Using hyperLOPIT to perform high-resolution mapping of the spatial proteome.


:The organization of eukaryotic cells into distinct subcompartments is vital for all functional processes, and aberrant protein localization is a hallmark of many diseases. Microscopy methods, although powerful, are usually low-throughput and dependent on the availability of fluorescent fusion proteins or highly specific and sensitive antibodies. One method that provides a global picture of the cell is localization of organelle proteins by isotope tagging (LOPIT), which combines biochemical cell fractionation using density gradient ultracentrifugation with multiplexed quantitative proteomics mass spectrometry, allowing simultaneous determination of the steady-state distribution of hundreds of proteins within organelles. Proteins are assigned to organelles based on the similarity of their gradient distribution to those of well-annotated organelle marker proteins. We have substantially re-developed our original LOPIT protocol (published by Nature Protocols in 2006) to enable the subcellular localization of thousands of proteins per experiment (hyperLOPIT), including spatial resolution at the suborganelle and large protein complex level. This Protocol Extension article integrates all elements of the hyperLOPIT pipeline, including an additional enrichment strategy for chromatin, extended multiplexing capacity of isobaric mass tags, state-of-the-art mass spectrometry methods and multivariate machine-learning approaches for analysis of spatial proteomics data. We have also created an open-source infrastructure to support analysis of quantitative mass-spectrometry-based spatial proteomics data ( and an accompanying interactive visualization framework (http://www. The procedure we outline here is applicable to any cell culture system and requires ∼1 week to complete sample preparation steps, ∼2 d for mass spectrometry data acquisition and 1-2 d for data analysis and downstream informatics.


Nat Protoc


Nature protocols


Mulvey CM,Breckels LM,Geladaki A,Britovšek NK,Nightingale DJH,Christoforou A,Elzek M,Deery MJ,Gatto L,Lilley KS




Has Abstract


2017-06-01 00:00:00














  • A high-yield double-purification proteomics strategy for the identification of SUMO sites.

    abstract::The small ubiquitin-like modifier (SUMO) is a protein modifier that is post-translationally coupled to thousands of lysines in more than a thousand proteins. An understanding of which lysines are modified by SUMO is critical in unraveling its function as a master regulator of all nuclear processes, as well as its invo...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Hendriks IA,Vertegaal AC

    更新日期:2016-09-01 00:00:00

  • Nucleotide resolution profiling of m7G tRNA modification by TRAC-Seq.

    abstract::Precise identification of sites of RNA modification is key to studying the functional role of such modifications in the regulation of gene expression and for elucidating relevance to diverse physiological processes. tRNA reduction and cleavage sequencing (TRAC-Seq) is a chemically based approach for the unbiased globa...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Lin S,Liu Q,Jiang YZ,Gregory RI

    更新日期:2019-11-01 00:00:00

  • Microwave-assisted synthesis of triple-helical, collagen-mimetic lipopeptides.

    abstract::Collagen-mimetic peptides and lipopeptides are widely used as substrates for matrix degrading enzymes, as new biomaterials for tissue engineering, as drug delivery systems and so on. However, the preparation and subsequent purification of these peptides and their fatty-acid conjugates are really challenging. Herein, w...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Banerjee J,Hanson AJ,Muhonen WW,Shabb JB,Mallik S

    更新日期:2010-01-01 00:00:00

  • Assaying stem cell mechanobiology on microfabricated elastomeric substrates with geometrically modulated rigidity.

    abstract::We describe the use of a microfabricated cell culture substrate, consisting of a uniform array of closely spaced, vertical, elastomeric microposts, to study the effects of substrate rigidity on cell function. Elastomeric micropost substrates are micromolded from silicon masters comprised of microposts of different hei...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Yang MT,Fu J,Wang YK,Desai RA,Chen CS

    更新日期:2011-02-01 00:00:00

  • A whole-tissue RNA-seq toolkit for organism-wide studies of gene expression with PME-seq.

    abstract::The immune system operates at the scale of the whole organism in mammals. We currently lack experimental approaches to systematically track and study organism-wide molecular processes in mice. Here we describe an integrated toolkit for measuring gene expression in whole tissues, 3-prime mRNA extension sequencing, that...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Pandey S,Takahama M,Gruenbaum A,Zewde M,Cheronis K,Chevrier N

    更新日期:2020-04-01 00:00:00

  • A simple and rapid nonviral approach to efficiently transfect primary tissue-derived cells using polyethylenimine.

    abstract::This protocol outlines steps for optimizing the transfection of adherent primary mammalian cells using the readily available off-the-shelf cationic polymer, 25-kDa branched polyethylenimine (bPEI25). Transfection efficiency of cationic polymers varies among cell lines and is highly dependent on the conditions and envi...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Hsu CY,Uludağ H

    更新日期:2012-04-19 00:00:00

  • Multiparameter screening method for developing optimized red-fluorescent proteins.

    abstract::Genetically encoded fluorescent proteins (FPs) are highly utilized in cell biology research to study proteins of interest or signal processes using biosensors. To perform well in specific applications, these FPs require a multitude of tailored properties. It is for this reason that they need to be optimized by using m...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Bindels DS,Postma M,Haarbosch L,van Weeren L,Gadella TWJ Jr

    更新日期:2020-02-01 00:00:00

  • CRISPR-Cas9-mediated genome editing in apple and grapevine.

    abstract::The CRISPR-Cas9 genome-editing tool and the availability of whole-genome sequences from plant species have revolutionized our ability to introduce targeted mutations into important crop plants, both to explore genetic changes and to introduce new functionalities. Here, we describe protocols adapting the CRISPR-Cas9 sy...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Osakabe Y,Liang Z,Ren C,Nishitani C,Osakabe K,Wada M,Komori S,Malnoy M,Velasco R,Poli M,Jung MH,Koo OJ,Viola R,Nagamangala Kanchiswamy C

    更新日期:2018-12-01 00:00:00

  • Synthesis of oligo(poly(ethylene glycol) fumarate).

    abstract::This protocol describes the synthesis of oligo(poly(ethylene glycol) fumarate) (OPF; 1-35 kDa; a polymer useful for tissue engineering applications) by a one-pot reaction of poly(ethylene glycol) (PEG) and fumaryl chloride. The procedure involves three parts: dichloromethane and PEG are first dried; the reaction step ...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Kinard LA,Kasper FK,Mikos AG

    更新日期:2012-05-31 00:00:00

  • Synthesis and use of an amphiphilic dendrimer for siRNA delivery into primary immune cells.

    abstract::Using siRNAs to genetically manipulate immune cells is important to both basic immunological studies and therapeutic applications. However, siRNA delivery is challenging because primary immune cells are often sensitive to the delivery materials and generate immune responses. We have recently developed an amphiphilic d...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Chen J,Ellert-Miklaszewska A,Garofalo S,Dey AK,Tang J,Jiang Y,Clément F,Marche PN,Liu X,Kaminska B,Santoni A,Limatola C,Rossi JJ,Zhou J,Peng L

    更新日期:2021-01-01 00:00:00

  • 3D mouse embryonic stem cell culture for generating inner ear organoids.

    abstract::This protocol describes a culture system in which inner-ear sensory tissue is produced from mouse embryonic stem (ES) cells under chemically defined conditions. This model is amenable to basic and translational investigations into inner ear biology and regeneration. In this protocol, mouse ES cells are aggregated in 9...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Koehler KR,Hashino E

    更新日期:2014-01-01 00:00:00

  • Generation of a humanized mouse model with both human immune system and liver cells to model hepatitis C virus infection and liver immunopathogenesis.

    abstract::Establishing a small animal model that accurately recapitulates hepatotropic pathogens, including hepatitis C virus (HCV) infection and immunopathogenesis, is essential for the study of hepatitis virus-induced liver disease and for therapeutics development. This protocol describes our recently developed humanized mous...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Bility MT,Zhang L,Washburn ML,Curtis TA,Kovalev GI,Su L

    更新日期:2012-09-01 00:00:00

  • Cloning of ground-state intestinal stem cells from endoscopic biopsy samples.

    abstract::'Adult' or 'somatic' stem cells harbor an intrinsic ability to regenerate tissues. Heterogeneity of such stem cells along the gastrointestinal tract yields the known segmental specificity of this organ and may contribute to the pathology of certain enteric conditions. Here we detail technology for the generation of 'l...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Duleba M,Yamamoto Y,Neupane R,Rao W,Xie J,Qi Y,Liew AA,Niroula S,Zhang Y,Mahalingam R,Wang S,Goller K,Ajani JA,Vincent M,Ho KY,Hou JK,Hyams JS,Sylvester FA,Crum CP,McKeon F,Xian W

    更新日期:2020-05-01 00:00:00

  • 3D computational reconstruction of tissues with hollow spherical morphologies using single-cell gene expression data.

    abstract::Single-cell gene expression analysis has contributed to a better understanding of the transcriptional heterogeneity in a variety of model systems, including those used in research in developmental, cancer and stem cell biology. Nowadays, technological advances facilitate the generation of large gene expression data se...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Durruthy-Durruthy R,Gottlieb A,Heller S

    更新日期:2015-03-01 00:00:00

  • A general pipeline for quality and statistical assessment of protein interaction data using R and Bioconductor.

    abstract::The systematic mapping of protein interactions by bait-prey techniques, including affinity purification-mass spectrometry or the yeast two-hybrid system, contributes a unique and relevant perspective on the comprehensive picture of cellular machines. We describe here a protocol for statistical analysis of node-and-edg...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Chiang T,Scholtens D

    更新日期:2009-01-01 00:00:00

  • Single-molecule mRNA detection and counting in mammalian tissue.

    abstract::We present a protocol for visualizing and quantifying single mRNA molecules in mammalian (mouse and human) tissues. In the approach described here, sets of about 50 short oligonucleotides, each labeled with a single fluorophore, are hybridized to target mRNAs in tissue sections. Each set binds to a single mRNA molecul...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Lyubimova A,Itzkovitz S,Junker JP,Fan ZP,Wu X,van Oudenaarden A

    更新日期:2013-09-01 00:00:00

  • Isolation of DNA from small amounts of elephant ivory.

    abstract::This protocol describes a method for the extraction of DNA from elephant ivory. These techniques are being used to assign geographic origin to poached ivory by comparing the ivory genotype to a geographic-based gene frequency map, developed separately. The method has three components: ivory pulverization, decalcificat...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Mailand C,Wasser SK

    更新日期:2007-01-01 00:00:00

  • Fast, efficient and reproducible genetic transformation of Phaseolus spp. by Agrobacterium rhizogenes.

    abstract::This transformation procedure generates, with high efficiency (70-90%), hairy roots in cultivars, landraces and accessions of Phaseolus vulgaris (common bean) and other Phaseolus spp. Hairy roots rapidly develop after wounding young plantlets with Agrobacterium rhizogenes, at the cotyledon node, and keeping the plants...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Estrada-Navarrete G,Alvarado-Affantranger X,Olivares JE,Guillén G,Díaz-Camino C,Campos F,Quinto C,Gresshoff PM,Sanchez F

    更新日期:2007-01-01 00:00:00

  • Direct ester condensation catalyzed by bulky diarylammonium pentafluorobenzenesulfonates.

    abstract::A protocol for ester condensation between equimolar amounts of carboxylic acids and alcohols catalyzed by bulky diarylammonium pentafluorobenzenesulfonate is described. We also present procedures for the synthesis of N-(2,6-diisopropylphenyl)-N-mesitylammonium pentafluorobenzenesulfonate. The present ester condensatio...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Sakakura A,Nakagawa S,Ishihara K

    更新日期:2007-01-01 00:00:00

  • Statistical analysis of cell migration in 3D using the anisotropic persistent random walk model.

    abstract::Cell migration through 3D extracellular matrices (ECMs) is crucial to the normal development of tissues and organs and in disease processes, yet adequate analytical tools to characterize 3D migration are lacking. The motility of eukaryotic cells on 2D substrates in the absence of gradients has long been described usin...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Wu PH,Giri A,Wirtz D

    更新日期:2015-03-01 00:00:00

  • Fabrication of functional three-dimensional tissues by stacking cell sheets in vitro.

    abstract::The fabrication of 3D tissues retaining the original functions of tissues/organs in vitro is crucial for optimal tissue engineering and regenerative medicine. The fabrication of 3D tissues also contributes to the establishment of in vitro tissue/organ models for drug screening. Our laboratory has developed a fabricati...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Haraguchi Y,Shimizu T,Sasagawa T,Sekine H,Sakaguchi K,Kikuchi T,Sekine W,Sekiya S,Yamato M,Umezu M,Okano T

    更新日期:2012-04-05 00:00:00

  • Microfluidic, marker-free isolation of circulating tumor cells from blood samples.

    abstract::The ability to isolate and analyze rare circulating tumor cells (CTCs) has the potential to further our understanding of cancer metastasis and enhance the care of cancer patients. In this protocol, we describe the procedure for isolating rare CTCs from blood samples by using tumor antigen-independent microfluidic CTC-...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Karabacak NM,Spuhler PS,Fachin F,Lim EJ,Pai V,Ozkumur E,Martel JM,Kojic N,Smith K,Chen PI,Yang J,Hwang H,Morgan B,Trautwein J,Barber TA,Stott SL,Maheswaran S,Kapur R,Haber DA,Toner M

    更新日期:2014-03-01 00:00:00

  • Tissue-specific and cell type-specific RNA interference in vivo.

    abstract::RNA interference (RNAi) is an efficient method for silencing genes in cultured cells. Here we describe a simple RNAi approach for silencing genes in a cell type-specific and tissue-specific way in vivo. The approach, which mimics the means by which naturally occurring 'microRNA's are generated, uses a tissue-specific ...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Rao MK,Wilkinson MF

    更新日期:2006-01-01 00:00:00

  • Fabrication of nanopores with ultrashort single-walled carbon nanotubes inserted in a lipid bilayer.

    abstract::We describe a protocol for the insertion of ultrashort single-walled carbon nanotubes (SWCNTs) to form nanopores in a Montal-Mueller lipid bilayer. The SWCNTs are designed to bind to a specific analyte of interest; binding will result in the reduction of current in single-channel recording experiments. The first stage...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Liu L,Xie J,Li T,Wu HC

    更新日期:2015-11-01 00:00:00

  • Vibrational spectroscopic image analysis of biological material using multivariate curve resolution-alternating least squares (MCR-ALS).

    abstract::Raman and Fourier transform IR (FTIR) microspectroscopic images of biological material (tissue sections) contain detailed information about their chemical composition. The challenge lies in identifying changes in chemical composition, as well as locating and assigning these changes to different conditions (pathology, ...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Felten J,Hall H,Jaumot J,Tauler R,de Juan A,Gorzsás A

    更新日期:2015-02-01 00:00:00

  • Labeling of biotin antibodies with horseradish peroxidase using cyanuric chloride.

    abstract::In this report, we describe a two-step protocol for labeling of an affinity-purified antibody to biotin with horseradish peroxidase (HRP) using cyanuric chloride (CC) as a bridge. The enzyme was first modified with CC, and following chromatography on a PD-10 column, the activated HRP was incubated with the antibody to...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Abuknesha RA,Jeganathan F,Wu J,Baalawy Z

    更新日期:2009-01-01 00:00:00

  • Tracking recent adaptive evolution in microbial species using TimeZone.

    abstract::An important goal of the analysis of sequenced genomes of microbial pathogens is to improve the therapy of infectious diseases. In this context, a major challenge is to detect genomic-level evolutionary changes that increase microbial virulence. TimeZone, a genome analysis software package, is designed to detect footp...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Chattopadhyay S,Paul S,Dykhuizen DE,Sokurenko EV

    更新日期:2013-04-01 00:00:00

  • Synthesis of oligo-RNAs with photocaged adenosine 2'-hydroxyls.

    abstract::This protocol describes a general method for the preparation of RNAs in which the reactivity or hydrogen-bonding properties of the molecule are modified in a photoreversible fashion by use of a caging strategy. A single caged adenosine, modified at the 2' position as a nitro-benzyl ether, can be incorporated into shor...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Chaulk SG,MacMillan AM

    更新日期:2007-01-01 00:00:00

  • Real-time single-molecule coimmunoprecipitation of weak protein-protein interactions.

    abstract::Coimmunoprecipitation (co-IP) analysis is a useful method for studying protein-protein interactions. It currently involves electrophoresis and western blotting, which are not optimized for detecting weak and transient interactions. In this protocol we describe an advanced version of co-IP analysis that uses real-time,...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Lee HW,Ryu JY,Yoo J,Choi B,Kim K,Yoon TY

    更新日期:2013-10-01 00:00:00

  • Generating high-purity cardiac and endothelial derivatives from patterned mesoderm using human pluripotent stem cells.

    abstract::Human pluripotent stem cells (hPSCs) provide a valuable model for the study of human development and a means to generate a scalable source of cells for therapeutic applications. This protocol specifies cell fate efficiently into cardiac and endothelial lineages from hPSCs. The protocol takes 2 weeks to complete and re...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Palpant NJ,Pabon L,Friedman CE,Roberts M,Hadland B,Zaunbrecher RJ,Bernstein I,Zheng Y,Murry CE

    更新日期:2017-01-01 00:00:00