Selective Enrichment of Azide-Containing Peptides from Complex Mixtures

Nessen, Merel A.; Kramer, Gertjan; Back, Jaap Willem; Baskin, Jeremy M.; Smeenk, Linde E. J.; Koning, Leo J. de; Maarseveen, Jan H. van; Jong, Luitzen de; Bertozzi, Carolyn R.; Hiemstra, Henk; Koster, Chris G. de

doi:10.1021/pr900257z

Cited by 96 publications

(87 citation statements)

References 45 publications

(105 reference statements)

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“…Thus, even if Anl were incorporated 10× more slowly at internal sites than at N-terminal sites, one would expect to detect comparable numbers of N-terminal and internal modifications. Consistent with this argument are the results of Nessen et al (50), who reported an analysis of peptides isolated from proteome-wide labeling in E. coli and found 10-fold more Aha modifications at internal sites than at N termini.…”

Section: Discussionsupporting

confidence: 64%

Mutant methionyl-tRNA synthetase from bacteria enables site-selective N-terminal labeling of proteins expressed in mammalian cells

Ngo

Schuman

Tirrell

2013

Proc. Natl. Acad. Sci. U.S.A.

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Newly synthesized cellular proteins can be tagged with a variety of metabolic labels that distinguish them from preexisting proteins and allow them to be identified and tracked. Many such labels are incorporated into proteins via the endogenous cellular machinery and can be used in numerous cell types and organisms. Though broad applicability has advantages, we aimed to develop a strategy to restrict protein labeling to specified mammalian cells that express a transgene. Here we report that heterologous expression of a mutant methionyl-tRNA synthetase from Escherichia coli permits incorporation of azidonorleucine (Anl) into proteins made in mammalian (HEK293) cells. Anl is incorporated site-selectively at N-terminal positions (in competition with initiator methionines) and is not found at internal sites. Site selectivity is enabled by the fact that the bacterial synthetase aminoacylates mammalian initiator tRNA, but not elongator tRNA. N-terminally labeled proteins can be selectively conjugated to a variety of useful probes; here we demonstrate use of this system in enrichment and visualization of proteins made during various stages of the cell cycle. N-terminal incorporation of Anl may also be used to engineer modified proteins for therapeutic and other applications.

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Section: Discussionsupporting

confidence: 64%

Mutant methionyl-tRNA synthetase from bacteria enables site-selective N-terminal labeling of proteins expressed in mammalian cells

Ngo

Schuman

Tirrell

2013

Proc. Natl. Acad. Sci. U.S.A.

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“…These include selective enrichment using affinity purification with biotinylated cross-linkers (15)(16)(17) and click chemistry with alkyne-tagged (18) or azide-tagged (19,20) cross-linkers. In addition, Staudinger ligation has recently been shown to be effective for selective enrichment of azidetagged cross-linked peptides (21).…”

Section: Dsso Contains Two Symmetric Collision-induced Dissociation (mentioning

confidence: 99%

Development of a Novel Cross-linking Strategy for Fast and Accurate Identification of Cross-linked Peptides of Protein Complexes

Kao¹,

Chiu²,

Vellucci³

et al. 2011

Molecular & Cellular Proteomics

366

570

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Knowledge of elaborate structures of protein complexes is fundamental for understanding their functions and regulations. Although cross-linking coupled with mass spectrometry (MS) has been presented as a feasible strategy for structural elucidation of large multisubunit protein complexes, this method has proven challenging because of technical difficulties in unambiguous identification of cross-linked peptides and determination of cross-linked sites by MS analysis. In this work, we developed a novel cross-linking strategy using a newly designed MS-cleavable cross-linker, disuccinimidyl sulfoxide (DSSO). DSSO contains two symmetric collision-induced dissociation (CID)-cleavable sites that allow effective identification of DSSO-cross-linked peptides based on their distinct fragmentation patterns unique to cross-linking types (i.e. interlink, intralink, and dead end). The CID-induced separation of interlinked peptides in MS/MS permits MS 3 analysis of single peptide chain fragment ions with defined modifications (due to DSSO remnants) for easy interpretation and unambiguous identification using existing database searching tools. Integration of data analyses from three generated data sets (MS, MS/MS, and MS Proteins form stable and dynamic multisubunit complexes under different physiological conditions to maintain cell viability and normal cell homeostasis. Detailed knowledge of protein interactions and protein complex structures is fundamental to understanding how individual proteins function within a complex and how the complex functions as a whole. However, structural elucidation of large multisubunit protein complexes has been difficult because of a lack of technologies that can effectively handle their dynamic and heterogeneous nature. Traditional methods such as nuclear magnetic resonance (NMR) analysis and x-ray crystallography can yield detailed information on protein structures; however, NMR spectroscopy requires large quantities of pure protein in a specific solvent, whereas x-ray crystallography is often limited by the crystallization process.In recent years, chemical cross-linking coupled with mass spectrometry (MS) has become a powerful method for studying protein interactions (1-3). Chemical cross-linking stabilizes protein interactions through the formation of covalent bonds and allows the detection of stable, weak, and/or transient protein-protein interactions in native cells or tissues (4 -9). In addition to capturing protein interacting partners, many studies have shown that chemical cross-linking can yield low resolution structural information about the constraints within a molecule (2, 3, 10) or protein complex (11-13). The application of chemical cross-linking, enzymatic digestion, and subsequent mass spectrometric and computational analyses for the elucidation of three-dimensional protein structures offers distinct advantages over traditional methods because of its speed, sensitivity, and versatility. Identification of cross-linked peptides provides distance constraints that aid in constructing...

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“…In such cases, affinity purification of newly synthesized proteins has been recently facilitated by pulse labeling with noncanonical amino acids. Specifically, the replacement of methionine by its azide-bearing analog azidohomoalanine (AHA) allows the enrichment of newly synthesized proteins via click-chemistry using alkyne-functionalized beads (26,27). The combination of this enrichment approach with stable isotope labeling methods allows the quantitative analysis of proteome dynamics, providing insight into the regulation of protein synthesis (28).…”

mentioning

confidence: 99%

Rapid Temporal Dynamics of Transcription, Protein Synthesis, and Secretion during Macrophage Activation

Eichelbaum

Krijgsveld

2014

Molecular & Cellular Proteomics

107

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Macrophages provide the first line of host defense with their capacity to react to an array of cytokines and bacterial components requiring tight regulation of protein expression and secretion to invoke a properly tuned innate immune response. To capture the dynamics of this system, we introduce a novel method combining pulsed stable isotope labeling with amino acids in cell culture (SILAC) with pulse labeling using the methionine analog azidohomoalanine that allows the enrichment of newly synthesized proteins via click-chemistry followed by their identification and quantification by mass spectrometry. We show that this permits the analysis of proteome changes on a rapid time scale, as evidenced by the detection of 4852 newly synthesized proteins after only a 20-min SILAC pulse. We have applied this methodology to study proteome response during macrophage activation in a time-course manner. We have combined this with full proteome, transcriptome, and secretome analyses, producing an integrative analysis of the first 3 h of lipopolysaccharide-induced macrophage activation. We observed the rapid induction of multiple processes well known to TLR4 signaling, as well as anti-inflammatory proteins and proteins not previously associated with immune response. By correlating transcriptional, translational, and secretory events, we derived novel mechanistic principles of processes specifically induced by lipopolysaccharides, including ectodomain shedding and proteolytic processing of transmembrane and extracellular proteins and protein secretion independent of transcription. In conclusion, we demonstrate that the combination of pulsed azidohomoalanine and pulsed SILAC permits the detailed characterization of proteomic events on a rapid time scale. We anticipate that this approach will be very useful in probing the immediate effects of cellular stimuli and will provide mechanistic insight into cellular perturbation in multiple biological systems.

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Selective Enrichment of Azide-Containing Peptides from Complex Mixtures

Cited by 96 publications

References 45 publications

Mutant methionyl-tRNA synthetase from bacteria enables site-selective N-terminal labeling of proteins expressed in mammalian cells

Mutant methionyl-tRNA synthetase from bacteria enables site-selective N-terminal labeling of proteins expressed in mammalian cells

Development of a Novel Cross-linking Strategy for Fast and Accurate Identification of Cross-linked Peptides of Protein Complexes

Rapid Temporal Dynamics of Transcription, Protein Synthesis, and Secretion during Macrophage Activation

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