Assessing the Relationship Between Mass Window Width and Retention Time Scheduling on Protein Coverage for Data-Independent Acquisition

Li, Wenxue; Chi, Hao; Šalovská, Barbora; Wu, Chongde; Sun, Liangliang; Rosenberger, George; Liu, Yansheng

doi:10.1007/s13361-019-02243-1

Cited by 33 publications

(50 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For both SW948 and RKO cell lines, the SILAC RPMI-1640 media lacking L-Arginine, L-Lysine (Thermo Scientific, # 88365) was used instead, with the same basic configuration. The Heavy L-Arginine-HCl (13C6, 15N4, purity >98%, #CCN250P1), and L-Lysine-2HCl (13C6, 15N2, purity >98%, # CCN1800P1) were purchased from Cortecnet and spiked into the culturing medium in the same manner as described previously for DMEM or RPMI medium 26,48 . Before…”

Section: Pulsed Silac Experimentmentioning

confidence: 99%

“…Label-free and labeled cells of HeLa cells, including HeLa_7 and HeLa_8, SW948, RKO, were harvested and digested, mainly as previously described 48,49 . Cells were washed three times by precooled PBS, harvested, and snap-frozen by liquid nitrogen.…”

Section: Deltasilac Proteomic Sample Preparationmentioning

confidence: 99%

“…After harvesting all dishes, samples of HeLa_7 and HeLa_8, as well as samples from SW948 and RKO, were processed for tryptic digestion. The cell pellets were ultrasonically lysed by sonication at 4 °C for 2 min using a VialTweeter device (Hielscher-Ultrasound Technology) 25,48,49 and then centrifuged at 18,000 × g for 1 hour to remove the insoluble material. A total of 700 μg supernatant proteins (determined by BioRad Bradford assay) were transferred to clean Eppendorf tubes.…”

Section: Deltasilac Proteomic Sample Preparationmentioning

confidence: 99%

“…For each proteomic and phosphoproteomic sample generated by DeltaSILAC, DIA-MS analysis was performed on 1 μg of peptides, as described previously 30,48 .…”

Section: Dia Mass Spectrometrymentioning

confidence: 99%

“…DIA-MS data analyses were performed using Spectronaut v13 51,52 . For both proteomic and phosphoproteomic measurements, the hybrid assay libraries were respectively generated, which were based on both DIA measurements as well as data-dependent acquisitions (DDA) on relevant samples method described previously 48 ), as well as the optimized pSILAC-DIA workflow 25,27 . To generate the library for pSILAC DIA-MS datasets, the default settings for Pulsar search of Spectronaut was used with modification in the Labeling setting: a) "Labelling Applied" option was enabled, b) SILAC labels ("Arg10" and "Lys8") were specified in the second channel.…”

Section: Mass Spectrometry Data Analysesmentioning

confidence: 99%

See 4 more Smart Citations

Global impact of phosphorylation on protein endurance

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

Post-translational modifications such as phosphorylation can have profound effects on the physicochemical and biological properties of proteins. However, high-throughput and systematic approaches have not yet been developed to assess the effects of specific modification types and sites on protein lifetime, which represents a key parameter for understanding signaling rewiring and drug development. Here we describe a proteomic method, DeltaSILAC, to quantify the impact of site-specific phosphorylation on the endurance of thousands of proteins in live cells. Being configured on the reproducible data-independent acquisition mass spectrometry (DIA-MS), the pulse labeling approach using stable isotope-labeled amino acids in cells (SILAC), together with a novel peptide-level matching strategy, this multiplexed assay revealed the global delaying effect of phosphorylation on protein turnover in growing cancer cells. Further, we identified local sequence and structural features in proximity to the phosphorylated sites that could be associated with protein endurance alterations. We found that phosphorylated sites accelerating protein turnover are functionally selected for cell fitness and evolutionarily conserved. DeltaSILAC provides a generalizable approach for prioritizing the effects of phosphorylation sites on protein lifetime in the context of cell signaling and disease biology, which is highly complementary to existing methods. Finally, DeltaSILAC is widely applicable to diverse posttranslational modification types and different cell systems.

show abstract

Section: Pulsed Silac Experimentmentioning

confidence: 99%

Section: Deltasilac Proteomic Sample Preparationmentioning

confidence: 99%

Section: Deltasilac Proteomic Sample Preparationmentioning

confidence: 99%

“…For each proteomic and phosphoproteomic sample generated by DeltaSILAC, DIA-MS analysis was performed on 1 μg of peptides, as described previously 30,48 .…”

Section: Dia Mass Spectrometrymentioning

confidence: 99%