Fall into Proteomics webinar series, Day 2 - Quantitative Proteomics

Presented By: Dustin Frost, PhD & Joao A. Paulo, PhD, MMS Speaker Biography: Dustin Frost received his Bachelor’s Degree in Chemistry and his Ph.D. in Pharmaceutical Sciences from the University of Wisconsin–Madison. During his graduate and post-doctoral studies, he designed, synthesized, and applied novel isobaric chemical tags for highly multiplexed quantitative proteomics on Thermo Scientific™ Orbitrap™ LC-MS platforms. In 2021, Dustin joined R&D at Thermo Fisher Scientific in Rockford, IL where he leads the development of Thermo Scientific™ TMTpro reagents and related technologies for high-throughput quantitative proteomics. Joao Paulo received his Master of Medical Sciences in Clinical Investigation from Harvard Medical School and his Ph.D. in Biochemistry from Brown University. Currently, Joao is an Instructor in Cell Biology at Harvard Medical School and serves as the Co-Director of the Thermo Fisher Center for Multiplexed Proteomics. His research focuses primarily on mass spectrometry-based applications aimed at understanding molecular mechanisms and cell signaling pathways. He created the Triple Knockout (TKO) standard, a diagnostic tool used to address ion interference in isobaric labeling experiments. He continues to develop, optimize, and promote TMT-based workflows for efficient and high-throughput profiling of proteome and post-translational modifications and through numerous collaborations. Webinar: Fall into Proteomics webinar series, Day 2 - Quantitative Proteomics Webinar Abstract: Expanding TMTpro reagents to 32-plex for high-throughput quantitative proteomics on Orbitrap platforms Dustin Frost, Ph.D. Tandem Mass Tag™ (TMT™) reagents enable simultaneous identification and relative quantification of thousands of proteins from many samples in a single LC-MS/MS acquisition on Thermo Scientific™ Orbitrap™ platforms. This seminar will highlight the benefits of multiplexing for high-throughput quantitative proteomics applications and introduce the latest advancement in multiplex isobaric tagging technology, the Thermo Scientific™ TMTpro 32plex reagent set. To nearly double the multiplexing of the TMTpro reagent, we developed an additional set of structurally identical isotopologues that substitute one 2H stable isotope into the reporter group to impart a 3 mDa mass difference between the new variants and the existing 18-plex set of tags. We determined the Orbitrap resolving power required to distinguish reporter ions in MS/MS spectra, benchmarked performance for bulk proteome analysis at higher throughput with fewer missing values, characterized chromatographic behavior of the deuterated variants, and established approaches to maximize quantitative performance using the expanded reagent set. For Research Use Only. Not for use in diagnostic procedures. TMT and Tandem Mass Tag are trademarks of Proteome Sciences. Expanding Quantitative Proteome Profiling with TMTpro 32-plex Sample Multiplexing Joao A. Paulo, PhD, MMS Mass spectrometry-based sample multiplexing with isobaric tags permits the development of high-throughput and precise quantitative biological assays with proteome-wide coverage and minimal missing values. Here, we nearly doubled the multiplexing capability of the TMTpro reagent set to up to 35-plex through the incorporation of a single deuterium isotope into the reporter group. We acknowledge that substituting deuterium frequently results in suboptimal peak co-elution which can compromise the accuracy of reporter ion-based quantification. To counteract the deuterium effect on quantitation, we implemented a strategy that necessitated the segregation of non-deuterium and deuterium-containing channels into distinct sub-plexes during normalization procedures with reassembly through a common bridge channel. We highlight several applications of the expanded TMTpro reagent set throughout its development. TMT and Tandem Mass Tag are trademarks of Proteome Sciences. For Research Use Only. Not for use in diagnostic procedures. Labroots on Social: Facebook:   / labrootsinc   Twitter:   / labroots   LinkedIn:   / labroots   Instagram:   / labrootsinc   Pinterest:   / labroots   SnapChat: labroots_inc