Abstract:
:Analysis of the sequence and nature of protein N termini has many applications. Defining the termini of proteins for proteome annotation in the Human Proteome Project is of increasing importance. Terminomics analysis of protease cleavage sites in degradomics for substrate discovery is a key new application. Here we describe the step-by-step procedures for performing terminal amine isotopic labeling of substrates (TAILS), a 2- to 3-d (depending on method of labeling) high-throughput method to identify and distinguish protease-generated neo-N termini from mature protein N termini with all natural modifications with high confidence. TAILS uses negative selection to enrich for all N-terminal peptides and uses primary amine labeling-based quantification as the discriminating factor. Labeling is versatile and suited to many applications, including biochemical and cell culture analyses in vitro; in vivo analyses using tissue samples from animal and human sources can also be readily performed. At the protein level, N-terminal and lysine amines are blocked by dimethylation (formaldehyde/sodium cyanoborohydride) and isotopically labeled by incorporating heavy and light dimethylation reagents or stable isotope labeling with amino acids in cell culture labels. Alternatively, easy multiplex sample analysis can be achieved using amine blocking and labeling with isobaric tags for relative and absolute quantification, also known as iTRAQ. After tryptic digestion, N-terminal peptide separation is achieved using a high-molecular-weight dendritic polyglycerol aldehyde polymer that binds internal tryptic and C-terminal peptides that now have N-terminal alpha amines. The unbound naturally blocked (acetylation, cyclization, methylation and so on) or labeled mature N-terminal and neo-N-terminal peptides are recovered by ultrafiltration and analyzed by tandem mass spectrometry (MS/MS). Hierarchical substrate winnowing discriminates substrates from the background proteolysis products and non-cleaved proteins by peptide isotope quantification and bioinformatics search criteria.
journal_name
Nat Protocjournal_title
Nature protocolsauthors
Kleifeld O,Doucet A,Prudova A,auf dem Keller U,Gioia M,Kizhakkedathu JN,Overall CMdoi
10.1038/nprot.2011.382subject
Has Abstractpub_date
2011-09-22 00:00:00pages
1578-611issue
10eissn
1754-2189issn
1750-2799pii
nprot.2011.382journal_volume
6pub_type
杂志文章相关文献
Nature Protocols文献大全abstract::Characterization of the cellular participants in tissue immune responses is crucial to understanding infection, cancer, autoimmunity, allergy, graft rejection and other immunological processes. Previous reports indicate that leukocytes in lung vasculature fail to be completely removed by perfusion. Several studies sug...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2014.005
更新日期:2014-01-01 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::In recent years, many mouse models have been developed to mark and trace the fate of adult cell populations using fluorescent proteins. High-resolution visualization of such fluorescent markers in their physiological setting is thus an important aspect of adult stem cell research. Here we describe a protocol to produc...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2011.365
更新日期:2011-07-28 00:00:00
abstract::We describe procedures for the synthesis of a fluorescent pyrimidine analog and its site-specific incorporation into a DNA oligomer. The 5'-protected and 3'-activated nucleoside 4 is synthesized in three steps with an overall yield of 40%. Site-specific incorporation into a DNA oligomer occurs with greater than 88% co...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2006.464
更新日期:2007-01-01 00:00:00
abstract::The ability to rapidly generate large panels of antigen-specific human antibodies in a rodent would enable the efficient discovery of novel therapeutically useful antibodies. We have developed a system wherein human antigen-specific antibody-secreting plasmablasts can be enriched in vivo, in a severe combined immunode...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2014.104
更新日期:2014-07-01 00:00:00
abstract::Optogenetic tools provide users the ability to photocontrol the activity of cells. Commonly, activation is achieved by expression of proteins from photosynthetic organisms, for example, microbial opsins (e.g., ChR2). Alternatively, a sister approach, synthetic optogenetics, enables photocontrol over proteins of mammal...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/s41596-018-0118-2
更新日期:2019-03-01 00:00:00
abstract::This protocol describes a method for converting short single-stranded and double-stranded DNA into libraries compatible with high-throughput sequencing using Illumina technology. This method has primarily been developed to improve sequence retrieval from ancient DNA, but it is also applicable to the sequencing of shor...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2013.038
更新日期:2013-04-01 00:00:00
abstract::Protein digestion using a dedicated protease represents a key element in a typical mass spectrometry (MS)-based shotgun proteomics experiment. Up to now, digestion has been predominantly performed with trypsin, mainly because of its high specificity, widespread availability and ease of use. Lately, it has become appar...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2016.057
更新日期:2016-05-01 00:00:00
abstract::This protocol details a subset of assays developed within the touchscreen platform to measure various aspects of executive function in rodents. Three main procedures are included: extinction, measuring the rate and extent of curtailing a response that was previously, but is no longer, associated with reward; reversal ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2013.123
更新日期:2013-10-01 00:00:00
abstract::The adeno-associated virus (AAV) is one of the most promising viral vectors for human gene therapy. As with any potential therapeutic system, a thorough understanding of it at the in vitro and in vivo levels is required. Over the years, numerous methods have been developed to better characterize AAV vectors. These met...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2006.207
更新日期:2006-01-01 00:00:00
abstract::Human embryonic stem (hES) cells are self-renewing, pluripotent cells that are valuable research tools and hold promise for use in regenerative medicine. Most hES cell lines are derived from cryopreserved human embryos that were created during in vitro fertilization (IVF) and are in excess of clinical need. Embryos th...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2008.60
更新日期:2008-01-01 00:00:00
abstract::This protocol describes an optimized method for direct in vitro monitoring of homo- and heterotypic axon-axon interactions involved in the developmental assembly of neural circuits. The assay exploits a classical example of heterotypic axonal interactions by modeling the sequential extension of spinal motor and somato...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2011.442
更新日期:2012-01-26 00:00:00
abstract::Biominerals serve as critical structures of living systems and play important roles in biochemical processes. Understanding their crystallization mechanisms is therefore central to many areas of biology, biogeoscience, and biochemistry. Some biominerals, such as bone and dentin, are hierarchical nanocomposite structur...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/s41596-018-0027-4
更新日期:2018-09-01 00:00:00
abstract::Cell-to-cell chemical signaling plays multiple roles in coordinating the activity of the functional elements of an organism, with these elements ranging from a three-neuron reflex circuit to the entire animal. In recent years, single-cell mass spectrometry (MS) has enabled the discovery of cell-to-cell signaling molec...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2007.277
更新日期:2007-01-01 00:00:00
abstract::Studies on colonic cells in the lamina propria (LP) of mice are important for understanding the cellular and immune responses in the gut, especially in inflammatory bowel diseases (such as morbus crohn and colitis ulcerosa). This protocol details a method to isolate LP cells and characterize freshly isolated cells by ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2007.315
更新日期:2007-01-01 00:00:00
abstract::Rapid evaluation of microbial cell-surface carbohydrates is essential to understanding the mechanisms by which bacteria use glycans to establish pathogenic or symbiotic relationships. Microbial glycan analysis is complicated both by the vast diversity of possible carbohydrate structures and by their dynamic nature. Ba...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2006.76
更新日期:2006-01-01 00:00:00
abstract::Determining enantiomeric excess (e.e.) in chiral compounds is key to development of chiral catalyst auxiliaries and chiral drugs. Here we describe a sensitive and robust fluorescence-based assay for determining e.e. in mixtures of enantiomers of 1,2- and 1,3-diols, chiral amines, amino alcohols, and amino-acid esters....
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/s41596-020-0329-1
更新日期:2020-07-01 00:00:00
abstract::The protein called 'small ubiquitin-like modifier' (SUMO) is post-translationally linked to target proteins at the ɛ-amino group of lysine residues. This 'SUMOylation' alters the behavior of the target protein, a change that is utilized to regulate diverse cellular processes. Understanding the target-specific conseque...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2015.095
更新日期:2015-09-01 00:00:00
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
doi:10.1038/nprot.2015.022
更新日期:2015-03-01 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::Small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS) are techniques used to extract structural parameters and determine the overall structures and shapes of biological macromolecules, complexes and assemblies in solution. The scattering intensities measured from a sample contain contributions ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2016.113
更新日期:2016-11-01 00:00:00
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::Electrochemically active bacteria (EAB) have the ability to transfer electrons to electron acceptors located outside the cell, and they are widely present in diverse environments. In spite of their important roles in geochemical cycles, environmental remediation and electricity generation, so far, only a limited numbe...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2013.173
更新日期:2014-01-01 00:00:00
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
doi:10.1038/nprot.2016.082
更新日期:2016-09-01 00:00:00
abstract::Ex vivo perfusion systems offer a reliable, reproducible method for studying acute physiological responses of an organ to various environmental manipulations. Unlike in vitro culture systems, the cellular organization, compartmentalization and three-dimensional structure of ex vivo-perfused organs are maintained. Thes...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2012.144
更新日期:2013-01-01 00:00:00
abstract::The elucidation of protein-protein interaction networks is a crucial task in the postgenomic era. In this protocol, we describe our approach to discover protein-protein interactions using the surface plasmon resonance technique coupled to mass spectrometry (MS). A peptide or a protein is immobilized on a sensor chip a...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2009.84
更新日期:2009-01-01 00:00:00
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
doi:10.1038/s41596-019-0226-7
更新日期:2019-11-01 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 world is on the cusp of a post-antibiotic era, but researchers and medical doctors have found a way forward-by looking back at how infections were treated before the advent of antibiotics, namely using phage therapy. Although bacteriophages (phages) continue to lack drug approval in Western medicine, an increasing...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/s41596-020-0346-0
更新日期:2020-09-01 00:00:00
abstract::Telomeres have emerged as crucial cellular elements in aging and various diseases including cancer. To measure the average length of telomere repeats in cells, we describe our protocols that use fluorescent in situ hybridization (FISH) with labeled peptide nucleic acid (PNA) probes specific for telomere repeats in com...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2006.263
更新日期:2006-01-01 00:00:00