Abstract
Successful high-throughput characterization of intact proteins from complex biological samples by mass spectrometry requires instrumentation capable of high mass resolving power, mass accuracy, sensitivity, and spectral acquisition rate. These limitations often necessitate the performance of hundreds of LC-MS/MS experiments to obtain reasonable coverage of the targeted proteome, which is still typically limited to molecular weights below 30 kDa. The National High Magnetic Field Laboratory (NHMFL) recently installed a 21 T FT-ICR mass spectrometer, which is part of the NHMFL FT-ICR User Facility and available to all qualified users. Here we demonstrate top-down LC-21 T FT-ICR MS/MS of intact proteins derived from human colorectal cancer cell lysate. We identified a combined total of 684 unique protein entries observed as 3238 unique proteoforms at a 1% false discovery rate, based on rapid, data-dependent acquisition of collision-induced and electron-transfer dissociation tandem mass spectra from just 40 LC-MS/MS experiments. Our identifications included 372 proteoforms with molecular weights over 30 kDa detected at isotopic resolution, which substantially extends the accessible mass range for high-throughput top-down LC-MS/MS.
Original language | English (US) |
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Pages (from-to) | 1087-1096 |
Number of pages | 10 |
Journal | Journal of Proteome Research |
Volume | 16 |
Issue number | 2 |
DOIs | |
State | Published - Feb 3 2017 |
Funding
We thank the Hunt Laboratory at the University of Virginia and Thermo Fisher Scientific (San Jose, CA) for help with ETD hardware and software and Chad Weisbrod for helpful discussion. This work was supported by the National Science Foundation Cooperative Agreement No. DMR-1157490 and the State of Florida as well as by the National Institute of General Medical Science (P41GM108569) for the National Resource for Translational and Developmental Proteomics (NRTDP) based at Northwestern University. Further support was provided by the computational resources and staff for the Quest high performance computing facility at Northwestern University, which is jointly supported by the Office of the Provost, the Office for Research, and Northwestern University Information Technology.
Keywords
- 21 tesla
- FT-ICR
- FTMS
- top-down proteomics
ASJC Scopus subject areas
- General Chemistry
- Biochemistry