Biological 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, the industry-leading, cloud-based platform for shotgun proteomic data analysis. We utilize multiple search algorithms, the use of which has repeatedly been shown to be both confirmatory and complementary, to provide maximum sensitivity while preserving specificity.
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, from raw mass spectrometer binary files, to peak list files, search parameter files, sequence libraries, search output files, parsed files and final reports. We believe you should be able to take your data and hand it to your local bioinformatic expert who should be able to reproduce our analytical data flow with minimal effort. Profiling reports consist of MS Excel spreadsheets detailing protein groups and relative expression values, peptides sequenced, sequencing events, and protein to peptide mappings. PDF reports include instructions on accessing and interacting with native data files, a glossary to aid in data interpretation and a full methods write-up suitable for publication in a peer-reviewed journal.
Instrumentation for Biological Mass Spectrometry
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), fragment mass resolution of 17,500 and low ppm mass accuracy. These capabilities allow the Q-Exactive to identify thousands of peptides mapping to over one thousand protein groups from less than one microgram of total protein in a standard two-hour LC-MS/MS assay. Because of the rapid sampling rate coupled with the high mass resolution and mass accuracy measurements, accurate quantitation is possible at the peptide level by measuring the MS1 peak areas for all identified peptides. Instrument design and capabilities are 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)
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
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
- Q-Exactive - quadrupole-Orbitrap mass spectrometer
- Easy-nLC 1000 - ultra-high pressure, nano-flow liquid chromatograph
- EASY Spray - heated, nano-electrospray ionization ion source
- 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. URL http://dx.doi.org/10.1074/mcp.m111.011015
- 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. URL http://dx.doi.org/10.1074/mcp.o112.019802
- 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. URL http://dx.doi.org/10.1002/prca.201200046
- 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. URL http://dx.doi.org/10.1021/ac901278y