Accurate Proteome-wide Label-free Quantification by Delayed Normalization and Maximal Peptide Ratio Extraction, Termed MaxLFQ

Cox, Jürgen; Hein, Marco Y.; Luber, Christian A.; Paron, Igor; Nagaraj, Nagarjuna; Mann, Matthias

doi:10.1074/mcp.m113.031591

Cited by 4,222 publications

(4,083 citation statements)

References 76 publications

Supporting

Mentioning

4,030

Contrasting

Unclassified

Order By: Relevance

“…Data-dependent LC-MS/MS analysis of extracted peptide mixtures after digestion was carried out on a Fusion tri-hybrid Orbitrap mass spectrometer (Thermo Fisher Scientific). Data from all five fractions for each sample were pooled and analyzed to identify peptides (at 1% FDR) and obtain Label-Free Quantitation (LFQ) with the software MaxQuant as previously described 5 . Data analysis and annotation was done with the Perseus package 30 and GraphPad Prism.…”

Section: Label Free Quantitative (Ms-lfq) Analyses By Mass Spectrometrymentioning

confidence: 99%

Translocon component Sec62 acts in endoplasmic reticulum turnover during stress recovery

et al. 2016

View full text Add to dashboard Cite

The endoplasmic reticulum (ER) is a site of protein biogenesis in eukaryotic cells. Perturbing ER homeostasis activates stress programs collectively called the unfolded protein response (UPR). The UPR enhances production of ER-resident chaperones and enzymes to reduce the burden of misfolded proteins. On resolution of ER stress, ill-defined, selective autophagic programs remove excess ER components. Here we identify Sec62, a constituent of the translocon complex regulating protein import in the mammalian ER, as an ER-resident autophagy receptor. Sec62 intervenes during recovery from ER stress to selectively deliver ER components to the autolysosomal system for clearance in a series of events that we name recovER-phagy. Sec62 contains a conserved LC3-interacting region in the C-terminal cytosolic domain that is required for its function in recovER-phagy, but is dispensable for its function in the protein translocation machinery. Our results identify Sec62 as a critical molecular component in maintenance and recovery of ER homeostasis. DOI: https://doi.org/10.1038/ncb3423Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-127515 Accepted Version Originally published at: Fumagalli, Fiorenza; Noak, Julia; Bergmann, Timothy J; Presmanes, Eduardo Cebollero; Pisoni, Giorgia Brambilla; Fasana, Elisa; Fregno, Ilaria; Galli, Carmela; Loi, Marisa; Solda, Tatiana; D'Antuono, Rocco; Raimondi, Andrea; Jung, Martin; Melnyk, Armin; Schorr, Stefan; Schreiber, Anne; Simonelli, Luca; Varani, Luca; Wilson-Zbinden, Caroline; Zerbe, Oliver; Hofmann, Kay; Peter, Matthias; Quadroni, Manfredo; Zimmermann, Richard; Molinari, Maurizio (2016 To define mechanisms that regulate the return of ER-resident chaperones and folding factors to their physiologic intracellular level after resolution of an ER stress, we established a protocol for reversible induction of UPR in cultured mammalian cells (Fig. 1a). Briefly, human embryonic kidney cells (HEK293) or mouse embryonic fibroblasts (MEF) were exposed for 12 h to non-toxic doses of cyclopiazonic acid (CPA), a reversible inhibitor of the sarco/endoplasmic reticulum calcium pump 6 . The return of ER-resident gene products at their pre-stress level was monitored during resolution of the UPR obtained upon CPA wash out ( CPA wash out initiated a recovery phase characterized by the rapid return of ER stress-induced transcripts at, or below, their pre-stress levels (Fig. 1b, recovery, T 1/2 average ≈ 1 h, blue line). The corresponding ER stress-induced proteins returned to their physiologic levels with much slower kinetics (Fig. 1c, d, T 1/2 average ≈ 10 h, blue). 3With the exception of Herp, which is rapidly turned over with intervention of proteasomes (Fig. 1c, d (Fig. 1g, 2a) and other membrane and luminal ER marker proteins such as Sec62 and Crt ( Fig. 2b and Extended data Fig. 3) in 0.5-1.5 µm diameter cytoplasmic puncta that rapidly disappeared upon BafA1 wash out (Extended data Fig. 4). Cytosolic puncta containing ER marker prot...

show abstract

Section: Label Free Quantitative (Ms-lfq) Analyses By Mass Spectrometrymentioning

confidence: 99%

Translocon component Sec62 acts in endoplasmic reticulum turnover during stress recovery

et al. 2016

View full text Add to dashboard Cite

show abstract

“…S1). LFQ values were normalized to adjust for any differences introduced as a result of sample handling or fractionation using the delayed normalization approach in maxquant (Cox et al ., 2014). Correlation plots for LFQ intensities revealed strong correlation between biological replicates within treatments, suggesting successful sample loading and normalization (Fig.…”

Section: Resultsmentioning

confidence: 99%

Quantitative analysis of the tomato nuclear proteome during Phytophthora capsici infection unveils regulators of immunity

et al. 2017

View full text Add to dashboard Cite

Summary Plant–pathogen interactions are complex associations driven by the interplay of host and microbe‐encoded factors. With secreted pathogen proteins (effectors) and immune signalling components found in the plant nucleus, this compartment is a battleground where susceptibility is specified. We hypothesized that, by defining changes in the nuclear proteome during infection, we can pinpoint vital components required for immunity or susceptibility.We tested this hypothesis by documenting dynamic changes in the tomato (Solanum lycopersicum) nuclear proteome during infection by the oomycete pathogen Phytophthora capsici. We enriched nuclei from infected and noninfected tissues and quantitatively assessed changes in the nuclear proteome. We then tested the role of candidate regulators in immunity through functional assays.We demonstrated that the host nuclear proteome dynamically changes during P. capsici infection. We observed that known nuclear immunity factors were differentially expressed and, based on this observation, selected a set of candidate regulators that we successfully implicated in immunity to P. capsici.Our work exemplifies a powerful strategy to gain rapid insight into important nuclear processes that underpin complex crop traits such as resistance. We have identified a large set of candidate nuclear factors that may underpin immunity to pathogens in crops.

show abstract

“…The match between runs option was also enabled with a match time window of 0.7 min and an alignment time window of 20 min. Relative, label‐free quantification of proteins was performed using the MaxLFQ algorithm (Cox et al., 2014) integrated into MaxQuant. The parameters were as follows: Minimum ratio count was set to 1, the FastLFQ option was enabled, LFQ minimum number of neighbors was set to 3, and the LFQ average number of neighbors was set to 6, as per default.…”

Section: Methodsmentioning

confidence: 99%

Oxidation resistance 1 is a novel senolytic target

et al. 2018

View full text Add to dashboard Cite

SummaryThe selective depletion of senescent cells (SCs) by small molecules, termed senolytic agents, is a promising therapeutic approach for treating age‐related diseases and chemotherapy‐ and radiotherapy‐induced side effects. Piperlongumine (PL) was recently identified as a novel senolytic agent. However, its mechanism of action and molecular targets in SCs was unknown and thus was investigated. Specifically, we used a PL‐based chemical probe to pull‐down PL‐binding proteins from live cells and then mass spectrometry‐based proteomic analysis to identify potential molecular targets of PL in SCs. One prominent target was oxidation resistance 1 (OXR1), an important antioxidant protein that regulates the expression of a variety of antioxidant enzymes. We found that OXR1 was upregulated in senescent human WI38 fibroblasts. PL bound to OXR1 directly and induced its degradation through the ubiquitin‐proteasome system in an SC‐specific manner. The knockdown of OXR1 expression by RNA interference significantly increased the production of reactive oxygen species in SCs in conjunction with the downregulation of antioxidant enzymes such as heme oxygenase 1, glutathione peroxidase 2, and catalase, but these effects were much less significant when OXR1 was knocked down in non‐SCs. More importantly, knocking down OXR1 selectively induced apoptosis in SCs and sensitized the cells to oxidative stress caused by hydrogen peroxide. These findings provide new insights into the mechanism by which SCs are highly resistant to oxidative stress and suggest that OXR1 is a novel senolytic target that can be further exploited for the development of new senolytic agents.

show abstract

Accurate Proteome-wide Label-free Quantification by Delayed Normalization and Maximal Peptide Ratio Extraction, Termed MaxLFQ

Cited by 4,222 publications

References 76 publications

Translocon component Sec62 acts in endoplasmic reticulum turnover during stress recovery

Translocon component Sec62 acts in endoplasmic reticulum turnover during stress recovery

Quantitative analysis of the tomato nuclear proteome during Phytophthora capsici infection unveils regulators of immunity

Oxidation resistance 1 is a novel senolytic target

Contact Info

Product

Resources

About