A nanostructure-initiator mass spectrometry-based enzyme activity assay

PDF Version Also Available for Download.

Description

We describe a Nanostructure-Initiator Mass Spectrometry (NIMS) enzymatic (Nimzyme) assay in which enzyme substrates are immobilized on the mass spectrometry surface by using fluorous-phase interactions. This 'soft' immobilization allows efficient desorption/ionization while also enabling the use of surface-washing steps to reduce signal suppression from complex biological samples, which results from the preferential retention of the tagged products and reactants. The Nimzyme assay is sensitive to subpicogram levels of enzyme, detects both addition and cleavage reactions (sialyltransferase and galactosidase), is applicable over a wide range of pHs and temperatures, and can measure activity directly from crude cell lysates. The ability of ... continued below

Creation Information

Siuzdak, Gary; Northen, Trent R.; Lee, Jinq-Chyi; Hoang, Linh; Raymond, Jason; Hwang, Der-Ren et al. March 10, 2008.

Context

This article is part of the collection entitled: Office of Scientific & Technical Information Technical Reports and was provided by UNT Libraries Government Documents Department to Digital Library, a digital repository hosted by the UNT Libraries. It has been viewed 37 times . More information about this article can be viewed below.

Who

People and organizations associated with either the creation of this article or its content.

Publisher

Provided By

UNT Libraries Government Documents Department

Serving as both a federal and a state depository library, the UNT Libraries Government Documents Department maintains millions of items in a variety of formats. The department is a member of the FDLP Content Partnerships Program and an Affiliated Archive of the National Archives.

Contact Us

What

Descriptive information to help identify this article. Follow the links below to find similar items on the Digital Library.

Description

We describe a Nanostructure-Initiator Mass Spectrometry (NIMS) enzymatic (Nimzyme) assay in which enzyme substrates are immobilized on the mass spectrometry surface by using fluorous-phase interactions. This 'soft' immobilization allows efficient desorption/ionization while also enabling the use of surface-washing steps to reduce signal suppression from complex biological samples, which results from the preferential retention of the tagged products and reactants. The Nimzyme assay is sensitive to subpicogram levels of enzyme, detects both addition and cleavage reactions (sialyltransferase and galactosidase), is applicable over a wide range of pHs and temperatures, and can measure activity directly from crude cell lysates. The ability of the Nimzyme assay to analyze complex mixtures is illustrated by identifying and directly characterizing {beta}-1,4-galactosidase activity from a thermophilic microbial community lysate. The optimal enzyme temperature and pH were found to be 65 C and 5.5, respectively, and the activity was inhibited by both phenylethyl-{beta}-d-thiogalactopyranoside and deoxygalactonojirimycin. Metagenomic analysis of the community suggests that the activity is from an uncultured, unsequenced {gamma}-proteobacterium. In general, this assay provides an efficient method for detection and characterization of enzymatic activities in complex biological mixtures prior to sequencing or cloning efforts. More generally, this approach may have important applications for screening both enzymatic and inhibitor libraries, constructing and screening glycan microarrays, and complementing fluorous-phase organic synthesis. The interest in leveraging mass spectrometry for studying enzyme activities in complex biological samples derives from its high sensitivity and specificity; however, signal suppression and significant sample preparation requirements limit its overall utility (1). Here we describe a Nanostructure-Initiator Mass Spectrometry (NIMS) enzymatic (Nimzyme) assay, which uses the fluorous liquid-coated surface of NIMS (2) to noncovalently attach enzyme substrates by means of fluorous tags. Enzymes play essential roles in a wide range of cellular processes and account for >20% of all drug targets (3). In addition, enzymes have found great utility in organic synthesis because they can efficiently catalyze chemical transformations that are difficult and inefficient to catalyze using conventional synthetic approaches. Furthermore, enzymatic transformations are particularly useful in reactions requiring multiple functional groups or stereo/regiochemically defined products (4). These properties make them particularly well suited for the synthesis and degradation of carbohydrates (5). Indeed, enzymatic approaches have found widespread applications in glycobiology (6, 7) and are of intense interest for the utilization of plant biomass for biofuels (8).

Source

  • Journal Name: Proceedings of the National Academy of Sciences

Language

Item Type

Identifier

Unique identifying numbers for this article in the Digital Library or other systems.

  • Report No.: LBNL-793E
  • Grant Number: DE-AC02-05CH11231
  • Office of Scientific & Technical Information Report Number: 935404
  • Archival Resource Key: ark:/67531/metadc894826

Collections

This article is part of the following collection of related materials.

Office of Scientific & Technical Information Technical Reports

Reports, articles and other documents harvested from the Office of Scientific and Technical Information.

Office of Scientific and Technical Information (OSTI) is the Department of Energy (DOE) office that collects, preserves, and disseminates DOE-sponsored research and development (R&D) results that are the outcomes of R&D projects or other funded activities at DOE labs and facilities nationwide and grantees at universities and other institutions.

What responsibilities do I have when using this article?

When

Dates and time periods associated with this article.

Creation Date

  • March 10, 2008

Added to The UNT Digital Library

  • Sept. 27, 2016, 1:39 a.m.

Description Last Updated

  • Oct. 3, 2016, 4:23 p.m.

Usage Statistics

When was this article last used?

Yesterday: 0
Past 30 days: 2
Total Uses: 37

Interact With This Article

Here are some suggestions for what to do next.

Start Reading

PDF Version Also Available for Download.

International Image Interoperability Framework

IIF Logo

We support the IIIF Presentation API

Siuzdak, Gary; Northen, Trent R.; Lee, Jinq-Chyi; Hoang, Linh; Raymond, Jason; Hwang, Der-Ren et al. A nanostructure-initiator mass spectrometry-based enzyme activity assay, article, March 10, 2008; Berkeley, California. (digital.library.unt.edu/ark:/67531/metadc894826/: accessed October 23, 2018), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT Libraries Government Documents Department.