Team:Macquarie Australia/WetLab/Protocols/MassSpec

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<li><a href="https://2014.igem.org/Team:Macquarie_Australia/WetLab/Protocols/SDSPAGE">SDS-PAGE</a></li>
<li><a href="https://2014.igem.org/Team:Macquarie_Australia/WetLab/Protocols/SDSPAGE">SDS-PAGE</a></li>
<li><a href="https://2014.igem.org/Team:Macquarie_Australia/WetLab/Protocols/Heat">Heat Shock</a></li>
<li><a href="https://2014.igem.org/Team:Macquarie_Australia/WetLab/Protocols/Heat">Heat Shock</a></li>
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<li><a href="https://2014.igem.org/Team:Macquarie_Australia/WetLab/Protocols/Operons">Induction of Operons</a></li>
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<h3>Mass Spectrometry</h3>
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<h3>Mass Spectrometry</h3><br/>
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<p>
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<ul style="list-style-type: decimal;">
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INSERT GLORIOUS CONTENT HERE
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<li>Samples need to be spotted on the 384 spot MALDI plate: Odd rows are used for samples, and even rows are for calibration standards. It is important that one sample spot in the odd row is coupled by a calibration spot in the even row.</li>
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<img src="http://i.imgur.com/JpeCE4X.png" width = 400 >
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<li>Samples are cleaned up with C18 Zip tips before spotting onto the plate. </li>
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<li>Start with a fresh Ziptip, using a 10 µL pipette, and slowly withdraw and dispense to waste 10 µL 90% (v/v) acetonitrile / 0.1 % TFA three times. Do not throw away the tip!</li>
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<li>Slowly withdraw and dispense to waste 10 µL 0.1 % TFA five times to equilibrate the C18 material in aqueous buffer.</li>
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<li>Slowly withdraw and dispense your first sample (10 µL) ten times. After this step the peptides from your digest are bound onto the C18 resin in the tip.</li>
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<li>Withdraw and discard to waste 10 µL of 0.1 % TFA five times. This washes away unwanted salts and contaminants. Make sure you expel all the liquid on the last wash.</li>
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<li>Withdraw 1 µL of matrix solution. This contains 70% acetonitrile so it is used to elute peptides in the presence of matrix. (4 mg / mL CHCA [α-cyano-4-hydroxycynnamic acid], 70 % MeCN, 0.1 % TFA). </li>
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<li>Spot the eluted peptide sample in matrix solution directly onto the MALDI target plate in an odd row, trying not to touch the surface of the plate with the tip. Record the location of your samples.</li>
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<li>New C18 tip is needed for each sample. </li>
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<li>BSA digest at 1pmol/µL is used as control. This does not required ziptipping.</li>
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<li>Next, prepare calibration standards (Pepmix and Matrix solutions at 1:1 ratio), and spot 1ul of this onto spots in even row next to your samples. These are your near-point calibration standards. </li>
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<li>Allow the plate to air-dry.</li>
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<li>Plates can be stored in 4<sup>o</sup>C fridge and in the dark for up to a week, ready for analysis.</li>
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<li>Samples are analyzed using the 4800 Plus MALDI Tof-Tof Analyzer.</li>
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</ul>
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<img src="https://static.igem.org/mediawiki/2014/e/ec/Massspec.png" />
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<p><b>Figure 1.</b> Location of samples (in blue) and standards (in red) on a 384 well MALDI target.</p>
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Latest revision as of 13:09, 17 October 2014

Protocols


Mass Spectrometry


  • Samples need to be spotted on the 384 spot MALDI plate: Odd rows are used for samples, and even rows are for calibration standards. It is important that one sample spot in the odd row is coupled by a calibration spot in the even row.
  • Samples are cleaned up with C18 Zip tips before spotting onto the plate.
  • Start with a fresh Ziptip, using a 10 µL pipette, and slowly withdraw and dispense to waste 10 µL 90% (v/v) acetonitrile / 0.1 % TFA three times. Do not throw away the tip!
  • Slowly withdraw and dispense to waste 10 µL 0.1 % TFA five times to equilibrate the C18 material in aqueous buffer.
  • Slowly withdraw and dispense your first sample (10 µL) ten times. After this step the peptides from your digest are bound onto the C18 resin in the tip.
  • Withdraw and discard to waste 10 µL of 0.1 % TFA five times. This washes away unwanted salts and contaminants. Make sure you expel all the liquid on the last wash.
  • Withdraw 1 µL of matrix solution. This contains 70% acetonitrile so it is used to elute peptides in the presence of matrix. (4 mg / mL CHCA [α-cyano-4-hydroxycynnamic acid], 70 % MeCN, 0.1 % TFA).
  • Spot the eluted peptide sample in matrix solution directly onto the MALDI target plate in an odd row, trying not to touch the surface of the plate with the tip. Record the location of your samples.
  • New C18 tip is needed for each sample.
  • BSA digest at 1pmol/µL is used as control. This does not required ziptipping.
  • Next, prepare calibration standards (Pepmix and Matrix solutions at 1:1 ratio), and spot 1ul of this onto spots in even row next to your samples. These are your near-point calibration standards.
  • Allow the plate to air-dry.
  • Plates can be stored in 4oC fridge and in the dark for up to a week, ready for analysis.
  • Samples are analyzed using the 4800 Plus MALDI Tof-Tof Analyzer.

Figure 1. Location of samples (in blue) and standards (in red) on a 384 well MALDI target.

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