Data-independent acquisition method for ubiquitinome analysis reveals regulation of circadian biology
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Data-independent acquisition method for ubiquitinome analysis reveals regulation of circadian biology. / Hansen, Fynn M; Tanzer, Maria C; Brüning, Franziska; Bludau, Isabell; Stafford, Che; Schulman, Brenda A; Robles, Maria S; Karayel, Ozge; Mann, Matthias.
In: Nature Communications, Vol. 12, No. 1, 11.01.2021, p. 254.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Data-independent acquisition method for ubiquitinome analysis reveals regulation of circadian biology
AU - Hansen, Fynn M
AU - Tanzer, Maria C
AU - Brüning, Franziska
AU - Bludau, Isabell
AU - Stafford, Che
AU - Schulman, Brenda A
AU - Robles, Maria S
AU - Karayel, Ozge
AU - Mann, Matthias
PY - 2021/1/11
Y1 - 2021/1/11
N2 - Protein ubiquitination is involved in virtually all cellular processes. Enrichment strategies employing antibodies targeting ubiquitin-derived diGly remnants combined with mass spectrometry (MS) have enabled investigations of ubiquitin signaling at a large scale. However, so far the power of data independent acquisition (DIA) with regards to sensitivity in single run analysis and data completeness have not yet been explored. Here, we develop a sensitive workflow combining diGly antibody-based enrichment and optimized Orbitrap-based DIA with comprehensive spectral libraries together containing more than 90,000 diGly peptides. This approach identifies 35,000 diGly peptides in single measurements of proteasome inhibitor-treated cells - double the number and quantitative accuracy of data dependent acquisition. Applied to TNF signaling, the workflow comprehensively captures known sites while adding many novel ones. An in-depth, systems-wide investigation of ubiquitination across the circadian cycle uncovers hundreds of cycling ubiquitination sites and dozens of cycling ubiquitin clusters within individual membrane protein receptors and transporters, highlighting new connections between metabolism and circadian regulation.
AB - Protein ubiquitination is involved in virtually all cellular processes. Enrichment strategies employing antibodies targeting ubiquitin-derived diGly remnants combined with mass spectrometry (MS) have enabled investigations of ubiquitin signaling at a large scale. However, so far the power of data independent acquisition (DIA) with regards to sensitivity in single run analysis and data completeness have not yet been explored. Here, we develop a sensitive workflow combining diGly antibody-based enrichment and optimized Orbitrap-based DIA with comprehensive spectral libraries together containing more than 90,000 diGly peptides. This approach identifies 35,000 diGly peptides in single measurements of proteasome inhibitor-treated cells - double the number and quantitative accuracy of data dependent acquisition. Applied to TNF signaling, the workflow comprehensively captures known sites while adding many novel ones. An in-depth, systems-wide investigation of ubiquitination across the circadian cycle uncovers hundreds of cycling ubiquitination sites and dozens of cycling ubiquitin clusters within individual membrane protein receptors and transporters, highlighting new connections between metabolism and circadian regulation.
KW - Circadian Rhythm/physiology
KW - HEK293 Cells
KW - Humans
KW - Peptide Library
KW - Proteome/metabolism
KW - Proteomics
KW - Reproducibility of Results
KW - Signal Transduction
KW - Tumor Necrosis Factor-alpha/metabolism
KW - Ubiquitin/metabolism
KW - Ubiquitination
U2 - 10.1038/s41467-020-20509-1
DO - 10.1038/s41467-020-20509-1
M3 - Journal article
C2 - 33431886
VL - 12
SP - 254
JO - Nature Communications
JF - Nature Communications
SN - 2041-1723
IS - 1
ER -
ID: 259829170