An Aptly Positioned Azido Group in the Spacer of a Protein Cross‐Linker for Facile Mapping of Lysines in Close Proximity

Kasper, Piotr T.; Back, Jaap Willem; Vitale, Maxime R.; Hartog, Aloysius F.; Roseboom, Winfried; Koning, Leo J. de; Maarseveen, Jan H. van; Muijsers, Anton O.; Koster, Chris G. de; Jong, Luitzen de

doi:10.1002/cbic.200700150

Cited by 39 publications

(54 citation statements)

References 43 publications

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“…TCEP induces a set of competing reactions in azhal-containing peptides present in primary fractions, i.e. conversion of the azido group to an amine or hydroxyl group and cleavage of the peptide bond at the C-terminal side of azhal residues (9,16,17). The subsequent enrichment is based on a difference in retention time during the secondary chromatographic runs between TCEP-induced reaction products and the bulk of unmodified peptides that are present in the particular primary fraction subjected to TCEP treatment (9).…”

Section: Methodsmentioning

confidence: 99%

Proteome-wide Alterations in Escherichia coli Translation Rates upon Anaerobiosis

Kramer

Sprenger

Nessen

et al. 2010

Molecular & Cellular Proteomics

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Enzyme reprofiling in bacteria during adaptation from one environmental condition to another may be regulated by both transcription and translation. However, little is known about the contribution of translational regulation. Recently, we have developed a pulse labeling method using the methionine analog azidohomoalanine to determine the relative amounts of proteins synthesized by Escherichia coli in a brief time frame upon a change in environmental conditions. Here we present an extension of our analytical strategy, which entails measuring changes in total protein levels on the same time scale as new protein synthesis. This allows identification of stable and labile proteins and demonstrates that altered levels of most newly synthesized proteins are the result of a change in translation rate rather than degradation rate. With this extended strategy, average relative translation rates for 10 min immediately after a switch from aerobiosis to anaerobiosis were determined. The majority of proteins with increased synthesis rates upon an anaerobic switch are involved in glycolysis and pathways aimed at preventing glycolysis grinding to a halt by a cellular redox imbalance. Our method can be used to compare relative translation rates with relative mRNA levels at the same time. Discrepancies between these parameters may reveal genes whose expression is regulated by translation rather than by transcription. This may help unravel molecular mechanism underlying changes in translation rates, e.g. mediated by small regulatory RNAs. Molecular & Cellular Proteomics 9:2508 -2516, 2010.An integrated view of the molecular events underlying adaptation of bacteria to major environmental changes requires insight into both transcriptional and post-transcriptional regulation of gene expression. With the availability of annotated genome databases, much has been learned about global changes induced in mRNA levels and the transcription factors involved as well as about changes in steady state protein levels upon a switch in environmental conditions. However, little is known concerning genome-wide changes in protein synthesis and degradation rates and about the contributions of transcription and translation to the regulation of gene expression when bacteria adapt to major changes in the environment. An important reason for this is the fact that it is much easier, using genome-wide microarray analysis, to find candidate genes with expression levels regulated via RNA transcription or degradation than to identify candidate genes with expression levels regulated at the level of translation.Renewed interest in gene expression regulation at the posttranscriptional level in prokaryotes has been sparked recently by the discovery in 2001-2002 of the existence of large numbers of small regulatory RNAs (sRNAs) 1 in Escherichia coli and other bacteria (1-4). These sRNAs may regulate translation of numerous mRNAs (5) often mediated by the RNA chaperone Hfq (6). Although the function of many sRNAs is not yet known, several recently reviewed findings (...

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Section: Methodsmentioning

confidence: 99%

Proteome-wide Alterations in Escherichia coli Translation Rates upon Anaerobiosis

Kramer

Sprenger

Nessen

et al. 2010

Molecular & Cellular Proteomics

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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%

“…These include UV photocleavable (33), chemical cleavable (19), isotopically coded cleavable (24), and MS-cleavable reagents (16, 26, 34 -38). MS-cleavable cross-linkers have received considerable attention because the resulting cross-linked products can be identified based on their characteristic fragmentation behavior observed during MS analysis.…”

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

365

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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“…These include incorporating an affinity tag into the linker for peptide purification [7][8][9][10][11][12][13][14], as well as various isotope labeling strategies [15][16][17][18][19]. These methods, however, often yield complex, difficult to analyze MS/MS data, as the intermolecular cross-links consist of two peptides covalently joined, and product ions from both peptides are present in the spectrum [20].…”

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confidence: 99%

A Negative Ion Mass Spectrometry Approach to Identify Cross-Linked Peptides Utilizing Characteristic Disulfide Fragmentations

Calabrese

Good

Wang

et al. 2012

J. Am. Soc. Mass Spectrom.

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Chemical cross-linking combined with mass spectrometry (MS) is an analytical tool used to elucidate the topologies of proteins and protein complexes. However, identification of the low abundance cross-linked peptides and modification sites amongst a large quantity of proteolytic fragments remains challenging. In this work, we present a strategy to identify cross-linked peptides by negative ion MS for the first time. This approach is based around the facile cleavages of disulfide bonds in the negative mode, and allows identification of cross-linked products based on their characteristic fragmentations. MS 3 analysis of the cross-linked peptides allows for their sequencing and identification, with residue specific location of cross-linking sites. We demonstrate the applicability of the commercially available cystine based cross-linking reagent dithiobis(succinimidyl) propionate (DSP) and identify cross-linked peptides from ubiquitin. In each instance, the characteristic fragmentation behavior of the cross-linked species is described. The data presented here indicate that this negative ion approach may be a useful tool to characterize the structures of proteins and protein complexes, and provides the basis for the development of high throughput negative ion MS chemical cross-linking strategies.

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An Aptly Positioned Azido Group in the Spacer of a Protein Cross‐Linker for Facile Mapping of Lysines in Close Proximity

Cited by 39 publications

References 43 publications

Proteome-wide Alterations in Escherichia coli Translation Rates upon Anaerobiosis

Proteome-wide Alterations in Escherichia coli Translation Rates upon Anaerobiosis

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

A Negative Ion Mass Spectrometry Approach to Identify Cross-Linked Peptides Utilizing Characteristic Disulfide Fragmentations

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