24 research outputs found
Benchmarking common quantification strategies for large-scale phosphoproteomics
Quantitative phosphoproteomics has become a standard method in molecular and cell biology. Here, the authors compare performance and parameters of phosphoproteome quantification by LFQ, SILAC, and MS2-/MS3-based TMT and introduce a TMT-adapted algorithm for calculating phosphorylation site stoichiometry
Alternative Translation Initiation Generates a Functionally Distinct Isoform of the Stress-Activated Protein Kinase MK2
A Compact Quadrupole-Orbitrap Mass Spectrometer with FAIMS Interface Improves Proteome Coverage in Short LC Gradients
Causal integration of multi-omics data with prior knowledge to generate mechanistic hypotheses.
Multi-omics datasets can provide molecular insights beyond the sum of individual omics. Various tools have been recently developed to integrate such datasets, but there are limited strategies to systematically extract mechanistic hypotheses from them. Here, we present COSMOS (Causal Oriented Search of Multi-Omics Space), a method that integrates phosphoproteomics, transcriptomics, and metabolomics datasets. COSMOS combines extensive prior knowledge of signaling, metabolic, and gene regulatory networks with computational methods to estimate activities of transcription factors and kinases as well as network-level causal reasoning. COSMOS provides mechanistic hypotheses for experimental observations across multi-omics datasets. We applied COSMOS to a dataset comprising transcriptomics, phosphoproteomics, and metabolomics data from healthy and cancerous tissue from eleven clear cell renal cell carcinoma (ccRCC) patients. COSMOS was able to capture relevant crosstalks within and between multiple omics layers, such as known ccRCC drug targets. We expect that our freely available method will be broadly useful to extract mechanistic insights from multi-omics studies
Regulation of the golgi apparatus by p38 and JNK kinases during cellular stress responses
p38 and c-Jun N-terninal kinase (JNK) are activated in response to acute stress and inflammatory signals. Through modification of a plethora of substrates, these kinases profoundly re-shape cellular physiology for the optimal response to a harmful environment and/or an inflammatory state. Here, we utilized phospho-proteomics to identify several hundred substrates for both kinases. Our results indicate that the scale of signaling from p38 and JNK are of a similar magnitude. Among the many new targets, we highlight the regulation of the transcriptional regulators grb10-interacting GYF protein 1 and 2 (GIGYF1/2) by p38-dependent MAP kinase-activated protein kinase 2 (MK2) phosphorylation and 14â3â3 binding. We also show that the Golgi apparatus contains numerous substrates, and is a major target for regulation by p38 and JNK. When activated, these kinases mediate structural rearrangement of the Golgi apparatus, which positively affects protein flux through the secretory system. Our work expands on our knowledge about p38 and JNK signaling with important biological ramifications
Alternative translation initiation generates a functionally distinct isoform of the stress-activated kinase MK2
Proteomic investigation of Cbl and Cbl-b in neuroblastoma cell differentiation highlights roles for SHP-2 and CDK16
Integrated proximal proteomics reveals IRS2 as a determinant of cell survival in ALK-driven neuroblastoma
Oncogenic anaplastic lymphoma kinase (ALK) is one of the few druggable targets in neuroblastoma, and therapy resistance to ALK-targeting tyrosine kinase inhibitors (TKIs) comprises an inevitable clinical challenge. Therefore, a better understanding of the oncogenic signaling network rewiring driven by ALK is necessary to improve and guide future therapies. Here, we performed quantitative mass spectrometry-based proteomics on neuroblastoma cells treated with one of three clinically relevant ALK TKIs (crizotinib, LDK378, or lorlatinib) or an experimentally used ALK TKI (TAE684) to unravel aberrant ALK signaling pathways. Our integrated proximal proteomics (IPP) strategy included multiple signaling layers, such as the ALK interactome, phosphotyrosine interactome, phosphoproteome, and proteome. We identified the signaling adaptor protein IRS2 (insulin receptor substrate 2) as a major ALK target and an ALK TKI-sensitive signaling node in neuroblastoma cells driven by oncogenic ALK. TKI treatment decreased the recruitment of IRS2 to ALK and reduced the tyrosine phosphorylation of IRS2. Furthermore, siRNA-mediated depletion of ALK or IRS2 decreased the phosphorylation of the survival-promoting kinase Akt and of a downstream target, the transcription factor FoxO3, and reduced the viability of three ALK-driven neuroblastoma cell lines. Collectively, our IPP analysis provides insight into the proximal architecture of oncogenic ALK signaling by revealing IRS2 as an adaptor protein that links ALK to neuroblastoma cell survival through the Akt-FoxO3 signaling axis
Performance Evaluation of the Q Exactive HFâX for Shotgun Proteomics
Progress in proteomics
is mainly driven by advances in mass spectrometric
(MS) technologies. Here we benchmarked the performance of the latest
MS instrument in the benchtop Orbitrap series, the Q Exactive HF-X,
against its predecessor for proteomics applications. A new peak-picking
algorithm, a brighter ion source, and optimized ion transfers enable
productive MS/MS acquisition above 40 Hz at 7500 resolution. The hardware
and software improvements collectively resulted in improved peptide
and protein identifications across all comparable conditions, with
an increase of up to 50 percent at short LCâMS gradients, yielding
identification rates of more than 1000 unique peptides per minute.
Alternatively, the Q Exactive HF-X is capable of achieving the same
proteome coverage as its predecessor in approximately half the gradient
time or at 10-fold lower sample loads. The Q Exactive HF-X also enables
rapid phosphoproteomics with routine analysis of more than 5000 phosphopeptides
with short single-shot 15 min LCâMS/MS measurements, or 16âŻ700
phosphopeptides quantified across ten conditions in six gradient hours
using TMT10-plex and offline peptide fractionation. Finally, exciting
perspectives for data-independent acquisition are highlighted with
reproducible identification of 55âŻ000 unique peptides covering
5900 proteins in half an hour of MS analysis