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Higher Resolution Improves Top-Down Protein Identification Results on an Orbitrap Mass Spectrometer for Large (>40 kDa) Proteins

Reputable Mentor II
Reputable Mentor II
Horn DM, Viner R, Sharma S, Denisov E, Makarov A.
ASMS 2012 Poster
Purpose: We will demonstrate in this poster that high mass resolution (>200,000) is necessary for ultimate confidence in the identification of proteins above 40 kDa. A comparison between various resolution settings for both direct infusion and LC-MS/MS data are shown. Methods: Yeast enolase (47 kDa) was injected into a hybrid ion trap-Orbitrap mass spectrometer via direct infusion and electron transfer dissociation (ETD) MS/MS spectra were acquired at 60,000, 120,000, 240,000, and 480,000 resolution. Yeast enolase was also separated on a PLRP-S column and injected into the same mass spectrometer and HCD MS/MS data were acquired at 60,000, 120,000, and 240,000 resolution. Bovine serum albumin (66 kDa) was also introduced to the mass spectrometer via direct infusion and both higher-energy collisional dissociation (HCD) and ETD MS/MS spectra were acquired at 240,000 and 480,000 resolution. All data were reduced to monoisotopic masses using THRASH1 and identified using software optimized for analysis of intact protein data. Results: For the direct infusion data for enolase, the 240,000 resolution ETD data produced both the most sequence coverage as well as the best expectation value. This was primarily due to the resolution of peaks from those from other isotopic clusters as well as the low-abundance background chemical noise. For the LC-MS/MS data, the 120,000 resolution produced the most unique identifications primarily for proteins below 20 kDa. However, the 240,000 resolution data produced far more hits for intact enolase, primarily due to the improved success in the measurement of the precursor mass. In all, 4 different gene products were identified from the yeast enolase sample, including both enolase 1 (46642 Da) and enolase 2 (46754 Da). The 240,000 resolution data was the only dataset where both intact enolase forms were identified. Finally, intact BSA was fragmented using ETD at 480,000 resolution and the improved resolution led to additional sequence coverage for the HCD and ETD data.

Thermo Fisher Scientific
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‎10-15-2021 12:07 PM
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