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Using targeted quantification (PRM) to quantify proteins between wild type Col and acinus-2 pinin-1 mutant with 15N metabolic labeling

ABSTRACT: ACINUS and PININ genes regulate transcription and alternative splicing (Bi et al., 2021) and we hypothesize that some proteins should have altered expression in the double mutant. Therefore, MS experiments were designed to identify these altered proteins on a global scale using 15N metabolic labeling. Some of these altered proteins were subjected to targeted quantification.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Plant Cell, Whole Body

SUBMITTER: Shouling Xu

LAB HEAD: Shouling Xu

PROVIDER: PXD030096 | Pride | 2022-04-19

REPOSITORIES: Pride

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Quantitative proteomic comparison between wild type Col and acinus-2 pinin-1 mutant using 15N metabolic labeling

Project description:ACINUS and PININ genes regulate transcription and alternative splicing (Bi et al., 2021) and we hypothesize that some proteins should have altered expression in the double mutant. Therefore, MS experiments were designed to identify these altered proteins on a global scale using 15N metabolic labeling.

2022-02-04 | PXD030081 | Pride

Parallel Reaction Monitoring of 42 peptide pairs with 15N metabolic labeling

Project description:To examine the properties of 14N and 15N labeled peptides from 15 metabolic labeling samples, we set up targeted quantification using Parallel Reaction Monitoring (PRM) on a high resolution, high accuracy mass spectrometer. We first examined the co-elution of light and heavy peptide forms. Furthermore, we examined the fragment spectra of the light and heavy peptides and compared the pattern of the fragments.

2022-06-09 | PXD030112 | Pride

Quantitative proteomic comparison between wild type Col and sec-5 mutant using 15N metabolic labeling

Project description:SECRET AGENT (SEC) is an O-GlcNAc transferase involved in O-GlcNAcylation of many proteins. However, sec-5 null allele mutant phenotype is very subtle. To examine if there are proteins that show altered expression in the sec-5 mutant compared to wild-type Col, we designed MS experiments to identify these candidates on a global scale using 15N metabolic labeling.

2022-02-04 | PXD030575 | Pride

Relative quantification of 15N labeled acinus-2 pinin-1 and 14N labeled Col through Data Dependent Acquisition to obtain peptide information for targeted Parallel Reaction Monitoring experiment

Project description:To examine the properties of 14N and 15N labeled peptides using Parallel Reaction Monitoring (PRM) on a high resolution, high accuracy mass spectrometer we first set up this DDA experiment. The purpose of this experiment was to choose peptides that are good candidates for targeted quantification. The retention times, mass, and charge from 42 candidate peptides were acquired for targeted quantification.

2022-06-09 | PXD030098 | Pride

15N Stable-isotope-labeling in Arabidopsis (SILIA) quantitative MS analysis of the AtACINUS interactome

Project description:AtACINUS protein is involved in regulation of alternative transcription and splicing(AS). Identifying interaction partners and protein complex compositions for AtACINUS can produce valuable information on the mechanisms by which they regulate transcription and AS, as well as post-translational modifications on AtACINUS. A homozygous 35S::AtACINUS-GFP/acinus-2 plant was selected for similar protein expression level to the endogenous AtACINUS protein of wild-type plants using a native α-AtACINUS antibody. We isolated putative AtACINUS interaction partners from young Arabidopsis seedlings using the native α-AtACINUS antibody. Plants expressing TAP-GFP under 35S promoter were used as controls.

2020-12-02 | PXD020700 | Pride

Proteomic comparison of Chlamydomonas wildtype and calredoxin knock-down mutants via 14N/15N metabolic labeling

Project description:Calcium (Ca2+) and redox signaling play important roles in acclimation processes from archaea to eukaryotic organisms. Herein we characterized a unique protein from Chlamydomonas reinhardtii that has the competence to integrate Ca2+- and redox-related signaling. This protein, designated as calredoxin (CRX), combines four Ca2+-binding EF-hands and a thioredoxin (TRX) domain. A crystal structure of CRX, at 1.6 Å resolution, revealed an unusual calmodulin-fold of the Ca2+-binding EF-hands, which is functionally linked via an inter-domain communication path with the enzymatically active TRX domain. CRX is chloroplast-localized and interacted with a chloroplast 2-Cys-peroxiredoxin (PRX1). Ca2+-binding to CRX is critical for its TRX activity and for efficient binding and reduction of PRX1. Thereby, CRX represents a new class of Ca2+-dependent "sensor-responder" proteins. Genetically engineered Chlamydomonas strains with strongly diminished amounts of CRX revealed altered photosynthetic electron transfer and were affected in oxidative stress response underpinning a function of CRX in stress acclimation.

2016-10-12 | PXD003759 | Pride

A type 2C protein phosphatase activates high-affinity nitrate uptake by directly dephosphorylating NRT2.1 in Arabidopsis

Project description:Nitrate transporter NRT2.1, which plays a central role in high-affinity nitrate uptake in roots, is activated at the post-translational level in response to nitrogen (N) starvation. However, critical enzymes required for post-translational activation of NRT2.1 remain to be identified. Here, we show that a type 2C protein phosphatase, designated CEPD-induced phosphatase (CEPH), activates high-affinity nitrate uptake by directly dephosphorylating S501 of NRT2.1, a residue that functions as a negative phospho-switch. CEPH is predominantly expressed in epidermal and cortex cells in roots and up-regulated by N starvation via a CEPDL2/CEPD1/2-mediated long-distance signaling from shoots. Loss of CEPH leads to a marked decrease in high-affinity nitrate uptake, tissue nitrate content, and plant biomass. Collectively, our results identify CEPH as a crucial enzyme in N starvation-dependent activation of NRT2.1, providing molecular and mechanistic insights into how plants regulate high-affinity nitrate uptake at the post-translational level in response to the N environment.

2021-03-02 | PXD022343 | Pride

Thylakoid membranes from Arabidopsis LC-MSMS

Project description:The small heat shock protein (sHsp) chaperones are crucial for cell survival and can prevent aggregation of client proteins that partially unfold under destabilizing conditions. Most investigations on the chaperone activity sHsps are based on a limited set of thermosensitive model substrate client proteins since the endogenous targets are often not known. There is a high diversity among sHsps, with a single conserved -sandwich fold domain defining the family, the α-crystallin domain, whereas the N-terminal and C-terminal regions are highly variable in length and sequence among various sHsps and conserved only within orthologues. The endogenous targets are probably also varying among various sHsps, cellular compartments, cell type and organism. Here we have investigated Hsp21, a non-metazoan sHsp expressed in the chloroplasts in green plants, which experience huge environmental fluctuations not least in temperature. We describe how Hsp21 can also interact with the chloroplast thylakoid membranes, both when isolated thylakoid membranes are incubated with Hsp21 protein and when plants are heat-stressed. The amount of Hsp21 associated with the thylakoid membranes was precisely determined by quantitative mass spectrometry after metabolic 15N-isotope labelling, of either recombinantly expressed and purified Hsp21 protein or intact Arabidopsis thaliana plants. We found that Hsp21 is among few proteins that become associated with the thylakoid membranes in heat-stressed plants, and that approximately two thirds of the pool of chloroplast Hsp21 is affected. We conclude that for a complete picture of the role of sHsps in plant stress resistance also their association with the membranes should be considered

2017-06-21 | PXD006734 | Pride

Chlamydomonas reinhardtii quantification of Mannosidase 1A and Xylosyltransferase1A

Project description:We have identified insertional mutants of mannosidase 1A and xylosyltransferase 1A in Chlamydomonas reinhardtii. To verify a knockout/knockdown of the enzymes in the insertional mutant strains, PRM measurements were performed. Mass spectrometric analyses of intact N-glycopeptides revealed that disruption of the genes altered the N-glycan composition of the alga (in single as well as double mutant).

2018-02-27 | PXD005257 | Pride

Transcription profiling of yeast wild type and ggc1-delta strains after treatment with rapmycin

Project description:This experiment was a 4-part timeseries (or, 2x2x4 factorial design) designed to provide additional biological insight into the role of ggc1 in the rapamycin response of Saccharomyces cerevisiae. Treated and untreated cultures were compared at 0, 1, 2, and 4 hours of growth, in both WT and ggc1-delta backgrounds.

2010-01-07 | E-TABM-638 | biostudies-arrayexpress

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