An official website of the United States government
A Highly Efficient Click Linker for Enrichment of Alkyne‐Tagged Proteins in Living Cells
Abstract Alkyne tags have been widely used for the enrichment of labeled proteins to enable their profiling at a proteome‐wide scale by mass spectrometry. The key component in the enrichment process is an azido‐terminated cleavable linker for capturing the labeled proteins/peptides via click reaction. Herein, we report a new efficient click linker (APAbiotin) featuring an acid‐cleavable acetal linkage end‐caped with a highly reactive picolyl azido head and a biotin handle for anchoring onto streptavidin‐coated supports. Using an amine‐reactive probe to profile the proteome structural changes in livingS. cerevisiaecells within 5 minutes of heat shock, we demonstrated that the linker allowed identification of >9400 labeled sites, among which 457–1656 with significantly altered reactivity upon heat shock. This study represented the first chemical labeling mass spectrometry (CL–MS)‐based profiling of proteome structural changes in living cells in response to external stimuli. Data are available via ProteomeXchange with identifier PXD051279.
more »
« less
- Award ID(s):
- 2005199
- PAR ID:
- 10514829
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Helvetica Chimica Acta
- Volume:
- 107
- Issue:
- 6
- ISSN:
- 0018-019X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
Abstract Azanone (HNO) is a reactive nitrogen species with pronounced biological activity and high therapeutic potential for cardiovascular dysfunction. A critical barrier to understanding the biology of HNO and furthering clinical development is the quantification and real‐time monitoring of its delivery in living systems. Herein, we describe the design and synthesis of the first chemiluminescent probe for HNO,HNOCL‐1, which can detect HNO generated from concentrations of Angeli's salt as low as 138 nmwith high selectivity based on the reaction with a phosphine group to form a self‐cleavable azaylide intermediate. We have capitalized on this high sensitivity to develop a generalizable kinetics‐based approach, which provides real‐time quantitative measurements of HNO concentration at the picomolar level.HNOCL‐1can monitor dynamics of HNO delivery in living cells and tissues, demonstrating the versatility of this method for tracking HNO in living systems.more » « less
-
Abstract Activity‐based protein profiling (ABPP) is a chemical proteomic method for investigating functional states of proteins in native biological settings. By quantifying changes in probe binding states of active and regulatory protein sites, ABPP reveals functional information on protein regulation and can be configured in competitive settings to determine global selectivity profiles of tool compounds and drugs in lysates, cells, and animals. Chemical probes used for ABPP analyses can target protein families with conserved enzymatic or structural features or can broadly profile the proteome using electrophiles with reactivity towards functional groups on amino acid side chains. The latter approach has provided insights to protein sites involved in allosteric regulation and non‐enzymatic functions. This review introduces quantitative ABPP workflows and discusses electrophilic groups used for ABPP profiling of functional sites in the proteome with an emphasis on tyrosine residues.more » « less
-
Proteins must be hydrated to function. Desiccation, a common event in an increasing number of ecosystems, can drive proteome-wide unfolding and aggregation. For cells to survive, proteins must disaggregate and retain their function upon rehydration. The molecular determinants that underlie protein desiccation resistance remain unknown. Here, we use mass spectrometry to show that some proteins possess an innate ability to survive dehydration and subsequent rehydration. Structural analysis correlates the ability of proteins to resist desiccation with their surface area chemistry. Remarkably, highly resistant proteins are responsible for the production of the cell's building blocks - amino acids, metabolites, and sugars. Conversely, those proteins that are desiccation-sensitive are responsible for ribosome biogenesis. As a result, the rehydrated proteome is preferentially enriched with metabolite and small molecule producers and depleted of ribosomes - the cell's heaviest consumers. We propose this functional bias allows cells to kickstart their metabolism and promote cell survival upon rehydration.more » « less
-
Abstract Histone acetyltransferases (HATs, also known as lysine acetyltransferases, KATs) catalyze acetylation of their cognate protein substrates using acetyl‐CoA (Ac‐CoA) as a cofactor and are involved in various physiological and pathological processes. Advances in mass spectrometry‐based proteomics have allowed the discovery of thousands of acetylated proteins and the specific acetylated lysine sites. However, due to the rapid dynamics and functional redundancy of HAT activities, and the limitation of using antibodies to capture acetylated lysines, it is challenging to systematically and precisely define both the substrates and sites directly acetylated by a given HAT. Here, we describe a chemoproteomic approach to identify and profile protein substrates of individual HAT enzymes on the proteomic scale. The approach involves protein engineering to enlarge the Ac‐CoA binding pocket of the HAT of interest, such that a mutant form is generated that can use functionalized acyl‐CoAs as a cofactor surrogate to bioorthogonally label its protein substrates. The acylated protein substrates can then be chemoselectively conjugated either with a fluorescent probe (for imaging detection) or with a biotin handle (for streptavidin pulldown and chemoproteomic identification). This modular chemical biology approach has been successfully implemented to identify protein substrates of p300, GCN5, and HAT1, and it is expected that this method can be applied to profile and identify the sub‐acetylomes of many other HAT enzymes. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Labeling HAT protein substrates with azide/alkyne‐biotin Alternate Protocol: Labeling protein substrates of HATs with azide/alkyne‐TAMRA for in‐gel visualization Support Protocol 1: Expression and purification of HAT mutants Support Protocol 2: Synthesis of Ac‐CoA surrogates Basic Protocol 2: Streptavidin enrichment of biotinylated HAT substrates Basic Protocol 3: Chemoproteomic identification of HAT substrates Basic Protocol 4: Validation of specific HAT substrates with western blottingmore » « less
