Technologies

The instrumentation at Nextgen Sciences includes LTQ, hybrid LTQ-Orbitrap and Quantum Ultra triple quadrupole mass spectrometers (Hyperlink Thermo-Finnigan mass spec page). The LTQ and LTQ-Orbitrap instruments are part of the peptide/protein identification and label/label free quantitation workflows. The LTQ is a fast scanning linear ion trap mass spectrometer capable of acquiring low resolution data that can be used to determine peptide/protein sequence and sites of post-translational modification. The LTQ-Orbitrap is a hybrid instrument that couples that fast scanning capabilities of the LTQ with the high resolution mass measurement of the Orbitrap mass analyzer. The high resolution of the Orbitrap mass anlayzer can be used to measure with great accuracy precursor ion, and if desired product ion mass to charge ratios (m/z). The accurate mass measurement capability of the Orbitrap mass analyzer significantly increases the confidence in the assignment of peptide sequence information. The data from the Orbitrap is ideally suited to the determination of hitherto unknown peptide sequences using de novo sequencing methods.

When coupled with bioinformatics tools the data from the LTQ and the LTQ-Orbitrap form the core of our high-through-put protein sequencing capabilities. The Quantum Ultra is a triple quadrupole mass spectrometer used for MRM assay development and sample testing. The unique design of the Quantum ultra mass analyzers enables acquisition of data in a high resolution mode thus reducing interferences when acquiring data for multiple MRM transitions. All our mass spectrometers are interfaced with nano scale chromatography using either Waters or Agilent systems that can be configured to enable several modes of chromatography. The LC and MS instruments are coupled using nanospray ionization typically performed at a flow rate of several hundred nanolitres/minute.

Ref: "Performance of a linear ion trap-orbitrap hybrid for peptide analysis" J.R. Yates, D. Cociorva, L. Liao, V. Zabrouskov. Analytical Chemistry, 2006, 78, 493-500.

Ref: "Quantitative, multiplexed Assays for Low Abundance Proteins in Plasma by Targeted Mass Spectrometry and Stable Isotope Dilution" H. Keshishian, T. Addona, M. Burgess, E. Kuhn and S.A. Carr. Molecular and Cellular Proteomics 2007, 6, 2212-2229.

Protein level separations of complex mixtures are achieved using multiple modes of chromatography including Size Exclusion Chromatography (SEC), Strong Cation Exchange (SCX), Hydrophobic Liquid Interaction Liquid Chromatography (HILIC) and Electrostatic Repulsion-Hydrophilic Interaction Chromatography (ERLIC). These modes of chromatography can be hyphenated with mass spectrometry or used in an off-line manner for preparative fractionation or purification.

Ref: "Hydrophilic Interaction Chromatography Reduces the Complexity of the Phosphproteome and Improves Global Phosphopeptide Isolation and Detection" D.E. McNulty and R.S. Annan. Molecular and Cellular Proteomics, 7, 971-980, 2008

Ref: "Protein Quantitation through Targeted Mass Spectrometry: The Way Out of Biomarker Purgatory" S. A. Carr and L. Anderson. Clinical Chemistry, 2008, 54, 1749-1752.

Gel electrophoresis is a useful means of reducing sample complexity and/or removing common biological reagents that can interfere with chemical analysis. As a quantitative tool 2D electrophoresis provides a simple means of evaluating the differences between different samples where limited information is available on the sample e.g. no sequenced genome.

Depletion technology is used to remove high abundance proteins from biological samples. As a result of the overwhelmingly high abundance of certain proteins in biological samples, such as albumin in plasma, analysis by LC/MS of low abundance proteins can be hindered. Depletion of the high abundance proteins is a method for providing access to the lower abundance proteins in a sample. There are several commercial products available for depletion of different degrees of abundant proteins.

Solid phase extraction (SPE) is a common tool for the removal of interferences prior to LC/MS analysis. We have adopted several SPE strategies for clean-up of tryptic digests prior to phosphoproteomic studies or as part of quantitative workflows.

Immuno-purification is a method for enriching low level target proteins or peptides from a complex sample prior to LC-MS/MS analysis. The specific interaction between an antibody and an antigen is used to enrich the target antigen. Poor antibody specificity can be negated by the high selectivity of the mass spectrometer.

Ref: "Differences among techniques for high-abundant protein depletion" N. Zolotarjova, J. Martosella, G. Nicol, J. Bailey, B.E. Boyes, W.C. Barrett. Proteomics 2005, 5, 3304-3313.

Ref: "Strategic use of Immunoprecipitation and LC/MS/MS for Trace-Level Protein Quantitation: Myosin Light Chain 1, a Biomarker of Cardiac Necrosis" M.J. Berna, Y. Zhen, D.E. Watson, J.E. Hale, B.L. Ackermann. Analytical Chemistry, 2007, 79, 4199-4205.

We primarily utilize the Mascot and SEQUEST search engines for mass spectrometry database searching, and the Scaffold analysis software for post-processing and dataset assembly. The Thermo Scientific SIEVE software is utilized for dMS analysis, the LCQuan software for quantitative data assembly and reporting, and the prototype SRM Workflow software to assist in development of targeted assays. Project-centric statistical analysis, reporting, and data visualization solutions are developed utilizing proprietary .NET and Python code libraries, with results stored in SQLServer 2008 relational databases. A number of other openly-available software tools and resources are used for specific tasks, including the Trans-Proteomic Pipeline (TPP) tools, the ArrayTrack™ statistical analysis package, and the R statistical programming language.