CPMSC Facility: Instruments
LTQ-Orbitrap Velos is a high accuracy dual cell linear trap combined with the high resolution Orbitrap mass analyzer from ThermoFisher Scientific. This provides excellent speed and reliability for the identification of samples while minimizing the false detection rate (FDR). The ultra-high resolution provides certainty in analytical results by enabling molecular weight determination for intact proteins and in-depth analysis of isobaric species with HCD and CID fragmentation capability.
The components of this include:
• Ion source: The CPMSC facility has two ion sources, the nanospray ESI and HESI
• S-lens: A stacked ion ring to guide the ions into a narrow tight beam using RF
• Orbitrap: Performs as a mass analyzer and detector. The ions in the Orbitrap are retained by electric field trapping, and varying RF is applied to separate the ions based on their oscillation frequencies. Due to this setup, the mass accuracy and resolution of the Orbitrap can be compared to an FT-ICR, as both techniques are frequency-based measurement.
The perform characteristics are
• Mass resolution of 60,000 at m/z 400 at a scan rate of 1 Hz, minimum resolution 7,500 maximum resolution >100,000 at m/z 400
• Mass accuracy of < 10 ppm with external calibration and optimization, < 1 ppm using internal calibration
The Orbitrap has a wide range of inlet capabilities, the samples can be introduced by direct infusion or through a LC. The LTQ-Orbitrap at the facility is coupled to a Waters nano-Acquity UPLC to achieve minimum waste generation and reproducible separations. It is also possible to perform two dimensional separations to increase the depth of coverage. The first dimension is an Ion Exchange column with a silica matrix of particle size 5 µm, column length 20 mm, internal dimensions 180 µm and pH range 2-7. The second dimension is a C18 column with a silica matrix of particle size 5 µm, column length 20 mm, internal dimensions 180 µm and pH range 2-8.
The data from the Orbitrap is analyzed using the Proteome Discoverer software. PD uses a node based workflow for a wide range of proteomics workflows, from protein and peptide identification to PTM analysis.
Synapt G2-Si from Waters is a high-definition MS system that uses the extra dimension of ion mobility separations to maximize selectivity and confidence in results by combining step wave with QuanTOF and tri-wave technologies. Synapt G2-Si delivers high sensitivity with its step wave ion optics technology and axis design, which actively eliminates neutral contaminants. Using the data independent MSe acquisition and the high resolution QuanTOF mass analyzer, facility staff can carry out comprehensive identification and quantification of compounds at very low concentration. The ion mobility efficiently separates ions on the basis of size, shape, and charge before transferring them to QuanTOF for high resolution mass analysis. Using the Synapt in Ion Mobility Mode, HDMSe allows the addition of an orthogonal method of separation based on drift time, reducing the sample complexity and allowing increase of the dynamic range of the instrument. Extremely complex samples analyzed using this method show an increased level of peptide and protein identifications, costing only a bit more time and sample.
The Synapt is coupled with a Waters M-class Acquity system for highly reproducible chromatographic separations. The UPLC column is C18 with a particle size 5 µm, column length 20 mm, internal dimensions 180 µm and pH range 2-8.
The data from the Synapt are analyzed using the PLGS algorithm or Progenesis QI based on the service requested.
Our newest instrument is the 5800 MALDI TOF-TOF from AB Sciex. The MALDI is capable of generating data from samples which have limited solubility in water or organic solvents, and it is more robust at handling samples with salts or detergents. It can be used for basic mass determination of small molecules, peptides, or intact proteins, as well as other macromolecules. It also has fragmentation capability either by post-source decay or an internal CID cell, yielding fragmentation information which aids in structure elucidation. It can be coupled with an off-line nanoLC and automated spotter robot for proteomics experiments, and we are also in the process of gathering information on the use of this instrument for such advanced projects as tissue imaging.
The Mass Spec Core has various pieces of equipment handy for sample prep, including an incubator, a SpeedVac, and a capillary laser puller. For more information on ancillary equipment, please contact the Mass Spec Core staff.