Abstract

Protein ubiquitylation is involved in a plethora of cellular processes. While antibodies directed at ubiquitin remnants (K-ɛ-GG) have improved the ability to monitor ubiquitylation using mass spectrometry, methods for highly multiplexed measurement of ubiquitylation in tissues and primary cells using sub-milligram amounts of sample remains a challenge. Here, we present a highly sensitive, rapid and multiplexed protocol termed UbiFast for quantifying ~10,000 ubiquitylation sites from as little as 500 μg peptide per sample from cells or tissue in a TMT10plex in ca. 5 h. High-field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) is used to improve quantitative accuracy for posttranslational modification analysis. We use the approach to rediscover substrates of the E3 ligase targeting drug lenalidomide and to identify proteins modulated by ubiquitylation in models of basal and luminal human breast cancer. The sensitivity and speed of the UbiFast method makes it suitable for large-scale studies in primary tissue samples.

Comprehensive protein ubiquitylation profiling by mass spectrometry typically requires large sample amounts, limiting its applicability to tissue samples. Here, the authors present an optimized proteomics method that enables multiplexed ubiquitylome analysis of cells and tumor tissue samples.

Details

Title
Rapid and deep-scale ubiquitylation profiling for biology and translational research
Author
Udeshi, Namrata D 1   VIAFID ORCID Logo  ; Mani, Deepak C 1 ; Satpathy Shankha 1   VIAFID ORCID Logo  ; Fereshetian Shaunt 1 ; Gasser, Jessica A 2 ; Svinkina Tanya 1 ; Olive, Meagan E 1 ; Ebert, Benjamin L 3   VIAFID ORCID Logo  ; Mertins Philipp 4   VIAFID ORCID Logo  ; Carr, Steven A 1   VIAFID ORCID Logo 

 Broad Institute of MIT and Harvard, Cambridge, USA (GRID:grid.66859.34) 
 Broad Institute of MIT and Harvard, Cambridge, USA (GRID:grid.66859.34); Brigham and Women’s Hospital, Division of Hematology, Boston, USA (GRID:grid.62560.37) (ISNI:0000 0004 0378 8294); Dana-Farber Cancer Institute, Department of Medical Oncology, Boston, USA (GRID:grid.65499.37) (ISNI:0000 0001 2106 9910) 
 Broad Institute of MIT and Harvard, Cambridge, USA (GRID:grid.66859.34); Brigham and Women’s Hospital, Division of Hematology, Boston, USA (GRID:grid.62560.37) (ISNI:0000 0004 0378 8294); Dana-Farber Cancer Institute, Department of Medical Oncology, Boston, USA (GRID:grid.65499.37) (ISNI:0000 0001 2106 9910); Dana-Farber Cancer Institute, Howard Hughes Medical Institute, Boston, USA (GRID:grid.65499.37) (ISNI:0000 0001 2106 9910) 
 Broad Institute of MIT and Harvard, Cambridge, USA (GRID:grid.66859.34); Max Delbrück Center for Molecular Medicine in the Helmholtz Society, Berlin, Germany (GRID:grid.419491.0) (ISNI:0000 0001 1014 0849); Berlin Institute of Health, Berlin, Germany (GRID:grid.484013.a) 
Publication year
2020
Publication date
2020
Publisher
Nature Publishing Group
e-ISSN
20411723
Source type
Scholarly Journal
Language of publication
English
ProQuest document ID
2343269982
Copyright
This work is published under http://creativecommons.org/licenses/by/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.