Rictor Phosphorylation on the THR-1135 Site Does Not Require Mammalian Target of Rapamycin Complex 2

available in PMC 2012 January 1.In animal cells, growth factors coordinate cell proliferation and survival by regulating the phosphoinositide 3-kinase/Akt signaling pathway. Deregulation of this signaling pathway is common in a variety of human cancers. The PI3K-dependent signaling kinase complex defined as mammalian target of rapamycin complex 2 (mTORC2) functions as a regulatory Ser-473 kinase of Akt. We find that activation of mTORC2 by growth factor signaling is linked to the specific phosphorylation of its component rictor on Thr-1135. The phosphorylation of this site is induced by the growth factor stimulation and expression of the oncogenic forms of ras or PI3K. Rictor phosphorylation is sensitive to the inhibition of PI3K, mTOR, or expression of integrin-linked kinase. The substitution of wild-type rictor with its specific phospho-mutants in rictor null mouse embryonic fibroblasts did not alter the growth factor–dependent phosphorylation of Akt, indicating that the rictor Thr-1135 phosphorylation is not critical in the regulation of the mTORC2 kinase activity. We found that this rictor phosphorylation takes place in the mTORC2-deficient cells, suggesting that this modification might play a role in the regulation of not only mTORC2 but also the mTORC2-independent function of rictor. Mol Cancer Res; 8(6); 896–906.University of Texas M.D. Anderson Cancer Center (Fellow Trust fund)American Cancer Society (M.D. Anderson Cancer Center Breast Specialized Programs of Research Excellence (RSG-09-026-01CCG01))National Institutes of Health (U.S.) (NIH grant CA133522)National Institutes of Health (U.S.) (NIH grant AI104389

A large peptidome dataset improves HLA class I epitope prediction across most of the human population

Published in final edited form as: Nat Biotechnol. 2020 February ; 38(2): 199–209. doi:10.1038/s41587-019-0322-9.Prediction of HLA epitopes is important for the development of cancer immunotherapies and vaccines. However, current prediction algorithms have limited predictive power, in part because they were not trained on high-quality epitope datasets covering a broad range of HLA alleles. To enable prediction of endogenous HLA class I-associated peptides across a large fraction of the human population, we used mass spectrometry to profile >185,000 peptides eluted from 95 HLA-A, -B, -C and -G mono-allelic cell lines. We identified canonical peptide motifs per HLA allele, unique and shared binding submotifs across alleles and distinct motifs associated with different peptide lengths. By integrating these data with transcript abundance and peptide processing, we developed HLAthena, providing allele-and-length-specific and pan-allele-pan-length prediction models for endogenous peptide presentation. These models predicted endogenous HLA class I-associated ligands with 1.5-fold improvement in positive predictive value compared with existing tools and correctly identified >75% of HLA-bound peptides that were observed experimentally in 11 patient-derived tumor cell lines.P01 CA229092 - NCI NIH HHS; P50 CA101942 - NCI NIH HHS; T32 HG002295 - NHGRI NIH HHS; T32 CA009172 - NCI NIH HHS; U24 CA224331 - NCI NIH HHS; R21 CA216772 - NCI NIH HHS; R01 CA155010 - NCI NIH HHS; U01 CA214125 - NCI NIH HHS; T32 CA207021 - NCI NIH HHS; R01 HL103532 - NHLBI NIH HHS; U24 CA210986 - NCI NIH HHSAccepted manuscrip

Multi-site assessment of the precision and reproducibility of multiple reaction monitoring–based measurements of proteins in plasma

Verification of candidate biomarkers relies upon specific, quantitative assays optimized for selective detection of target proteins, and is increasingly viewed as a critical step in the discovery pipeline that bridges unbiased biomarker discovery to preclinical validation. Although individual laboratories have demonstrated that multiple reaction monitoring (MRM) coupled with isotope dilution mass spectrometry can quantify candidate protein biomarkers in plasma, reproducibility and transferability of these assays between laboratories have not been demonstrated. We describe a multilaboratory study to assess reproducibility, recovery, linear dynamic range and limits of detection and quantification of multiplexed, MRM-based assays, conducted by NCI-CPTAC. Using common materials and standardized protocols, we demonstrate that these assays can be highly reproducible within and across laboratories and instrument platforms, and are sensitive to low µg/ml protein concentrations in unfractionated plasma. We provide data and benchmarks against which individual laboratories can compare their performance and evaluate new technologies for biomarker verification in plasma

Deep proteomics identifies shared molecular pathway alterations in synapses of patients with schizophrenia and bipolar disorder and mouse model

Summary: Synaptic dysfunction is implicated in the pathophysiology of schizophrenia (SCZ) and bipolar disorder (BP). We use quantitative mass spectrometry to carry out deep, unbiased proteomic profiling of synapses purified from the dorsolateral prefrontal cortex of 35 cases of SCZ, 35 cases of BP, and 35 controls. Compared with controls, SCZ and BP synapses show substantial and similar proteomic alterations. Network analyses reveal upregulation of proteins associated with autophagy and certain vesicle transport pathways and downregulation of proteins related to synaptic, mitochondrial, and ribosomal function in the synapses of individuals with SCZ or BP. Some of the same pathways are similarly dysregulated in the synaptic proteome of mutant mice deficient in Akap11, a recently discovered shared risk gene for SCZ and BP. Our work provides biological insights into molecular dysfunction at the synapse in SCZ and BP and serves as a resource for understanding the pathophysiology of these disorders

Automated Microchromatography Enables Multiplexing of Immunoaffinity Enrichment of Peptides to Greater than 150 for Targeted MS-Based Assays

Immunoaffinity enrichment of peptides coupled with analysis by stable isotope dilution multiple reaction mass spectrometry has been shown to have analytical performance and detection limits suitable for many biomarker verification studies and biological applications. Prior studies have shown that antipeptide antibodies can be multiplexed up to 50 in a single assay without significant loss of performance. Achieving higher multiplex levels is relevant to all studies involving precious biological material as this minimizes the amount of sample that must be consumed to measure a given set of analytes and reduces the assay cost per analyte. Here we developed automated methods employing the Agilent AssayMAP Bravo microchromatography platform and used these methods to characterize the performance of immunoaffinity enrichment of peptides up to multiplex levels of 172. Median capture efficiency for the target peptides remained high (88%) even at levels of 150-plex and declined to 70% at 172-plex compared to antibody performance observed at standard lower multiplex levels (<i>n</i> = 25). Subsequently, we developed and analytically characterized a multiplexed immuno-multiple reaction monitoring-mass spectrometry (immuno-MRM-MS) assay (<i>n</i> = 110) and applied it to measure candidate protein biomarkers of cardiovascular disease in plasma of patients undergoing planned myocardial infarction. The median lower limit of detection of all peptides was 71.5 amol/μL (nM), and the coefficient of variation (CV) was less than 15% at the lower limit of quantification. The results demonstrate that high multiplexed immuno-MRM-MS assays are readily achievable using the optimized sample processing and peptide capture methods described here