Analysis of protein complexes in Arabidopsis leaves using size exclusion chromatography and label-free protein correlation profiling

Aryal, Uma K.; McBride, Zachary; Chen, Donglai; Xie, Jun; Szymanski, Daniel B.

doi:10.1016/j.jprot.2017.06.004

Cited by 50 publications

(89 citation statements)

References 94 publications

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“…SEC samples were analyzed by LC-MS/MS as described by Aryal et al , 2017 ( 55 ). Briefly, using an Eksigent nano-LC 425 HPLC (Dublin, CA), 5 μl of peptides were resolved over a 90 min gradient from 0 to 35% acetonitrile in 0.1% FA prior to analysis on an AB Sciex quadrupole time-of-flight (QqTOF) TripleTOF 5600, (Framingham, MA).…”

Section: Methodsmentioning

confidence: 99%

Global Analysis of Membrane-associated Protein Oligomerization Using Protein Correlation Profiling

McBride

Chen

Reick

et al. 2017

Molecular & Cellular Proteomics

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Membrane-associated proteins are required for essential processes like transport, organelle biogenesis, and signaling. Many are expected to function as part of an oligomeric protein complex. However, membrane-associated proteins are challenging to work with, and large-scale data sets on the oligomerization state of this important class of proteins is missing. Here we combined cell fractionation of Arabidopsis leaves with nondenaturing detergent solubilization and LC/MS-based profiling of size exclusion chromatography fractions to measure the apparent masses of >1350 membrane-associated proteins. Our method identified proteins from all of the major organelles, with more than 50% of them predicted to be part of a stable complex. The plasma membrane was the most highly enriched in large protein complexes compared with other organelles. Hundreds of novel protein complexes were identified. Over 150 proteins had a complicated localization pattern, and were clearly partitioned between cytosolic and membrane-associated pools. A subset of these dual localized proteins had oligomerization states that differed based on localization. Our data set is an important resource for the community that includes new functionally relevant data for membrane-localized protein complexes that could not be predicted based on sequence alone. Our method enables the analysis of protein complex localization and dynamics, and is a first step in the development of a method in which LC/MS profile data can be used to predict the composition of membrane-associated protein complexes.

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Section: Methodsmentioning

confidence: 99%

Global Analysis of Membrane-associated Protein Oligomerization Using Protein Correlation Profiling

McBride

Chen

Reick

et al. 2017

Molecular & Cellular Proteomics

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“…Elution profile for individual protein was normalized and smoothed by using scale command in R Language. The approximate apparent mass (Mapp) of all proteins identified in our dataset was calculated similar as the previous publication [26]. After that, the ratio of Mapp to the predicted monomeric mass (Mmono) was calculated.…”

Section: Discussionmentioning

confidence: 99%

“…After fractionation, samples were analyzed using LC/MS/MS. The value of Rapp, the ratio of Mapp (proteins apparent molecular mass, Figure S2) to Mmono (the predicted monomeric mass), was evaluated to estimate whether a protein involved a stable complex on SEC column [26], [27]. Rapp>=2 is used to classify proteins to be not oligomeric but within a complex, while Rapp≤0.5 suggests protein degradation during the protein extraction process (Figure 1B).…”

Section: Workflow For Protein Complexes Identification In Synechocystismentioning

confidence: 99%

Global Landscape of Native Protein Complexes inSynechocystissp. PCC 6803

Wang

Yang

et al. 2020

Preprint

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Synechocystis sp. PCC 6803 is a model organism for studying photosynthesis, energy metabolism, and environmental stress. Though known as the first fully sequenced phototrophic organism, Synechocystis sp. PCC 6803 still has almost half of its proteome without functional annotations. In this study, we obtained 291 protein complexes, including 24,092 protein-protein interactions (PPIs) among 2,062 proteins by using co-fractionation and LC/MS/MS. The additional level of PPIs information not only revealed the roles of photosynthesis in metabolism, cell motility, DNA repair, cell division, and other physiological processes, but also showed how protein functions vary from bacteria to higher plants due to the changed interaction partner. It also allows us to uncover functions of hypothetical proteins, such as Sll0445, Sll0446, Sll0447 participating in photosynthesis and cell motility, and Sll1334 regulating the expression of fatty acid. Here we presented the most extensive protein interaction data in Synechocystis so far, which might provide critical insights into the fundamental molecular mechanism in Cyanobacterium.

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“…Prompted by these observations, we decided to use SEC to fractionate proteinmetabolite complexes and determine interaction partners using the chromatographic coelution criterion. Similar attempts were proven to be successful for identifying protein-protein complexes in native cellular extracts and for monitoring interactions between proteins and drugs (Aryal, McBride, Chen, Xie, & Szymanski, 2017;Chan et al, 2012;Havugimana et al, 2012). In doing this, we developed protein-metabolite interactions using a sizeseparation method that we dubbed PROMIS.…”

Section: Of 18mentioning

confidence: 99%

PROMIS: Global Analysis of PROtein‐Metabolite Interactions

et al. 2019

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Small molecules are not only intermediates of metabolism, but also play important roles in signaling and in controlling cellular metabolism, growth, and development. Although a few systematic studies have been conducted, the true extent of protein–small molecule interactions in biological systems remains unknown. PROtein–metabolite interactions using size separation (PROMIS) is a method for studying protein–small molecule interactions in a non‐targeted, proteome‐ and metabolome‐wide manner. This approach uses size‐exclusion chromatography followed by proteomics and metabolomics liquid chromatography–mass spectrometry analysis of the collected fractions. Assuming that small molecules bound to proteins would co‐fractionate together, we found numerous small molecules co‐eluting with proteins, strongly suggesting the formation of stable complexes. Using PROMIS, we identified known small molecule–protein complexes, such as between enzymes and cofactors, and also found novel interactions. © 2019 The Authors. Basic Protocol 1: Preparation of native cell lysate from plant material Support Protocol: Bradford assay to determine protein concentration Basic Protocol 2: Separation of molecular complexes using size‐exclusion chromatography Basic Protocol 3: Simultaneous extraction of proteins and metabolites using single‐step extraction protocol Basic Protocol 4: Metabolomics analysis Basic Protocol 5: Proteomics analysis

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Analysis of protein complexes in Arabidopsis leaves using size exclusion chromatography and label-free protein correlation profiling

Cited by 50 publications

References 94 publications

Global Analysis of Membrane-associated Protein Oligomerization Using Protein Correlation Profiling

Global Analysis of Membrane-associated Protein Oligomerization Using Protein Correlation Profiling

Global Landscape of Native Protein Complexes inSynechocystissp. PCC 6803

PROMIS: Global Analysis of PROtein‐Metabolite Interactions

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