Protein Mass Spectrometry Assays

The main method in proteins and biomolecules research as a whole is analytical method of mass spectrometry. Bioproximity operates two state-of-the-art mass spectrometers, a dual pressure linear ion trap and a quadrupole-Orbitrap. Our assays are performed using nano-flow high performance liquid chromatography interfaced via electrospray ionization (ESI). Nanoflow HPLC yields a 10-1,000 fold improvement in sensitivity vs. capillary- or analytical-flow HPLC conducted at microliter per minute flow rates.

All mass spec data analyses are conducted using Proteome Cluster,

Your data is securely stored in redundant locations. Data is analyzed using industry-standard, open source algorithms to guarantee ease of reproducibility. All data components are available to you via Proteome Cluster, peptides sequenced.


For providing protein identification service with the use of our mass spec service and sequencing/characterization of proteins you can visit the following pages: Proteomic Profiling, GeLC-MS/MS analysis and Protein Characterization.

Instrumentation for Biological Mass Spectrometry

Thermo Q-Exactive

Instrumentation for Biological Mass Spectrometry - Thermo Q-Exactive
Unbiased Discovery

This mass spectrometer combines a quadrupole mass filter with an Orbitrap analyzer is able to select ions "virtually instantaneously," fragment peptides via Higher energy Collision Dissociation (HCD) and measure both precusor and fragment ions at high mass resolution and high mass accuracy. In a typical discovery experiment the Q-Exactive performs 10 tandem MS scans per second, with precursor mass resolution of 70,000 (at 200 m/z), described here (1).

Targeted Proteomic Quantification

The Q-Exactive not only excels at global shotgun proteomics, discovery-style assays, it delivers "similar or better performances in terms of selectivity, dynamic range, and sensitivity" (2) compared to the standard selected reaction monitoring (SRM) technique, until now the gold standard for targeted proteomic assays. Two modes are available for targeted assays. Single ion monitoring (SIM) takes advantage of the high resolution and mass accuracy (HR/AM) capabilities of the instrument to permit accurate quantitation of peptides in low- to medium-complexity mixtures with limits of quantitation comparable to SRM in the low attomole range. Parallel reaction monitoring (PRM) offers increased specificity with a minimal loss of sensitivity by quantifying the fragment ions. The multiplexing capabilities of the instrument permit accurate, sensitive quantitation of hundreds of peptides in a short, one-hour LC-PRM-MS/MS assay.

Ultra-High Pressure Liquid Chromatography (UHPLC)
Instrumentation for Biological Mass Spectrometry - Ultra-High Pressure Liquid Chromatography (UHPLC)
We have interfaced a Thermo Easy-nLC 1000 UHPLC to the Q-Exactive via the EASY Spray heated, nano-electrospray ionization source. The combination of UHPLC with a source with integrated column heater permits use of very long, 50 cm columns with very small, 2 micron diameter chromatographic media. As a result, very long gradients can be run which still maintain very high chromatographic resolution, meaning narrow peptide peak widths and high peptide concentrations at the point of electrospray ionization. A single four-hour LC-MS/MS assay utilizing this setup permits identification of about 4,000 protein groups from about one microgram of peptide digest (3). This depth of coverage is sufficient for many applications, including complete or nearly complete coverage of simple eukaryotes such as yeast and most prokaryotes.

Thermo LTQ Velos

Instrumentation for Biological Mass Spectrometry - Thermo LTQ Velos

The LTQ Velos is a dual-pressure linear ion trap mass spectrometer (4). We have interfaced it with a Thermo Easy-nLC II HPLC for high resolution chromatographic separations at nanoliter per minute flow rates for optimal sensitivity. The Velos utilizes the same S-lens as the Q-Exactive to focus the ion cloud as it enters the mass spectrometer and improve overall sensitivity. The linear ion trap enables very fast sequencing speed of 15 MS/MS scans in 1.8 seconds. Collision induced fragmentation (CID) is used to fragment peptides for sequence analysis. CID produces peptide ladders from both N- and C-terminal ends of the peptide, allowing for complementary and confirmatory sequence interpretation resulting in high confidence assignments.

Links of Interest

  1. Q-Exactive - quadrupole-Orbitrap mass spectrometer
  2. Easy-nLC 1000 - ultra-high pressure, nano-flow liquid chromatograph
  3. EASY Spray - heated, nano-electrospray ionization ion source

References

  1. Michalski, A., Damoc, E., Hauschild, J.-P., Lange, O., Wieghaus, A., Makarov, A., Nagaraj, N., Cox, J., Mann, M., Horning, S., Jun. 2011.
    Mass spectrometry-based proteomics using q exactive, a high-performance benchtop quadrupole orbitrap mass spectrometer. Molecular & Cellular Proteomics.
    DOI
  2. Gallien, S., Duriez, E., Crone, C., Kellmann, M., Moehring, T., Domon, B., Dec. 2012.
    Targeted proteomic quantification on quadrupole-orbitrap mass spectrometer. Molecular & cellular proteomics : MCP 11 (12), 1709-1723.
    DOI
  3. Wiśniewski, J. R., Duś, K., Mann, M., Apr. 2013.
    Proteomic workflow for analysis of archival formalin-fixed and paraffin-embedded clinical samples to a depth of 10 000 proteins. Prot. Clin. Appl. 7 (3-4), 225-233.
    DOI
  4. Second, T. P. P., Blethrow, J. D., Schwartz, J. C., Merrihew, G. E., MacCoss, M. J., Swaney, D. L., Russell, J. D., Coon, J. J., Zabrouskov, V., Sep. 2009.
    Dual-pressure linear ion trap mass spectrometer improving the analysis of complex protein mixtures. Analytical chemistry 81 (18), 7757-7765.
    DOI