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TMT Quantification for Shotgun Proteomics on a Trapped-Ion-Mobility quadrupole-Time-Of- Flight mass spectrometer (TIMS-QTOF) powered by Parallel-Accumulation and Serial- Fragmentation (PASEF)
EP30381
Poster Title: TMT Quantification for Shotgun Proteomics on a Trapped-Ion-Mobility quadrupole-Time-Of- Flight mass spectrometer (TIMS-QTOF) powered by Parallel-Accumulation and Serial- Fragmentation (PASEF)
Submitted on 24 Jul 2019
Author(s): Matt Willetts1, Shourjo Ghose1 Heiner Koch2, Scarlet Koch2, Markus Lubeck2, Gary Kruppa1
Affiliations: 1Bruker Daltonics, Billerica, MA, USA 2Bruker Daltonik GmbH, 28359 Bremen, Germany
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Poster Information
Abstract: To investigate the performance of the timsTOF Pro for isobaric labelling experiments Flag-HA-ERCC8 was tandem affinity purified, aliquoted in ratios 10:4:1:1:4:10, and TMT labeled. To provide a background, mock Flag IP eluates from HeLa were aliquoted in equal ratios and labeled with TMT 126, 127, and 128.
ERCC8 IP peptides and mock Flag IP peptides were combined, desalted, and dried. Peptides were separated by nano-HPLC (nanoElute, Bruker Daltonics) on a 250 mm pulled emitter column (IonOpticks, Australia) and analyzed on a high resolution TIMS enabled QTOF instrument using the PASEF method (timsTOF Pro, Bruker Daltonics). 200 ng of sample were separated on 90 min, gradient (2-30% ACN). A PASEF cycle of 2.2 s was used equating to a 100 msMS scan followed by ten 100 msMSMS cycles each fragmenting up to 12 precursors. Feature extraction, database searching and quantitation were performed in PEAKS 8.5 (Bioinformatics Solutions Inc.)
Summary: Here we evaluate the performance of a unique dual trapped ion mobility spectrometer (TIMS) coupled to a QTOF (timsTOF Pro) for the analysis of isobaric labeling experiments. TIMS provides an additional dimension of separation and higher peak capacity which, when operated with the Parallel Accumulation Serial Fragmentation (PASEF) method, allows for operation at nearly 100 % duty cycle with very high sequencing speeds.References: Protein complex samples provided courtesy of Marto Lab, Dana-Farber Cancer Institute, Boston, MA.Report abuse »
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