Selective enrichment of newly synthesized proteins for quantitative secretome analysis

Eichelbaum, Katrin; Winter, Markus; Díaz, Mauricio Berriel; Herzig, Stephan; Krijgsveld, Jeroen

doi:10.1038/nbt.2356

Cited by 233 publications

(270 citation statements)

References 47 publications

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“…AHA contains an azide group enabling capture of proteins via click chemistry (Dieterich et al, 2006). Combining AHA with SILAC enables relatively short pulse times (Eichelbaum and Krijgsveld, 2014;Eichelbaum et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

Kinetic Analysis of Protein Stability Reveals Age-Dependent Degradation

McShane

Sin

Zauber

et al. 2016

Cell

290

293

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Do young and old protein molecules have the same probability to be degraded? We addressed this question using metabolic pulse-chase labeling and quantitative mass spectrometry to obtain degradation profiles for thousands of proteins. We find that over 10% of proteins are degraded non-exponentially. Specifically, proteins are less stable in the first few hours of their life and stabilize with age. Degradation profiles are conserved and similar in two cell types.Many non-exponentially degraded (NED) proteins are subunits of complexes that are produced in super-stoichiometric amounts relative to their exponentially degraded (ED) counterparts.Within complexes, NED proteins have larger interaction interfaces and assemble earlier than ED subunits. Amplifying genes encoding NED proteins increases their initial degradation.Consistently, decay profiles can predict protein level attenuation in aneuploid cells. Together, our data show that non-exponential degradation is common, conserved and has important consequences for complex formation and regulation of protein abundance.3

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Section: Introductionmentioning

confidence: 99%

Kinetic Analysis of Protein Stability Reveals Age-Dependent Degradation

McShane

Sin

Zauber

et al. 2016

Cell

290

293

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“…The enrichment from conditioned media was performed as described previously (29). Cells were lysed in urea buffer for 15 min and then sonicated (three times for 10 s each time with 1 min of cooling in between), agitated for 5 min, and centrifuged and then subjected to the catalytic reaction using 200 l of agarose resin slurry.…”

Section: Methodsmentioning

confidence: 99%

“…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). Recently, we have demonstrated the utility of combining pulsed AHA and pulsed SILAC labeling for the detection and quantification of newly synthesized proteins in the secretomes of a range of cell types, including macrophages (29).…”

mentioning

confidence: 99%

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

Eichelbaum

Krijgsveld

2014

Molecular & Cellular Proteomics

Self Cite

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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“…23 More importantly, the enrichment of these newly synthesized proteins is possible via an alkyne-bearing biotinylated tag and a 'click chemistry' reaction. Although other metabolic labeling techniques such as pulsed-SILAC 42 have been used to label and analyze newly synthesized proteins, the lack of enrichment of the labeled proteins would limit their applications in the study of autophagy, for which the protein synthesis machinery is largely suppressed. The enrichment of low-abundant proteins by BONCAT also eliminates the necessity of extensive peptide fractioning.…”

Section: Discussionmentioning

confidence: 99%

“…The enrichment of low-abundant proteins by BONCAT also eliminates the necessity of extensive peptide fractioning. 42 The advantage of BONCAT in de novo protein enrichment and identification enables it to be applied in studying the events that occur at early or intermediate stages of biological processes, such as autophagy, which require a short period of labeling.…”

Section: Discussionmentioning

confidence: 99%

Quantitative chemical proteomics profiling of de novo protein synthesis during starvation-mediated autophagy

et al. 2016

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Autophagy is an intracellular degradation mechanism in response to nutrient starvation. Via autophagy, some nonessential cellular constituents are degraded in a lysosome-dependent manner to generate biomolecules that can be utilized for maintaining the metabolic homeostasis. Although it is known that under starvation the global protein synthesis is significantly reduced mainly due to suppression of MTOR (mechanistic target of rapamycin serine/threonine kinase), emerging evidence demonstrates that de novo protein synthesis is involved in the autophagic process. However, characterizing these de novo proteins has been an issue with current techniques. Here, we developed a novel method to identify newly synthesized proteins during starvation-mediated autophagy by combining bio-orthogonal noncanonical amino acid tagging (BONCAT) and isobaric tags for relative and absolute quantitation (iTRAQ TM ). Using bio-orthogonal metabolic tagging, L-azidohomoalanine (AHA) was incorporated into newly synthesized proteins which were then enriched with avidin beads after a click reaction between alkyne-bearing biotin and AHA's bio-orthogonal azide moiety. The enriched proteins were subjected to iTRAQ labeling for protein identification and quantification using liquid chromatography-tandem mass spectrometry (LC-MS/ MS). Via the above approach, we identified and quantified a total of 1176 proteins and among them 711 proteins were found to meet our defined criteria as de novo synthesized proteins during starvationmediated autophagy. The characterized functional profiles of the 711 newly synthesized proteins by bioinformatics analysis suggest their roles in ensuring the prosurvival outcome of autophagy. Finally, we performed validation assays for some selected proteins and found that knockdown of some genes has a significant impact on starvation-induced autophagy. Thus, we think that the BONCAT-iTRAQ approach is effective in the identification of newly synthesized proteins and provides useful insights to the molecular mechanisms and biological functions of autophagy.

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Selective enrichment of newly synthesized proteins for quantitative secretome analysis

Cited by 233 publications

References 47 publications

Kinetic Analysis of Protein Stability Reveals Age-Dependent Degradation

Kinetic Analysis of Protein Stability Reveals Age-Dependent Degradation

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

Quantitative chemical proteomics profiling of de novo protein synthesis during starvation-mediated autophagy

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