,206 proteins, of which 64 percent were membrane proteins. "The combined method provides a streamlined protocol for rapid and sensitive membrane proteome mapping," according to the abstract.


Journal:Journal of Proteome Research , Nov. 9 [Epub ahead of print]
Title: Improved membrane proteomics coverage of human embryonic stem cells by peptide IPG-IEF
Authors: LR McQuade; U Schmidt; D Pascovici; T Stojanov; MS Baker

The authors recently demonstrated the advantages of using peptide isoelectric focusing in the first dimension on immobilized pH gradients followed by reversed-phase chromatography and MS/MS to increase membrane proteome coverage. In the current study, they aim at achieving a similar level of membrane proteome coverage by modifying reported methods and restricting the number of characterized human embryonic stem cells to 107 cells. From 260 micrograms of human embryonic stems cell membrane-enriched fraction, the authors identified 2,292 non-redundant proteins with two or more high-accuracy peptide matches. The false discovery rate was 0.32 percent. Gene ontology-mapping predicted 1,279 to be membrane proteins, of which 395 are predicted to be derived from the plasma membrane compartment.


Journal:Nucleic Acids Research , Nov. 9 [Epub ahead of print]
Title: dbDEPC: a database of differentially expressed proteins in human cancers
Authors: H Li; G Ding; C Wang; L Xie; Y Li

Described is a database of differentially expressed proteins in human cancers "with the goal of collecting curated cancer proteomics data, providing a resource for information on protein-level expression changes, and exploring protein profile differences among different cancers," according to the abstract. The database currently contains 1,803 proteins differentially expressed in 15 cancers, curated from 65 MS-based experiments in peer-reviewed publications. They also integrated data from low-throughput experiments from the same literature and cancer-associated genes from external databases.


Journal:Journal of Proteomics, Nov. 7 [Epub ahead of print]
Title: Combining low-and high-energy tandem mass spectra for optimized peptide quantification with isobaric tags
Authors: L Dayon; C Pasquerello; C Hoogland; JC Sanchez; A Scherl

MS-MS analyses of isobarically labeled peptides with hybrid ion trap Orbitrap instruments have been carried out largely with higher-energy C-trap dissociation or pulsed q dissociation, according to the abstract. While HCD provides good fragmentation of the reporter ions, peptide sequence ion recovery is generally poor compared to collision-induced dissociation, the authors said. Described here is an approach where CID is combined with HCD. The approach, they said, "ensures efficiently both identification and relative quantification of proteins." Tandem mass tags were used to label digests of human plasma and LC-MS/MS was performed with an LTQ-OT platform. Different HCD collision energies were tested, and a program was developed "to merge the peptide sequence ion m/z range from CID spectra and the reporter ion m/z range from HCD spectra, and alternatively to separate both spectral data into different files," according to the abstract.

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Journal: Journal of Visualized Experiments: JoVE, Nov. 6
Title: Digital microfluidics for automated proteomic processing
Authors: MJ Jebrail; VN Luk; SC Shih; R Fobel; AH Ng; H Yang; SL Freire; AR Wheeler

A new digital microfluidics-based method integrating several processing step used in clinical proteomics is reported. Droplets are manipulated on an open space. The droplets are positioned on top of an array of electrodes coated by a dielectric layer. "When an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric," according to the abstract. These charges act as electrostatic handles that can be used to control droplet position. "By biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface," according to the abstract. "Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays."


Journal:Proteomics, Nov. 6 [Epub ahead of print]
Title: An optimized procedure for the capture, fractionation, and proteomic analysis of proteins using hydrogel nanoparticles
Authors: A Rainczuk; K Meehan; DL Steer; PG Stanton; DM Robertson; An Stephens

The method uses dual size inclusion/affinity hydrogel nanoparticles to capture and comparatively analyze low molecule mass proteins directly from biological samples. It facilitates charge- and size-dependent protein binding, direct analysis by mass spectrometry, and other means, and is highly reproducible, according to the abstract.


Journal:Journal of Proteomics , Nov. 4 [Epub ahead of print]
Title: Automated imaging MS: Toward high-throughput imaging mass spectrometry
Authors: LA McDonnell; A van Remoortere; RJ van Zeijl; H Dalebout; MR Bladergroen; AM Deelder

According to the abstract, the low-throughput of imaging mass spectrometry has been a limiting factor in the clinical potential of using the technology to describe tissue by its constituent proteins and peptides and to link this with established histological features. The authors report an automated set-up, "consisting of a controlled environment sample storage chamber, a sample loading robot, and a MALDI TOF/TOF mass spectrometer, all controlled by a single-user interface," they said in the abstract.


Journal:Journal of Proteome Research, Nov. 4 [Epub ahead of print]
Title: MSQuant, an open source platform for mass spectrometry-based quantitative proteomics
Authors: P Mortensen; JW Gouw; JV Olsen; SE Ong; KT Rigbolt; J Bunkenborg; J Cox; L Foster; AJ Heck; B Blagoev; JS Andersen; M Mann

MS Quant is an open source software environment which allows visualization and validation of peptide identifications directly on the raw mass spec data. It iteratively recalibrates MS data, "thereby significantly increasing mass accuracy, leading to fewer false-positive peptide identifications," according to the abstract. Algorithms to improve data quality include and an MS3 score for peptide identification, and a post-translational modification score "that determines the probability that a modification such as phosphorylation is placed at a specific residue in an identified peptide," the authors said.


Journal:Analytical Chemistry, Nov. 2 [Epub ahead of print]
Title: Top-down identification of protein biomarkers in bacteria with unsequenced genomes
Authors: C Wynne; C Fenselau; PA Demirev; N Edwards

The authors apply high resolution MS/MS analysis on an Orbitrap and top-down bioinformatics "to identify major biomarker proteins observed in MALDI spectra of intact bacteria for which little genomic or protein sequence information is available," according to the abstract. "The strategy depends on [the] recognition of proteins with very high homology in related (sequenced) species, making it possible to place unsequenced organisms in their correct phylogenetic context."


Journal:Journal of Biomedicine & Biotechnology, 2010 [Epub Nov. 1]
Title: A proteomic approach for plasma biomarkers discovery with iTRAQ labeling and OFFGEL fractionation
Authors: E Ernoult; A Bourreau; E Gamelin; C Guette

The approach is used in order to handle the high dynamic range of human blood plasma. The strategy combines iTRAQ "as a reagent which improved the peptide ionization and peptide OFFGEl fractionation … to improve the proteome coverage of cellular extracts," according to the abstract. Immunodepletion and a bead-based library of combinatorial hexapeptide technology as prefractionation methods were compared. The resulting data suggested that both methods were complementary in terms of the number of proteins identified.

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Journal:Analytical Chemistry, November
Title: Integrated platform for proteome analysis with combination of protein and peptide separation via online digestion
Authors: H Yuan; L Zhang; C Hou; G Zhu; D Tao; Z Liang; Y Zhang

In the integrated platform described, proteins are first separated by a micro-column packed with mixed weak anion and weak cation exchange particles "under a series of salt steps, online digested by a trypsin immobilized microenzymatic reactor, trapped, and desalted by two parallel C8 precolumns, separated by microreversed-phase liquid chromatography under a linear gradient of organic modifier concentration, and finally identified by electrospray ionization-MS/MS," according to the abstract. The platform was evaluated by analysis of a mixture of myoglobin, cytochrome c, bovine serum albumin, and alpha-casein. "Compared to the methods by off-line protein fractionation and shotgun-based strategy, the analysis time, including sample preparation, digestion, desalting, separation, and detection, was shortened from [approximately] 30 [hours] to 5 [hours], and cytochrome c with abundance of 25 ng could be identified with improved sequence coverage," according to the abstract.


Journal:Journal of Proteome Research, November
Title: Avoiding nonspecific interactions in studies of the plasma proteome: practical solutions to prevention of nonspecific interactions for label-free detection of low-abundance plasma proteins
Authors: JL Richens; EA Lunt; D Sanger; G McKenzie; P O'Shea

Described is an examination of the effect of albumin interference on accurate label-free detection by protein microarrays. "Albumin was found to disrupt specific antigen-antibody binding interactions of low-abundance proteins," according to the abstract. The authors optimized procedures for accurate analysis to be performed without the need for prior treatment of samples. "The emphasis is placed on disrupting nonspecific interactions including both electrostatic (i.e., Colulombic) and electrodynamic (hydrophobic and other nonpolar based) interactions," according to the abstract.


Journal: Rapid Communications in Mass Spectrometry : RCM , November
Title: Precursor ion scans for the targeted detection of stabile-isotope-labeled peptides
Authors: M Colzani; WV Bienvenut; E Faes; M Quadroni

The authors investigated the "specific detection and identification of labeled proteins using acquisition methods based on Precursor Ion Scans on a triple-quadrupole ion trap MS," according to the abstract. PIS methods were set to detect unique immonium ions originating from labeled peptides. Compared with an untargeted analysis on the same instrument, the authors' approach provided a "several-fold increase" in the specificity of detection of labeled proteins over unlabeled proteins. "The technique was applied to the identification of proteins secreted by human cells into growth media containing bovine serum proteins, allowing the preferential detection of labeled cellular proteins over unlabeled bovine ones," according to the abstract. "However, compared with untargeted acquisitions on two different instruments, the PIS-based strategy showed some limitations in sensitivity."


Journal:Electrophoresis, November
Title: De novo peptide sequencing by tandem MS using complementary CID and electron transfer dissociation
Authors: A Bertsch; A Leinenbach; A Pervukhin; M Lubeck; R Hartmer; C Baessmann; YA Elnakady; R Müller; S Böcker; CG Huber; O Kohlbacher

A de novo sequencing algorithm is presented, and a "divide-and-conquer" approach is combined with an efficient mass decomposition algorithm "to exploit the complementary information contained in CID and ETD spectra," according to the abstract. The CompNovo algorithm was applied to the de novo sequencing of peptides derived from the protein extract of Sorangium cellulosum bacteria. The CID and ETD spectra contained 2,406 pairs, of which 675 full correct sequences were assigned, a success rate of 28.1 percent. "It is shown that the CompNovo algorithm yields significantly improved sequencing accuracy as compared with published approaches using only CID spectra or combined CID and ETD spectra," the authors said.


Journal:Nature Biotechnology, Oct. 30 [Epub ahead of print]
Title: Towards proteome scale antibody selections using phase display
Authors: M Mersmann; D Meier; J Mersmann; S Gräsland; S Helmsing; P Nilsson; M Hust; S Dübel

"In vitro antibody generation by panning a large universal gene library with phage display was employed to generate antibodies to over 60 different antigens," according to the abstract. A comparison of pannings on 20 different SH2 domains from the Structural Genomics Consortium was of particular interest. For the SH2 domains, two successive series of selections were done, 2,668 clones were analyzed, resulting in 347 primary hits in ELISA. Half of those hits were further analyzed. and more than 90 difference scFv antibodies to all antigens were identified.


Journal:Journal of Proteome Research, Oct. 26 [Epub ahead of print]
Title: A dynamic range compression and three-dimensional peptide fractionation analysis platform expands proteome coverage and the diagnostic potential of whole saliva
Authors: S Bandhakavi; MD Stone; G Onsongo; SK Van Riper; TJ Griffin

A comprehensive identification of proteins in whole saliva "is critical for appreciating its full diagnostic potential," according to the abstract, but the large dynamic range of proteins in the fluid poses challenges. To address this, the authors present a platform combining hexapeptide libraries for dynamic range compression with 3D peptide fractionation. Using their approach, they identified 2,340 proteins in whole saliva, which "represents the largest saliva proteomic dataset generated using a single analysis platform," they said.

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