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Christopher M Overall - One of the best experts on this subject based on the ideXlab platform.
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terminal amine Isotopic Labeling of substrates tails analysis reveals novel potential substrates of tumor suppressive matrix metalloproteinase 8 in oral tongue carcinoma
Oral Surgery Oral Medicine Oral Pathology and Oral Radiology, 2019Co-Authors: Krista Juurikka, Christopher M Overall, Pirjo Astrom, Antoine Dufour, Meeri Sutinen, Tuula SaloAbstract:Objectives Oral tongue squamous cell carcinoma (OTSCC)is an aggressive cancer with poor survival and increasing incidence. Matrix metalloproteinase-8 (MMP-8), unlike most MMPs, provides protective effects in various human cancers including OTSCC. Yet, the mechanism behind these effects remain unclear. Knowledge of the molecular basis of disease progression is crucial for developing novel treatments. Thus our study aimed to identify novel candidate substrates of MMP-8 to examine the mechanisms behind its tumor-suppressive actions in OTSCC. Findings We have previously generated stably MMP-8 overexpressing oral squamous cell carcinoma cell line (HSC-3 MMP-8+ cells) and showed that overexpression of MMP-8 significantly decreased the cell migration and invasion. However, the molecular mechanisms hampering the motility of these cells remained unrevealed. The current study aimed to unravel these mechanisms by subjecting the secretomes of MMP-8+ and control HSC-3 cells to Terminal Amine Isotopic Labeling of Substrates (TAILS) analysis to find novel candidate substrates for MMP-8. The analysis revealed cleaved proteins, including dysadherin, 60S ribosomal protein L13, kallikrein-5, lipolysis-stimulated lipoprotein receptor, matrix-remodeling-associated protein 7, POTE ankyrin domain family member E, stathmin 1 and tubulin alpha-1C chain, which were enriched in MMP-8+ secretomes. Dysadherin is known to promote metastasis of various cancers and decrease cell adhesion. MMP-8+ cells showed decreased levels of dysadherin, suggesting a cleavage by MMP-8 from the cell membrane. Moreover, the adhesion of MMP-8+ cells was enhanced which might affect the migration. Conclusions Several novel candidate substrates of MMP-8 were revealed by TAILS analysis. The potential substrates, including dysadherin, may play crucial role in the changed behavior of MMP-8+ cells and needs to be further explored for their potential role in OTSCC. The cleavage of tumor-promoting dysadherin from the cell membrane might be one of the mechanisms by which MMP-8 increases tumor cell adhesion and thereby suppresses migration.
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the human odontoblast cell layer and dental pulp proteomes and n terminomes
Journal of Dental Research, 2018Co-Authors: Simon R Abbey, Ulrich Eckhard, Nestor Solis, Giada Marino, Ian R Matthew, Christopher M OverallAbstract:The proteome and N-terminome of the human odontoblast cell layer were identified for the first time by shotgun proteomic and terminal amine Isotopic Labeling of substrates (TAILS) N-terminomic anal...
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identifying natural substrates for dipeptidyl peptidases 8 and 9 using terminal amine Isotopic Labeling of substrates tails reveals in vivo roles in cellular homeostasis and energy metabolism
Journal of Biological Chemistry, 2013Co-Authors: Claire H Wilson, Alain Doucet, Christopher M Overall, Dono Indarto, Lisa Pogson, Melissa R Pitman, Kym Mcnicholas, Ian R Menz, Catherine A AbbottAbstract:Abstract Dipeptidyl peptidases (DP) 8 and 9 are homologous, cytoplasmic N-terminal post-proline-cleaving enzymes that are anti-targets for the development of DP4 (DPPIV/CD26) inhibitors for treating type II diabetes. To date, DP8 and DP9 have been implicated in immune responses and cancer biology, but their pathophysiological functions and substrate repertoire remain unknown. This study utilizes terminal amine Isotopic Labeling of substrates (TAILS), an N-terminal positional proteomic approach, for the discovery of in vivo DP8 and DP9 substrates. In vivo roles for DP8 and DP9 in cellular metabolism and homeostasis were revealed via the identification of more than 29 candidate natural substrates and pathways affected by DP8/DP9 overexpression. Cleavage of 14 substrates was investigated in vitro; 9/14 substrates for both DP8 and DP9 were confirmed by MALDI-TOF MS, including two of high confidence, calreticulin and adenylate kinase 2. Adenylate kinase 2 plays key roles in cellular energy and nucleotide homeostasis. These results demonstrate remarkable in vivo substrate overlap between DP8/DP9, suggesting compensatory roles for these enzymes. This work provides the first global investigation into DP8 and DP9 substrates, providing a number of leads for future investigations into the biological roles and significance of DP8 and DP9 in human health and disease.
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Identifying and quantifying proteolytic events and the natural N terminome by terminal amine Isotopic Labeling of substrates
Nature Protocols, 2011Co-Authors: Oded Kleifeld, Alain Doucet, Anna Prudova, Jayachandran N Kizhakkedathu, Magda Gioia, Ulrich Auf Dem Keller, Christopher M OverallAbstract: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.
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identifying and quantifying proteolytic events and the natural n terminome by terminal amine Isotopic Labeling of substrates
Nature Protocols, 2011Co-Authors: Oded Kleifeld, Alain Doucet, Ulrich Auf Dem Keller, Anna Prudova, Jayachandran N Kizhakkedathu, Magda Gioia, Christopher M OverallAbstract:Identifying and quantifying proteolytic events and the natural N terminome by terminal amine Isotopic Labeling of substrates
Hideo Iwai - One of the best experts on this subject based on the ideXlab platform.
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Segmental Isotopic Labeling by asparaginyl endopeptidase-mediated protein ligation
Journal of Biomolecular NMR, 2018Co-Authors: Kornelia M. Mikula, Luisa Krumwiede, Andreas Plückthun, Hideo IwaiAbstract:Segmental Isotopic Labeling can facilitate NMR studies of large proteins, multi-domain proteins, and proteins with repetitive sequences by alleviating NMR signal overlaps. Segmental Isotopic Labeling also allows us to investigate an individual domain in the context of a full-length protein by NMR. Several established methods are available for segmental Isotopic Labeling such as intein-mediated ligation, but each has specific requirements and limitations. Here, we report an enzymatic approach using bacterially produced asparagine endopeptidase from Oldenlandia affinis for segmental Isotopic Labeling of a protein with repetitive sequences, a designed armadillo repeat protein, by overcoming some of the shortcomings of enzymatic ligation for segmental Isotopic Labeling.
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segmental Isotopic Labeling of a single domain globular protein without any refolding step by an asparaginyl endopeptidase
FEBS Letters, 2017Co-Authors: Kornelia M. Mikula, Igor Tascon, Jenni Tommila, Hideo IwaiAbstract:Asparaginyl endopeptidases (AEPs) catalyze head-to-tail backbone cyclization of naturally occurring cyclic peptides such as cyclotides, and have become an important peptide-engineering tool for macrocyclization and peptide ligation. Here, we report efficient protein ligation in trans by mimicking efficient backbone cyclization by an AEP without any excess of reactants. We demonstrate a practical application of segmental Isotopic Labeling for NMR studies of a single-domain globular protein without any refolding step using the recombinant AEP prepared from Escherichia coli. This simple protein ligation approach using an AEP could be applied for incorporation of various biophysical probes into proteins as well as post-translational production of full-length proteins.
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Segmental Isotopic Labeling of a 140 kDa dimeric multi-domain protein CheA from Escherichia coli by expressed protein ligation and protein trans-splicing
Journal of Biomolecular NMR, 2012Co-Authors: Yuichi Minato, Takumi Ueda, Asako Machiyama, Ichio Shimada, Hideo IwaiAbstract:Segmental Isotopic Labeling is a powerful Labeling tool to facilitate NMR studies of larger proteins by not only alleviating the signal overlap problem but also retaining features of uniform Isotopic Labeling. Although two approaches, expressed protein ligation (EPL) and protein trans -splicing (PTS), have been mainly used for segmental Isotopic Labeling, there has been no single example in which both approaches have been directly used with an identical protein. Here we applied both EPL and PTS methods to a 140 kDa dimeric multi-domain protein E. coli CheA, and successfully produced the ligated CheA dimer by both approaches. In EPL approach, extensive optimization of the ligation sites and the conditions were required to obtain sufficient amount for an NMR sample of CheA, because CheA contains a dimer forming domain and it was not possible to achieve high reactant concentrations (1–5 mM) of CheA fragments for the ideal EPL condition, thereby resulting in the low yield of segmentally labelled CheA dimer. PTS approach sufficiently produced segmentally labeled ligated CheA in vivo as well as in vitro without extensive optimizations. This is presumably because CheA has self-contained domains connected with long linkers, accommodating a seven-residue mutation without loss of the function, which was introduced by PTS to achieve the high yield. PTS approach was less laborious than EPL approach for the routine preparation of segmentally-isotope labeled CheA dimer. Both approaches remain to be further developed for facilitating preparations of segmental isotope-labelled samples without extensive optimizations for ligation.
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segmental Isotopic Labeling of a 140 kda dimeric multi domain protein chea from escherichia coli by expressed protein ligation and protein trans splicing
Journal of Biomolecular NMR, 2012Co-Authors: Yuichi Minato, Takumi Ueda, Asako Machiyama, Ichio Shimada, Hideo IwaiAbstract:Segmental Isotopic Labeling is a powerful Labeling tool to facilitate NMR studies of larger proteins by not only alleviating the signal overlap problem but also retaining features of uniform Isotopic Labeling. Although two approaches, expressed protein ligation (EPL) and protein trans-splicing (PTS), have been mainly used for segmental Isotopic Labeling, there has been no single example in which both approaches have been directly used with an identical protein. Here we applied both EPL and PTS methods to a 140 kDa dimeric multi-domain protein E. coli CheA, and successfully produced the ligated CheA dimer by both approaches. In EPL approach, extensive optimization of the ligation sites and the conditions were required to obtain sufficient amount for an NMR sample of CheA, because CheA contains a dimer forming domain and it was not possible to achieve high reactant concentrations (1–5 mM) of CheA fragments for the ideal EPL condition, thereby resulting in the low yield of segmentally labelled CheA dimer. PTS approach sufficiently produced segmentally labeled ligated CheA in vivo as well as in vitro without extensive optimizations. This is presumably because CheA has self-contained domains connected with long linkers, accommodating a seven-residue mutation without loss of the function, which was introduced by PTS to achieve the high yield. PTS approach was less laborious than EPL approach for the routine preparation of segmentally-isotope labeled CheA dimer. Both approaches remain to be further developed for facilitating preparations of segmental isotope-labelled samples without extensive optimizations for ligation.
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segmental Isotopic Labeling of multi domain and fusion proteins by protein trans splicing in vivo and in vitro
Nature Protocols, 2010Co-Authors: Mikko Muona, Sesilja A Aranko, Vytas Raulinaitis, Hideo IwaiAbstract:Segmental Isotopic Labeling is a powerful Labeling technique for reducing nuclear magnetic resonance (NMR) signal overlap, which is associated with larger proteins by incorporating stable isotopes into only one region of a protein for NMR detections. Segmental Isotopic Labeling can not only reduce complexities of NMR spectra but also retain possibilities to carry out sequential resonance assignments by triple-resonance NMR experiments. We described in vivo (i.e., in Escherichia coli) and in vitro protocols for segmental Isotopic Labeling of multi-domain and fusion proteins via protein trans-splicing (PTS) using split DnaE intein without any refolding steps or alpha-thioester modification. The advantage of PTS approach is that it can be carried out in vivo by time-delayed dual-expression system with two controllable promoters. A segmentally isotope-labeled protein can be expressed in Escherichia coli within 1 d once required vectors are constructed. The total preparation time of a segmentally labeled sample can be as short as 7-13 d depending on the protocol used.
Oded Kleifeld - One of the best experts on this subject based on the ideXlab platform.
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Identifying and quantifying proteolytic events and the natural N terminome by terminal amine Isotopic Labeling of substrates
Nature Protocols, 2011Co-Authors: Oded Kleifeld, Alain Doucet, Anna Prudova, Jayachandran N Kizhakkedathu, Magda Gioia, Ulrich Auf Dem Keller, Christopher M OverallAbstract: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.
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identifying and quantifying proteolytic events and the natural n terminome by terminal amine Isotopic Labeling of substrates
Nature Protocols, 2011Co-Authors: Oded Kleifeld, Alain Doucet, Ulrich Auf Dem Keller, Anna Prudova, Jayachandran N Kizhakkedathu, Magda Gioia, Christopher M OverallAbstract:Identifying and quantifying proteolytic events and the natural N terminome by terminal amine Isotopic Labeling of substrates
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Isotopic Labeling of terminal amines in complex samples identifies protein n termini and protease cleavage products
Nature Biotechnology, 2010Co-Authors: Oded Kleifeld, Alain Doucet, Ulrich Auf Dem Keller, Anna Prudova, Oliver Schilling, Rajesh K Kainthan, Amanda E Starr, Leonard J Foster, Jayachandran N Kizhakkedathu, Christopher M OverallAbstract:Effective proteome-wide strategies that distinguish the N-termini of proteins from the N-termini of their protease cleavage products would accelerate identification of the substrates of proteases with broad or unknown specificity. Our approach, named terminal amine Isotopic Labeling of substrates (TAILS), addresses this challenge by using dendritic polyglycerol aldehyde polymers that remove tryptic and C-terminal peptides. We analyze unbound naturally acetylated, cyclized or labeled N-termini from proteins and their protease cleavage products by tandem mass spectrometry, and use peptide isotope quantification to discriminate between the substrates of the protease of interest and the products of background proteolysis. We identify 731 acetylated and 132 cyclized N-termini, and 288 matrix metalloproteinase (MMP)-2 cleavage sites in mouse fibroblast secretomes. We further demonstrate the potential of our strategy to link proteases with defined biological pathways in complex samples by analyzing mouse inflammatory bronchoalveolar fluid and showing that expression of the poorly defined breast cancer protease MMP-11 in MCF-7 human breast cancer cells cleaves both endoplasmin and the immunomodulator and apoptosis inducer galectin-1.
David Cowburn - One of the best experts on this subject based on the ideXlab platform.
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Segmental Isotopic Labeling of Proteins for NMR Study Using Intein Technology.
Methods of Molecular Biology, 2016Co-Authors: David CowburnAbstract:Segmental Isotopic Labeling of samples for NMR studies is attractive for large complex biomacromolecular systems, especially for studies of function-related protein-ligand interactions and protein dynamics (Goto and Kay, Curr Opin Struct Biol 10: 585-592, 2000; Rosa et al., Molecules (Basel, Switzerland) 18: 440, 2013; Hiroaki, Expert Opin Drug Discovery 8: 523-536, 2013). Advantages of segmental Isotopic Labeling include selective examination of specific segment(s) within a protein by NMR, significantly reducing the spectral complexity for large proteins, and allowing for the application of a variety of solution-based NMR strategies. By utilizing intein techniques (Wood and Camarero, J Biol Chem 289: 14512-14519, 2014; Paulus, Annu Rev Biochem 69: 447-496, 2000), two related approaches can generally be used in the segmental Isotopic Labeling of proteins: expressed protein ligation (Muir, Annu Rev Biochem 72: 249289, 2003) and protein trans - splicing (Shah et al., J Am Chem Soc 134: 11338-11341, 2012). Here, we describe general implementation and latest improvements of expressed protein ligation method for the production of segmental Isotopic labeled NMR samples.
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Segmental Isotopic Labeling of proteins for nuclear magnetic resonance.
Methods in Enzymology, 2009Co-Authors: Rong Xu, David CowburnAbstract:Abstract Nuclear magnetic resonance (NMR) spectroscopy has emerged as one of the principle techniques of structural biology. It is not only a powerful method for elucidating the three‐dimensional structures under near physiological conditions but also a convenient method for studying protein‐ligand interactions and protein dynamics. A major drawback of macromolecular NMR is its size limitation, caused by slower tumbling rates and greater complexity of the spectra as size increases. Segmental Isotopic Labeling allows for specific segment(s) within a protein to be selectively examined by NMR, thus significantly reducing the spectral complexity for large proteins and allowing for the application of a variety of solution‐based NMR strategies. Two related approaches are generally used in the segmental Isotopic Labeling of proteins: expressed protein ligation and protein trans ‐splicing. Here, we describe the methodology and recent application of expressed protein ligation and protein trans ‐splicing for NMR structural studies of proteins and protein complexes. We also describe the protocol used in our lab for the segmental Isotopic Labeling of a 50‐kDa protein Csk (C‐terminal Src kinase) using expressed protein ligation methods.
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autoregulation of a bacterial σ factor explored by using segmental Isotopic Labeling and nmr
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: David Cowburn, Julio A Camarero, Alexander Shekhtman, Elizabeth A Campbell, Mark Chlenov, Tanja M Gruber, Donald A Bryant, Seth A DarstAbstract:Bacterial σ factors combine with the catalytic core RNA polymerase to direct the process of transcription initiation through sequence-specific interactions with the −10 and −35 elements of promoter DNA. In the absence of core RNA polymerase, the DNA-binding function of σ is autoinhibited by its own N-terminal 90 amino acids (region 1.1), putatively by a direct interaction with conserved region 4.2, which binds the −35 promoter element. In the present work, this mechanism of autoinhibition was studied by using a combination of NMR spectroscopy and segmental Isotopic Labeling of a σ70-like subunit from Thermotoga maritima. Our data argue strongly against a high-affinity interaction between these two domains. Instead we suggest that autoinhibition of DNA binding occurs through an indirect steric and/or electrostatic mechanism. More generally, the present work illustrates the power of segmental Isotopic Labeling for probing molecular interactions in large proteins by NMR.
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chemical ligation of folded recombinant proteins segmental Isotopic Labeling of domains for nmr studies
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Brenda Ayers, David Cowburn, Tom W MuirAbstract:A convenient in vitro chemical ligation strategy has been developed that allows folded recombinant proteins to be joined together. This strategy permits segmental, selective Isotopic Labeling of the product. The src homology type 3 and 2 domains (SH3 and SH2) of Abelson protein tyrosine kinase, which constitute the regulatory apparatus of the protein, were individually prepared in reactive forms that can be ligated together under normal protein-folding conditions to form a normal peptide bond at the ligation junction. This strategy was used to prepare NMR sample quantities of the Abelson protein tyrosine kinase-SH(32) domain pair, in which only one of the domains was labeled with 15N. Mass spectrometry and NMR analyses were used to confirm the structure of the ligated protein, which was also shown to have appropriate ligand-binding properties. The ability to prepare recombinant proteins with selectively labeled segments having a single-site mutation, by using a combination of expression of fusion proteins and chemical ligation in vitro, will increase the size limits for protein structural determination in solution with NMR methods. In vitro chemical ligation of expressed protein domains will also provide a combinatorial approach to the synthesis of linked protein domains.
Alain Doucet - One of the best experts on this subject based on the ideXlab platform.
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identifying natural substrates for dipeptidyl peptidases 8 and 9 using terminal amine Isotopic Labeling of substrates tails reveals in vivo roles in cellular homeostasis and energy metabolism
Journal of Biological Chemistry, 2013Co-Authors: Claire H Wilson, Alain Doucet, Christopher M Overall, Dono Indarto, Lisa Pogson, Melissa R Pitman, Kym Mcnicholas, Ian R Menz, Catherine A AbbottAbstract:Abstract Dipeptidyl peptidases (DP) 8 and 9 are homologous, cytoplasmic N-terminal post-proline-cleaving enzymes that are anti-targets for the development of DP4 (DPPIV/CD26) inhibitors for treating type II diabetes. To date, DP8 and DP9 have been implicated in immune responses and cancer biology, but their pathophysiological functions and substrate repertoire remain unknown. This study utilizes terminal amine Isotopic Labeling of substrates (TAILS), an N-terminal positional proteomic approach, for the discovery of in vivo DP8 and DP9 substrates. In vivo roles for DP8 and DP9 in cellular metabolism and homeostasis were revealed via the identification of more than 29 candidate natural substrates and pathways affected by DP8/DP9 overexpression. Cleavage of 14 substrates was investigated in vitro; 9/14 substrates for both DP8 and DP9 were confirmed by MALDI-TOF MS, including two of high confidence, calreticulin and adenylate kinase 2. Adenylate kinase 2 plays key roles in cellular energy and nucleotide homeostasis. These results demonstrate remarkable in vivo substrate overlap between DP8/DP9, suggesting compensatory roles for these enzymes. This work provides the first global investigation into DP8 and DP9 substrates, providing a number of leads for future investigations into the biological roles and significance of DP8 and DP9 in human health and disease.
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Identifying and quantifying proteolytic events and the natural N terminome by terminal amine Isotopic Labeling of substrates
Nature Protocols, 2011Co-Authors: Oded Kleifeld, Alain Doucet, Anna Prudova, Jayachandran N Kizhakkedathu, Magda Gioia, Ulrich Auf Dem Keller, Christopher M OverallAbstract: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.
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identifying and quantifying proteolytic events and the natural n terminome by terminal amine Isotopic Labeling of substrates
Nature Protocols, 2011Co-Authors: Oded Kleifeld, Alain Doucet, Ulrich Auf Dem Keller, Anna Prudova, Jayachandran N Kizhakkedathu, Magda Gioia, Christopher M OverallAbstract:Identifying and quantifying proteolytic events and the natural N terminome by terminal amine Isotopic Labeling of substrates
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Isotopic Labeling of terminal amines in complex samples identifies protein n termini and protease cleavage products
Nature Biotechnology, 2010Co-Authors: Oded Kleifeld, Alain Doucet, Ulrich Auf Dem Keller, Anna Prudova, Oliver Schilling, Rajesh K Kainthan, Amanda E Starr, Leonard J Foster, Jayachandran N Kizhakkedathu, Christopher M OverallAbstract:Effective proteome-wide strategies that distinguish the N-termini of proteins from the N-termini of their protease cleavage products would accelerate identification of the substrates of proteases with broad or unknown specificity. Our approach, named terminal amine Isotopic Labeling of substrates (TAILS), addresses this challenge by using dendritic polyglycerol aldehyde polymers that remove tryptic and C-terminal peptides. We analyze unbound naturally acetylated, cyclized or labeled N-termini from proteins and their protease cleavage products by tandem mass spectrometry, and use peptide isotope quantification to discriminate between the substrates of the protease of interest and the products of background proteolysis. We identify 731 acetylated and 132 cyclized N-termini, and 288 matrix metalloproteinase (MMP)-2 cleavage sites in mouse fibroblast secretomes. We further demonstrate the potential of our strategy to link proteases with defined biological pathways in complex samples by analyzing mouse inflammatory bronchoalveolar fluid and showing that expression of the poorly defined breast cancer protease MMP-11 in MCF-7 human breast cancer cells cleaves both endoplasmin and the immunomodulator and apoptosis inducer galectin-1.