Abstract：
:Top-down proteomics (TDP) by mass spectrometry (MS) is a technique by which intact proteins are analyzed. It has become increasingly popDesalting and concentrating GELFrEEular in translational research because of the value of characterizing distinct proteoforms of intact proteins. Compared to bottom-up proteomics (BUP) strategies, which measure digested peptide mixtures, TDP provides highly specific molecular information that avoids the bioinformatic challenge of protein inference. However, the technique has been difficult to implement widely because of inherent limitations of existing sample preparation methods and instrumentation. Recent improvements in proteoform pre-fractionation and the availability of high-resolution benchtop mass spectrometers have made it possible to use high-throughput TDP for the analysis of complex clinical samples. Here, we provide a comprehensive protocol for analysis of a common sample type in translational research: human peripheral blood mononuclear cells (PBMCs). The pipeline comprises multiple workflows that can be treated as modular by the reader and used for various applications. First, sample collection and cell preservation are described for two clinical biorepository storage schemes. Cell lysis and proteoform pre-fractionation by gel-eluted liquid fractionation entrapment electrophoresis are then described. Importantly, instrument setup and liquid chromatography-tandem MS are described for TDP analyses, which rely on high-resolution Fourier-transform MS. Finally, data processing and analysis are described using two different, application-dependent software tools: ProSight Lite for targeted analyses of one or a few proteoforms and TDPortal for high-throughput TDP in discovery mode. For a single sample, the minimum completion time of the entire experiment is 72 h.
journal_name
Nat Protocjournal_title
Nature protocolsauthors
Toby TK,Fornelli L,Srzentić K,DeHart CJ,Levitsky J,Friedewald J,Kelleher NLdoi
10.1038/s41596-018-0085-7subject
Has Abstractpub_date
2019-01-01 00:00:00pages
119-152issue
1eissn
1754-2189issn
1750-2799pii
10.1038/s41596-018-0085-7journal_volume
14pub_type
杂志文章相关文献
Nature Protocols文献大全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
doi：10.1038/s41596-019-0250-7
更新日期：2020-02-01 00:00:00
abstract：:The human stomach contains two primary domains: the corpus, which contains the fundic epithelium, and the antrum. Each of these domains has distinct cell types and functions, and therefore each presents with unique disease pathologies. Here, we detail two protocols to differentiate human pluripotent stem cells (hPSCs)...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/s41596-018-0080-z
更新日期：2019-01-01 00:00:00
abstract：:Freestanding plasmonic nanoparticle (NP) superlattice sheets are novel 2D nanomaterials with tailorable properties that enable their use for broad applications in sensing, anticounterfeit measures, ionic gating, nanophotonics and flat lenses. We recently developed a robust, yet general, two-step drying-mediated approa...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/s41596-019-0200-4
更新日期：2019-09-01 00:00:00
abstract：:This protocol measures externalization of aminophospholipids (APLs) to the outside of the plasma membrane using mass spectrometry (MS). APL externalization occurs in numerous events, and it is relevant for transplant medicine, immunity and cancer. In this protocol, externalized APLs are chemically modified by using a ...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2013.163
更新日期：2014-01-01 00:00:00
abstract：:The study of angiogenic endothelial cells (ECs) has in recent years greatly stimulated multiple fields of vascular biology research. A number of cellular models and numerous complex developmental, manipulatory and tumor animal models have been developed to study angiogenesis in vitro and in vivo. To connect the versat...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2009.96
更新日期：2009-01-01 00:00:00
abstract：:Improvement of cellular uptake and cellular localization is still one of the main obstacles to the development of antisense-antigene therapeutics, including peptide nucleic acid (PNA). Cell-penetrating peptides (CPPs) such as Tat peptide and polyarginine have been widely used to improve the cellular uptake of PNA and ...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2006.92
更新日期：2006-01-01 00:00:00
abstract：:Here, we describe a robust protocol for the reverse transfection of cells on small interfering (siRNA) arrays, which, in combination with multi-channel immunofluorescence or time-lapse microscopy, is suitable for genome-wide RNA interference (RNAi) screens in intact human cells. The automatic production of 48 'transfe...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2006.483
更新日期：2007-01-01 00:00:00
abstract：:The small ubiquitin-like modifiers (SUMOs) are posttranslationally conjugated to eukaryotic cellular proteins with generally unpredictable consequences. SUMO substrates are found in many cellular systems, and functional analysis has revealed that substrate SUMOylation often has an important role in their regulation. H...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2009.128
更新日期：2009-01-01 00:00:00
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
doi：10.1038/nprot.2009.195
更新日期：2010-01-01 00:00:00
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
doi：10.1038/nprot.2013.109
更新日期：2013-09-01 00:00:00
abstract：:Gram-negative and Gram-positive bacteria release a variety of membrane vesicles through different formation routes. Knowledge of the structure, molecular cargo and function of bacterial extracellular vesicles (BEVs) is primarily obtained from bacteria cultured in laboratory conditions. BEVs in human body fluids have b...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/s41596-019-0236-5
更新日期：2020-01-01 00:00:00
abstract：:This protocol describes pulsed-field gel electrophoresis (PFGE), a method developed for separation of large DNA molecules. Whereas standard DNA gel electrophoresis commonly resolves fragments up to approximately 50 kb in size, PFGE fractionates DNA molecules up to 10 Mb. The mechanism driving these separations exploit...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2007.94
更新日期：2007-01-01 00:00:00
abstract：:RNA production using in vivo transcription by Escherichia coli allows preparation of milligram quantities of RNA for biochemical, biophysical and structural investigations. We describe here a generic protocol for the overproduction and purification of recombinant RNA using liquid chromatography. The strategy utilizes ...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2009.67
更新日期：2009-01-01 00:00:00
abstract：:Iterative saturation mutagenesis (ISM) is a new and efficient method for the directed evolution of functional enzymes. It reduces the necessary molecular biological work and the screening effort drastically. It is based on a Cartesian view of the protein structure, performing iterative cycles of saturation mutagenesis...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2007.72
更新日期：2007-01-01 00:00:00
abstract：:The development of single-molecule switching (SMS) fluorescence microscopy (also called single-molecule localization microscopy) over the last decade has enabled researchers to image cell biological structures at unprecedented resolution. Using two opposing objectives in a so-called 4Pi geometry doubles the available ...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/s41596-020-00428-7
更新日期：2020-12-16 00:00:00
abstract：:The glycocalyx comprises glycosylated proteins and lipids and fcorms the outermost layer of cells. It is involved in fundamental inter- and intracellular processes, including non-self-cell and self-cell recognition, cell signaling, cellular structure maintenance, and immune protection. Characterization of the glycocal...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/s41596-020-0350-4
更新日期：2020-08-01 00:00:00
abstract：:The increasing multi-resistance of pathogenic bacteria requires the development of novel classes of antibiotics. Antimicrobial host defense peptides represent one promising class. Here we describe a protocol for screening large numbers of peptides against any microbe of interest. Peptides synthesized on a cellulose su...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2007.203
更新日期：2007-01-01 00:00:00
abstract：:This protocol describes a simple silver staining method used to visualize DNA fragments and other organic molecules with unsurpassed detail following traditional polyacrylamide gel electrophoresis (PAGE). Sensitivity rivals radioisotopic methods and DNA in the picogram range can be reliably detected. The described pro...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2007.330
更新日期：2007-01-01 00:00:00
abstract：:Limbal stem cells (LSCs) have an important role in the maintenance of the corneal surface epithelium, and autologous cultured limbal epithelial cell transplantations have contributed substantially to the treatment of the visually disabling condition known as LSC deficiency. In this protocol, we describe a method of es...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2010.115
更新日期：2010-08-01 00:00:00
abstract：:Dynamic interplay between intracellular organelles requires a particular functional apposition of membrane structures. The organelles involved come into close contact, but do not fuse, thereby giving rise to notable microdomains; these microdomains allow rapid communication between the organelles. Plasma membrane-asso...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2014.016
更新日期：2014-02-01 00:00:00
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
doi：10.1038/nprot.2009.26
更新日期：2009-01-01 00:00:00
abstract：:The forced swim test (FST) is one of the most commonly used animal models for assessing antidepressant-like behavior. This protocol details using the FST in rats, which takes place over 48 h and is followed by the video analysis of the behavior. The swim test involves the scoring of active (swimming and climbing) or p...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2012.044
更新日期：2012-05-03 00:00:00
abstract：:The transmembrane (TM) anchors of cell surface proteins have been one of the 'blind spots' in structural biology because they are generally very hydrophobic, sometimes dynamic, and thus difficult targets for structural characterization. A plethora of examples show these membrane anchors are not merely anchors but can ...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/s41596-019-0188-9
更新日期：2019-08-01 00:00:00
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
doi：10.1038/nprot.2009.6
更新日期：2009-01-01 00:00:00
abstract：:An important area of proteomics involves the need for quantification, whether relative or absolute. Many methods now exist for relative quantification, but to support biomarker proteomics and systems biology, absolute quantification rather than relative quantification is required. Absolute quantification usually invol...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2006.129
更新日期：2006-01-01 00:00:00
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
doi：10.1038/nprot.2014.100
更新日期：2014-01-01 00:00:00
abstract：:Small ubiquitin-like modifier (SUMO) post-translational modification (PTM) of proteins has a crucial role in the regulation of important cellular processes. This protocol describes the chemical synthesis of functional SUMO-peptide conjugates. The two crucial stages of this protocol are the solid-phase synthesis of pep...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2015.013
更新日期：2015-02-01 00:00:00
abstract：:The analysis of large microbiome data sets holds great promise for the delineation of the biological and metabolic functioning of living organisms and their role in the environment. In the midst of this genomic puzzle, viruses, especially those that infect microbial communities, represent a major reservoir of genetic ...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2017.063
更新日期：2017-08-01 00:00:00
abstract：:A fluorescence-based microarray technique that does not require target DNA labeling is detailed. This 'label-free' approach utilizes a cationic, water-soluble conjugated polymer PFBT (poly[9,9'-bis(6''-(N,N,N-trimethylammonium)hexyl)fluorene-co-alt-4,7-(2,1,3-benzothiadiazole) dibromide]), and neutral PNA (peptide nuc...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2007.307
更新日期：2007-01-01 00:00:00
abstract：:Detailed biochemical analysis of unmanipulated germinal center (GC) B cells has not been achieved. Previously, we designed and used a simple, economical and new magnetic bead separation scheme for the purification of 'untouched' mature GC and non-GC B cells from the spleens of immunized mice and reported the first bio...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2011.344
更新日期：2011-06-09 00:00:00
