Affinity Proteomics to Study Endogenous Protein Complexes: Pointers, Pitfalls, Preferences and Perspectives

LaCava, John; Molloy, Kelly R.; Taylor, Martin S.; Domanski, Michal; Chait, Brian T.; Rout, Michael P.

doi:10.2144/000114262

Cited by 57 publications

(51 citation statements)

References 168 publications

(214 reference statements)

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“…An excellent depletion of the target protein is typically ≥70% of the soluble fraction of the target protein, but >90% is desirable and may be achievable with careful parameterization of extraction conditions. Many such practical considerations are covered in reference 1 . Paramagnetic beads are manipulated using neodymium magnets in a specialized microcentrifuge tube holder, although homemade alternatives are viable.…”

Section: Discussionmentioning

confidence: 99%

“…7. Elute in either a native or denaturing manner; consider the strategy best for the downstream application 1 . NOTE: The details of native elution strategies will depend upon the affinity tag used (See Discussion).…”

Section: Cell Extract Preparationmentioning

confidence: 99%

“…Subject the samples to SDS-PAGE followed by protein staining using standard techniques 31 . MS may be used to characterize the sample composition 1 . Individual protein bands may be excised for identification or the entire sample may be characterized in a single analysis by electrophoresing the sample only briefly (4-6 mm into the gel) and processing all the proteins in the sample together as a 'gel plug.'…”

Section: Cell Extract Preparationmentioning

confidence: 99%

“…A typical application of the presented procedures is to stabilize and obtain a high yield and purity of endogenous protein complexes of interest for interactomic characterization 1 . It is understood that dynamic networks of both stably and transiently associated macromolecular complexes, chiefly comprised of proteins, orchestrate cellular processes 2,3 .…”

Section: Introductionmentioning

confidence: 99%

“…Diverse cell and molecular biological studies have utilized mechanical breakage of cells by milling at cryogenic temperatures for at least the last forty years [14][15][16][17][18][19] ; and molecular separations utilizing (super)paramagnetic beads have become increasingly common over the last two decades 20 . Combining these different technologies has served to synergistically improve the results that can be obtained in protein complex affinity capture experiments 1 , as evidenced by an extensive body of work produced by ourselves and the wider research community 7,9,11,18,19,[21][22][23] . Supporting results are presented in …”

Section: Introductionmentioning

confidence: 99%

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Protein Complex Affinity Capture from Cryomilled Mammalian Cells

LaCava

Jiang

Rout

2016

JoVE

Self Cite

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Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose-and Sepharose-based media. Primary benefits of magnetic media include typically lower nonspecific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

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Section: Discussionmentioning

confidence: 99%

Section: Cell Extract Preparationmentioning

confidence: 99%

Section: Cell Extract Preparationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Protein Complex Affinity Capture from Cryomilled Mammalian Cells

LaCava

Jiang

Rout

2016

JoVE

Self Cite

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Native Isolation of 3×HA‐Tagged Protein Complexes to Characterize Protein‐Protein Interactions

Lim¹,

Iftner²,

Simon³

2021

Current Protocols

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Co‐immunoprecipitation (Co‐IP) is a straightforward method that is widely used in studying direct protein‐protein interactions in physiological environments. This technique is based on the antigen‐antibody interaction: the protein of interest (bait) is captured by a specific antibody, followed by antibody‐bait precipitation. The proteins interacting with the bait protein (prey) co‐precipitate with the antibody‐bait complex from a cell lysate as an antibody‐bait/prey complex. Nowadays, a variety of surface‐functionalized materials with antibodies immobilized on agarose or magnetic beads are available, replacing the precipitation of antibodies and simplifying the application. However, unspecific binding of cellular proteins to matrix surfaces and/or antibodies has become a common issue. Unspecific binding that leads to false‐positive signals and a high background can hamper further analysis. Our protocol describes a strategy to tremendously reduce unspecific background when isolating native proteins and protein complexes. Instead of eluting our samples under denaturing conditions, we elute triple hemagglutinin (3×HA)‐tagged bait/prey complexes in their native form with a competitive peptide simulating the 3×HA tag of the bait protein. Matrix‐unspecific interacting proteins and Co‐IP antibodies remain on the matrix instead of being eluted under conventionally applied denaturing conditions. We optimized the elution by altering incubation time, eluent concentration, and temperature. These improvements result in more pure proteins. This strategy not only reduces background in SDS‐PAGE and western blot but also allows complex characterization in vitro. © 2021 Wiley Periodicals LLC.

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Native top‐down mass spectrometry for higher‐order structural characterization of proteins and complexes

Liu

Xia

2022

Mass Spectrometry Reviews

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Progress in structural biology research has led to a high demand for powerful and yet complementary analytical tools for structural characterization of proteins and protein complexes. This demand has significantly increased interest in native mass spectrometry (nMS), particularly native top‐down mass spectrometry (nTDMS) in the past decade. This review highlights recent advances in nTDMS for structural research of biological assemblies, with a particular focus on the extra multi‐layers of information enabled by TDMS. We include a short introduction of sample preparation and ionization to nMS, tandem fragmentation techniques as well as mass analyzers and software/analysis pipelines used for nTDMS. We highlight unique structural information offered by nTDMS and examples of its broad range of applications in proteins, protein‐ligand interactions (metal, cofactor/drug, DNA/RNA, and protein), therapeutic antibodies and antigen‐antibody complexes, membrane proteins, macromolecular machineries (ribosome, nucleosome, proteosome, and viruses), to endogenous protein complexes. The challenges, potential, along with perspectives of nTDMS methods for the analysis of proteins and protein assemblies in recombinant and biological samples are discussed.

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Affinity Proteomics to Study Endogenous Protein Complexes: Pointers, Pitfalls, Preferences and Perspectives

Cited by 57 publications

References 168 publications

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Native Isolation of 3×HA‐Tagged Protein Complexes to Characterize Protein‐Protein Interactions

Native top‐down mass spectrometry for higher‐order structural characterization of proteins and complexes

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