Recent advances in native mass spectrometry (MS) and denatured intact protein MS have made these techniques essential for biotherapeutic characterization. As MS analysis has increased in throughput and scale, new data analysis workflows are needed to provide rapid quantitation from large datasets. Here, we describe the UniDec processing pipeline (UPP) for the analysis of batched biotherapeutic intact MS data. UPP is built into the UniDec software package, which provides fast processing, deconvolution, and peak detection. The user and programming interfaces for UPP read a spreadsheet that contains the data file names, deconvolution parameters, and quantitation settings. After iterating through the spreadsheet and analyzing each file, it returns a spreadsheet of results and HTML reports. We demonstrate the use of UPP to measure the correct pairing percentage on a set of bispecific antibody data and to measure drug-to-antibody ratios from antibody-drug conjugates. Moreover, because the software is free and open-source, users can easily build on this platform to create customized workflows and calculations. Thus, UPP provides a flexible workflow that can be deployed in diverse settings and for a wide range of biotherapeutic applications.
Data Analysis , Software , Mass Spectrometry/methods , Workflow
KRAS, which is mutated in â¼30% of all cancers, activates the RAF-MEK-ERK signaling cascade. CRAF is required for growth of KRAS mutant lung tumors, but the requirement for CRAF kinase activity is unknown. Here, we show that subsets of KRAS mutant tumors are dependent on CRAF for growth. Kinase-dead but not dimer-defective CRAF rescues growth inhibition, suggesting that dimerization but not kinase activity is required. Quantitative proteomics demonstrates increased levels of CRAF:ARAF dimers in KRAS mutant cells, and depletion of both CRAF and ARAF rescues the CRAF-loss phenotype. Mechanistically, CRAF depletion causes sustained ERK activation and induction of cell-cycle arrest, while treatment with low-dose MEK or ERK inhibitor rescues the CRAF-loss phenotype. Our studies highlight the role of CRAF in regulating MAPK signal intensity to promote tumorigenesis downstream of mutant KRAS and suggest that disrupting CRAF dimerization or degrading CRAF may have therapeutic benefit.
Carcinogenesis/metabolism , Dimerization , Proto-Oncogene Proteins p21(ras)/metabolism , Animals , Carcinogenesis/drug effects , Cell Line, Tumor , Humans , MAP Kinase Signaling System/drug effects , Mice , Phosphorylation/physiology , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins B-raf/genetics , Signal Transduction/drug effects , Signal Transduction/physiology , ras Proteins/genetics
The ubiquitin conjugating enzyme UBE2W catalyzes non-canonical ubiquitination on the N-termini of proteins, although its substrate repertoire remains unclear. To identify endogenous N-terminally-ubiquitinated substrates, we discover four monoclonal antibodies that selectively recognize tryptic peptides with an N-terminal diglycine remnant, corresponding to sites of N-terminal ubiquitination. Importantly, these antibodies do not recognize isopeptide-linked diglycine (ubiquitin) modifications on lysine. We solve the structure of one such antibody bound to a Gly-Gly-Met peptide to reveal the molecular basis for its selective recognition. We use these antibodies in conjunction with mass spectrometry proteomics to map N-terminal ubiquitination sites on endogenous substrates of UBE2W. These substrates include UCHL1 and UCHL5, where N-terminal ubiquitination distinctly alters deubiquitinase (DUB) activity. This work describes an antibody toolkit for enrichment and global profiling of endogenous N-terminal ubiquitination sites, while revealing functionally relevant substrates of UBE2W.
Antibodies/chemistry , Peptides/chemistry , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitinated Proteins/metabolism , Amino Acid Sequence , Animals , Antibodies/immunology , Cells, Cultured , Crystallography, X-Ray/methods , Humans , Mass Spectrometry/methods , Protein Binding , Proteomics/methods , Rabbits , Ubiquitin-Conjugating Enzymes/chemistry , Ubiquitin-Conjugating Enzymes/immunology , Ubiquitination
Neddylation is the post-translational protein modification most closely related to ubiquitination. Whereas the ubiquitin-like protein NEDD8 is well studied for its role in activating cullin-RING E3 ubiquitin ligases, little is known about other substrates. We developed serial NEDD8-ubiquitin substrate profiling (sNUSP), a method that employs NEDD8 R74K knock-in HEK293 cells, allowing discrimination of endogenous NEDD8- and ubiquitin-modification sites by MS after Lys-C digestion and K-εGG-peptide enrichment. Using sNUSP, we identified 607 neddylation sites dynamically regulated by the neddylation inhibitor MLN4924 and the de-neddylating enzyme NEDP1, implying that many non-cullin proteins are neddylated. Among the candidates, we characterized lysine 112 of the actin regulator cofilin as a novel neddylation event. Global inhibition of neddylation in developing neurons leads to cytoskeletal defects, altered actin dynamics and neurite growth impairments, whereas site-specific neddylation of cofilin at K112 regulates neurite outgrowth, suggesting that cofilin neddylation contributes to the regulation of neuronal actin organization.
Actins/metabolism , Cofilin 1/metabolism , NEDD8 Protein/metabolism , Neurons/metabolism , Animals , Cell Line , Cells, Cultured , Gene Knock-In Techniques , HEK293 Cells , Humans , Mice , Mice, Inbred C57BL , NEDD8 Protein/genetics , Neurons/cytology , Point Mutation , Rats , Rats, Sprague-Dawley , Ubiquitin/metabolism , Ubiquitination
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
