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{{LSApp
{{LSApp
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   |name        = LC Multi Valve Systems
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   |name        = Hydrogen Deuterium Exchange
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   |image      = 2_valve_on_top.png
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   |image      = HDX_System.png‎
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   |type        = '''Multi Valves'''
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   |type        = '''SPECIAL'''
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   |id          = ABC
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   |id          = HD-X PAL
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   |description = HPLC Multi Vale Applications
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   |description = Automated Sample Prep for HD-X
}}
}}
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{{logo}}
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=== General Description ===
 +
Hydrogen Deuterium Exchange (HD-x) is an experimental technique to obtain structural data on proteins. It relies on the accurate measurement of the degree of labeling of the protein by deuterated hydrogen during a precisely measured labeling interval. The labeling reaction is stopped by addition of a quenching reagent, and loss of label between the quench step and analysis (usually by LCMS) is minimized by keeping the reactants at a temperature close to zero degrees Celsius. <br/><br/>
 +
The technique is difficult to perform manually due to the demands for precise timing and reproducing exactly the same experimental conditions. Automation solves both of these challenges. LEAP has developed a robotic solution to the sample Prep and introduction to the LCMS. It consists of a Twin PAL workstation configured as shown below. There are two cooled zones on the workstation: A cooled stack at 1oC for the samples and labeled reactants and a cooled chromatography module which contains the injection valve, the column selection valves and the columns themselves. These are maintained also at around 1degree C. The automation process is controlled using LEAP Shell software. <br/><br/>
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<a target="_blank" href="http://feeds.feedburner.com/~r/Leapwiki/~6/1"><img src="http://feeds.feedburner.com/Leapwiki.1.gif" alt="LEAP Wiki - New Pages" style="border:0"></a>
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=== Significant Markets  ===
 +
* Pharmaceutical - Drug Discovery
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<p style="margin-top:10px; margin-bottom:0; padding-bottom:0; text-align:center; line-height:0"><a target="_blank" href="http://feeds.feedburner.com/~r/Leapwiki/~6/1"><img src="http://feeds.feedburner.com/Leapwiki.1.gif" alt="LEAP Wiki - New Pages" style="border:0"></a></p><p style="margin-top:5px; padding-top:0; font-size:x-small; text-align:center"><a href="http://feedburner.google.com/fb/a/headlineanimator/install?id=2q84kdee5kmedl4f36gq0dskv4&amp;w=1" onclick="window.open(this.href, 'haHowto', 'width=520,height=600,toolbar=no,address=no,resizable=yes,scrollbars'); return false" target="_blank">&uarr; Grab this Headline Animator</a></p>
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* Academia      - Protein structural studies
 +
=== Protein Structure ===
 +
Protein function is strongly related to their tertiary structure and conformation. Chemical interactions which change this conformation can significantly affect protein functionality. This is important in studying disease states or effectiveness of drug candidates.
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=== Chronology ===
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There are 3 commonly used techniques to study protein structure:
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{| class="wikitable" border="1" style="font-size:85%"
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|-
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! Date
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! Year
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! Event
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|-
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| 15 May
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| 1973
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| NBS publishes a first request for a standard encryption algorithm
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|-
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| 27 August
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| 1974
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| NBS publishes a second request for encryption algorithms
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|-
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| 17 March
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| 1975
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| DES is publis
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| The [[Field-programmable gate array|FPGA]] based parallel machine [[Custom hardware attack#History|COPACOBANA]] of the University of Bochum and Kiel, Germany, breaks DES in 9 days at $10,000 hardware cost.<ref name="copacobana-2006"/>  Within a year software improvements reduced the average time to 6.4 days.
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|}
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1. '''Crystallography'''
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{| cellpadding="6" style="border:1px solid darkgray;"
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2. '''NMR '''
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|- align=left
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!width="20"|Code
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!width="195"|Application
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!width="85"|Control SW
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!width="140"|Key Hardware
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!width="205"|Key Benefits
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!width="235"|Restrictions
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!width="15"|GLP
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|- align=left
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| style="border:1px solid blue;"|'''M-001'''
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| style="border:1px solid blue;"|'''Dual Channel Serial Injection'''<br><br>Provides shorter cycle times by regenerating columns during next analytical run
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| style="border:1px solid blue;"|Analyst with ICC-CE
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| style="border:1px solid blue;"|* 6 port injection valve<BR>* 10 port Valve <br>* Gradient Pump<br>* Regeneration pump
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| style="border:1px solid blue;"|* Saving of regeneration time(Back Cut)<br>* Increase throughput at little cost<br> * Control is all within Analyst<br>* Compliance is not affected
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| style="border:1px solid blue;"|* Can not do "Look Ahead"<br>* Injections are not staggered<br>* Peaks of interest must come of early<br>* Columns may perform slightly differently on alternating samples
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| style="border:1px solid blue;"|Yes
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|}
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3. '''HD Exchange'''
 +
 +
HD Exchange is a good approach in cases where there is insufficient sample (NMR) or the protein will not crystallize. It relies on the accurate measurement of the degree of labeling of a protein by deuterated hydrogen during a precisely measured labeling interval.  The deuterium is exchanged with hydrogen atoms on the exposed amide backbone of the protein and depending on the which regions are exposed and how the protein is folded, differences are seen in the MS spectra of the digested protein when compared with non-labeled proteins.
 +
 +
Labeling time is important (exposure time of the protein to Deuterium) as is prevention of "back-exchange" after the labeling has occurred. This is the natural tendency of normal hydrogen to replace the deuterium after the label is removed. This is minimized by reducing the temperature of the mixture to close to zero degrees Celsius.
 +
 +
=== What is an HD-X experiment ? ===
 +
 +
Performing HD-X experiments '''manually''' involves setting up many labeling mixtures then quenching these mixtures at precisely timed intervals (by changing the pH) and then injecting them onto a chilled injection valve to the LCMS. In most cases a range of label times is used, from 10 seconds to several hours. Sources of error (apart from normal liquid handling inaccuracies) are timing errors, and degradation of sample due to "back-exchange". All of these sources of error are eliminated using the automated system described here.
 +
 +
=== Overview of H/D-x PAL System ===
 +
H/D-X PAL™ is an easy-to-use, system that provides an automated process for the scheduling and experimental execution of H/D-X experimental workflow.  By use of the advanced LEAP Shell scheduling software experimental design is simplified and reliable.  Synchronous reagent addition and sample labeling steps are automatically scheduled to increase throughput and produce high quality data.
 +
 +
=== What does the H/D-X PAL do? ===
 +
 +
[[Image:LineDrawing HD-x system.png]]
 +
 +
The instrument has two cooled zones for sample storage and labeling. The injection valve is chilled to 1 degree Celsius and includes a solvent pre-chiller and column selection valve for in-line protein digestion.
 +
 +
A typical experiment would consist of around a dozen time points for each protein sample, ranging from 10 seconds to 8 hours. The PAL system will schedule the experiments and perform each of the following 3 steps for each time point and sample:
 +
 +
'''Labeling'''    The protein is mixed with a Deuterium reagent and incubated for a precisely timed interval at room temperature.
 +
 +
'''Quenching'''  The labeling reaction is stopped by transferring a volume of the mixture to an excess of quenching reagent in a pre-cooled vessel.
 +
 +
'''Injection'''    A measured portion of the quenched reactants is injected into a multi-column, multi-valve LCMS system. This step is synchronized with the analytical system using hardware signals. The PAL will inject samples as the LCMS system becomes ready. Labeled and quenched reactants are held in the cooled stack until they have been injected and analyzed.
 +
 +
=== Software Control ===
 +
LEAP has developed a scheduling application using their proprietary software '''“LEAP Shell”''' which will automate the above labeling steps, as well as the injection to the MS and the associated chromatography.
 +
 +
 +
[[Image:Scr.Shot_-_Sample_List.png]]
 +
 +
'''LEAP Shell Screen Shot 1:''' User interface to set up the labelling experiment:
 +
 +
 +
[[Image:Scr.Shot_-_PAL_Method.png]]
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 +
'''LEAP Shell Screen Shot 2:''' Powerfull editor for the PAL Methods:
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 +
 +
[[Image:Scr.Shot_-_QueueList.png]]
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'''LEAP Shell Screen Shot 3:''' Run-time display of the labelling experiment with actual times:
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 +
 +
 +
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=== Useful links ===
 +
 +
[[Image:Www icon.png|40px]] [http://www.hxms.com/hxms.htm HXMS.com  A good general reference site on HD Exchange theory and practice ]
 +
 +
[[Image:Www icon.png|40px]] [http://www.hxms.com/asms/ ASMS Interest Group - Hydrogen Exchange and Covalent Labeling ]
 +
 +
[[Image:Www icon.png|40px]]  [http://www.leaptec.com/proteomics/hydrogen-deuterium-exchange--sample-prep.php LEAP Website H/D-x product description]
 +
 +
=== Publications ===
 +
[[image: Info icon.png|40px]] [[Media:Scripps_HDX.pdf | American Chemical Society 2006. Chalmers]]<br>
 +
[[image: Info icon.png|40px]] [[Media:PJW ASMS2006.pdf | Poster from ASMS 2006]]<br>
 +
[[image: Info icon.png|40px]] [[Media:ASMS 2006 Poster Final Wilcox.pdf‎ | Poster ASMS 2006 Washington Univ. In St Louis]]<br>
 +
 +
=== Other Reference material ===
 +
[[image:Download Icon.png|40px]] [[Media:LEAP HDXAppNoteVer1.pdf | LEAP HDX Application Note]]<br>
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[[image:Download Icon.png|40px]] [[Media:LEAPExsarFlyer.pdf| Collaboration between LEAP & ExSAR]]<br>
 +
[[image:Download Icon.png|40px]] [[Media:HDX_DataProcessing_SW_Comparison.pdf| Comparison of Data Processing software]]<br>
----
----
-
[[Image:LEAP Logo.png|frame|www.leaptec.com<br>www.leapwiki.com]]
+
{{logo}}
-
=== Contact LEAP ===
+
LEAP provides automated workstation instrumentation solutions based on the LEAP CTC PAL X, Y, Z syringe only autosampler robot from LEAP Technologies. This extremely flexible, precise, and adaptable liquid handling robotic platform is available in a variety of lengths and options depending on the requirements of your sample preparation and injections for your UHPLC, LC or GC chromatography.LEAP offers full support and service for the PAL platform in addition to being able to write custom macros, cycles, and scheduling to your applications. Please contact LEAP Technologies on how we can help you get maximized throughput with flexible pipetting automation solutions.
-
For additional information about this technique please contact LEAP Technologies for detailed information
+
=== Contact LEAP ===
 +
{{contact|topic=LEAP and the PAL Platform}}
-
* [http://www.leaptec.com/contact-us.php Contact the LEAP office]
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<br><br>
 +
[[Category:Application_Solutions]]

Revision as of 13:11, 29 September 2009

Hydrogen Deuterium Exchange
Application Type
  SPECIAL
Application ID
  HD-X PAL
Description
  Automated Sample Prep for HD-X
 ↳ leaptec.com  ↳ leapwiki.com
 ↳ leaptec.com
 ↳ leapwiki.com

Contents

General Description

Hydrogen Deuterium Exchange (HD-x) is an experimental technique to obtain structural data on proteins. It relies on the accurate measurement of the degree of labeling of the protein by deuterated hydrogen during a precisely measured labeling interval. The labeling reaction is stopped by addition of a quenching reagent, and loss of label between the quench step and analysis (usually by LCMS) is minimized by keeping the reactants at a temperature close to zero degrees Celsius.

The technique is difficult to perform manually due to the demands for precise timing and reproducing exactly the same experimental conditions. Automation solves both of these challenges. LEAP has developed a robotic solution to the sample Prep and introduction to the LCMS. It consists of a Twin PAL workstation configured as shown below. There are two cooled zones on the workstation: A cooled stack at 1oC for the samples and labeled reactants and a cooled chromatography module which contains the injection valve, the column selection valves and the columns themselves. These are maintained also at around 1degree C. The automation process is controlled using LEAP Shell software.

Significant Markets

  • Pharmaceutical - Drug Discovery
  • Academia - Protein structural studies

Protein Structure

Protein function is strongly related to their tertiary structure and conformation. Chemical interactions which change this conformation can significantly affect protein functionality. This is important in studying disease states or effectiveness of drug candidates.

There are 3 commonly used techniques to study protein structure:

1. Crystallography

2. NMR

3. HD Exchange

HD Exchange is a good approach in cases where there is insufficient sample (NMR) or the protein will not crystallize. It relies on the accurate measurement of the degree of labeling of a protein by deuterated hydrogen during a precisely measured labeling interval. The deuterium is exchanged with hydrogen atoms on the exposed amide backbone of the protein and depending on the which regions are exposed and how the protein is folded, differences are seen in the MS spectra of the digested protein when compared with non-labeled proteins.

Labeling time is important (exposure time of the protein to Deuterium) as is prevention of "back-exchange" after the labeling has occurred. This is the natural tendency of normal hydrogen to replace the deuterium after the label is removed. This is minimized by reducing the temperature of the mixture to close to zero degrees Celsius.

What is an HD-X experiment ?

Performing HD-X experiments manually involves setting up many labeling mixtures then quenching these mixtures at precisely timed intervals (by changing the pH) and then injecting them onto a chilled injection valve to the LCMS. In most cases a range of label times is used, from 10 seconds to several hours. Sources of error (apart from normal liquid handling inaccuracies) are timing errors, and degradation of sample due to "back-exchange". All of these sources of error are eliminated using the automated system described here.

Overview of H/D-x PAL System

H/D-X PAL™ is an easy-to-use, system that provides an automated process for the scheduling and experimental execution of H/D-X experimental workflow. By use of the advanced LEAP Shell scheduling software experimental design is simplified and reliable. Synchronous reagent addition and sample labeling steps are automatically scheduled to increase throughput and produce high quality data.

What does the H/D-X PAL do?

Image:LineDrawing HD-x system.png

The instrument has two cooled zones for sample storage and labeling. The injection valve is chilled to 1 degree Celsius and includes a solvent pre-chiller and column selection valve for in-line protein digestion.

A typical experiment would consist of around a dozen time points for each protein sample, ranging from 10 seconds to 8 hours. The PAL system will schedule the experiments and perform each of the following 3 steps for each time point and sample:

Labeling The protein is mixed with a Deuterium reagent and incubated for a precisely timed interval at room temperature.

Quenching The labeling reaction is stopped by transferring a volume of the mixture to an excess of quenching reagent in a pre-cooled vessel.

Injection A measured portion of the quenched reactants is injected into a multi-column, multi-valve LCMS system. This step is synchronized with the analytical system using hardware signals. The PAL will inject samples as the LCMS system becomes ready. Labeled and quenched reactants are held in the cooled stack until they have been injected and analyzed.

Software Control

LEAP has developed a scheduling application using their proprietary software “LEAP Shell” which will automate the above labeling steps, as well as the injection to the MS and the associated chromatography.


Image:Scr.Shot_-_Sample_List.png

LEAP Shell Screen Shot 1: User interface to set up the labelling experiment:


Image:Scr.Shot_-_PAL_Method.png

LEAP Shell Screen Shot 2: Powerfull editor for the PAL Methods:


Image:Scr.Shot_-_QueueList.png

LEAP Shell Screen Shot 3: Run-time display of the labelling experiment with actual times:



Useful links

HXMS.com A good general reference site on HD Exchange theory and practice

ASMS Interest Group - Hydrogen Exchange and Covalent Labeling

LEAP Website H/D-x product description

Publications

American Chemical Society 2006. Chalmers
Poster from ASMS 2006
Poster ASMS 2006 Washington Univ. In St Louis

Other Reference material

LEAP HDX Application Note
Collaboration between LEAP & ExSAR
Comparison of Data Processing software

 ↳ leaptec.com  ↳ leapwiki.com
 ↳ leaptec.com
 ↳ leapwiki.com

LEAP provides automated workstation instrumentation solutions based on the LEAP CTC PAL X, Y, Z syringe only autosampler robot from LEAP Technologies. This extremely flexible, precise, and adaptable liquid handling robotic platform is available in a variety of lengths and options depending on the requirements of your sample preparation and injections for your UHPLC, LC or GC chromatography.LEAP offers full support and service for the PAL platform in addition to being able to write custom macros, cycles, and scheduling to your applications. Please contact LEAP Technologies on how we can help you get maximized throughput with flexible pipetting automation solutions.

Contact LEAP

Contact LEAP

For additional information about LEAP and the PAL Platform, please contact LEAP Technologies.



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