Biomolecular Mass Spectrometry

Service Analyses

A. Molecular Mass Measuerment
B. Protein Identification/PTM determination
C. Non-covalent (native) MS


<h2 class="heading-underline">Molecular Mass Measurement</h2>

<table class="heading-underline" cellpadding="0" cellspacing="0" style="width:500px;border: 0px solid white;margin-left:auto;margin-right:auto;"><tbody><tr><td style="border: 0px solid white"><img alt="Molecular mass measurement" src="/biologicalsciences/images/molecularmass_1.png" /></td><td style="text-align: left;vertical-align:text-top;border: 0px solid white"><strong>Useful for:</strong><ul><li>Confirming correct synthesis/expression</li><li>Establishing purity</li><li>Detecting PTMs</li><li>Checking chemical derivitisation</li></ul></td></tr></tbody></table>

Using LC-MS, protein samples are desalted immediately prior to mass measurement.Protein Mass Measurement

With this approach, in most cases, buffer exchange prior to analysis is no longer required (N.B. any PEG or glycerol must still be removed before analysis).

Minimum sample volume is 20 uL at 20 uM concentration.

Users must also submit an aliquot of sample buffer (min. 100 uL for each sample) to enable dilution of samples if required.

Details of method used to measure concentration should be provided. Internal users have the option of using the VersaWave spectrophotometer in the MS Facility to determine concentration.

Some sensitive proteins are not amenable to LC-MS degrading during analysis.

In these cases we can buffer exchange samples into a volatile ammonium buffer by dialysis or using a buffer exchange spin column. This will be carried out by the Facility immediately prior to analysis.

Oligonucleotide mass measurement

Oligos require extensive purification before analysis to remove metal ions particularly sodium. The most successful methods of purification are dialysis against 200 mM ammonium acetate or using precipitation methods.

Minimum sample volume is 20 uL at 50-100 uM concentration.

Accurate mass measurement

Molecules below 2000 Da in mass can be determined to within 5 ppm.

Small organic molecules and peptides can be submitted in lyophilised form or in solution using an acceptable solvent system.

Acceptable solvent systems can be formulated from: water, acetonitrile, methanol, propan-2-ol, hexafluoropropan-2-ol and chloroform.

Solvents to be avoided are the less volatile ones such as dimethylformamide, and less stable ones such as tetrahydrofuran. DMSO is acceptable only if present at below 1% v/v.

Sample submission

Samples can be submitted to the service at any time by bringing them to the MS Facility in lab 9.107 of the Astbury building.

You should download and complete a request form  form to bring along with your sample (Please note that results of analysis cannot be made available until a grant code has been received).

Please note that by submitting samples to the MS Facility for analysis you agree to our conditions of service and publications policy. A copy of which can be found here.

Results for molecular mass analysis are usually available and emailed to you within 48 hours of submission depending on current demand.

Users external to the University can find additional information here. (link to contact and sample submission section)

Notes

The concentrations and volumes stated above are provided as a guide only.

We will still accept and attempt MS analysis on samples of lower (or unknown) concentration and/or volume.

Where analytes are only stable outside of their preferred buffer for short periods of time, buffer exchange can be performed in the mass spec lab immediately prior to analysis. Please contact us in advance to arrange this if required.

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<h2 class="heading-underline">Protein Identification</h2>

<table cellpadding="0" cellspacing="0" style="width:500px;border: 0px solid white;margin-left:auto;margin-right:auto;" class="heading-underline"><tbody><tr><td style="border: 0px solid white"><img alt="protein ID and PTM determination" src="/biologicalsciences/images/proteinid.png" /></td><td style="text-align: left;vertical-align:text-top;border: 0px solid white"><strong>Useful for:</strong><ul><li>Confirming protein identity</li><li>Identifying unknown bands on SDS-PAGE gels</li><li>Detecting and localising PTMs</li><li>Determining interacting partners in pull downs</li></ul></td></tr></tbody></table>

Samples for protein identification should be submitted in the form of either a 1D gel that has been Coomassie stained or in a suitable buffer solution.

Please note that we cannot perform intact molecular mass measurements on samples supplied in gels, these must be prepared in solution or lyophilised.

For gel samples, once the gel has been run, it should be handled inside a laminar flow hood where possible. This is to reduce contamination with keratin proteins that interfere with the identification.

Rinsing all boxes, eppendorfs, gel plates, scalpels etc. that come into contact with the gel with 70% ethanol can help to reduce the risk of keratin contamination.

Staining should be performed in clean gel boxes that have not been used for western blots.

Where possible Expedeon InstantBlue Comassie stain should be used as this is MS compatible (n.b. gels stained with Generon Quick Comassie stain will NOT be accepted for analysis).

Do not use excessively long staining times, bands should start to appear within 15 min depending upon the amount of material present.

The MS Facility can provide aliquots of Expedeon InstantBlue stain for internal users using the service for the first time.

If you require ID of bands detected using silver staining, please contact the Facility.

Please provide the entire gel to the Facility in water along with a photograph indicating which bands to excise for analysis. Please do not attempt to excise the bands yourself.

If you wish to submit a sample in solution please see below for the minimum sample amounts.

Minimum sample requirements

Gel Samples

The image below shows Coomassie-stained gel bands at different levels of protein loading:

Coomassie-stained gel bands

 

To be suitable for protein ID analysis, there must be a clearly defined band as shown for each loading level in the picture above.

Generally, the higher the loading on the gel the higher the sequence coverage achieved.

The band appearance at different loading levels will vary protein to protein.

Bands that are feint and/or diffuse, or require extreme settings during image acquisition to visualise are unlikely to give any sequencing results.

If we need to tilt or hold a gel up to the light to visualise a band, it is not suitable for analysis and will not be run. Ideally between 1 - 5 µg protein should be loaded where possible.

In-solution samples

Single proteins should be between 0.1-1mg mL-1 concentrations. For relatively simple protein mixtures (a few tens of proteins), 20-50 µg of total protein is required in volumes of between 50-100 µL.

For complex protein mixtures (100+ proteins) at least 300 µg of protein should be submitted.

Detergents, glycerol and very high salt concentration (0.2 M+) should be avoided where possible.

Sample submission deadlines

Protein ID is run on a weekly basis depending on demand.

Processing begins each Tuesday and bands should be submitted no later than 9.30am on the Tuesday morning.

Due to the demand on this service we are having to strictly enforce this deadline. Any samples submitted after this time will be held over until the following week.

Samples should be brought to the Mass Spec Facility in lab 9.107 of the Astbury building. You should download and complete a Protein ID analysis request form to bring along with your sample.

Please note that by submitting samples to the MS Facility for analysis you agree to our conditions of service and publications policy. A copy of which can be found here.

Results for Protein ID are usually available by the Tuesday following the start of sample processing.

Users external to the University can find additional information in the sample submission section.

Notes

When submitting samples, you should provide the name of the species used to express the protein as well as the expected species of the expressed protein.

If the expected protein sequence differs from the wild type sequence in any way (e.g. inclusion of His tag or presence of TEV cleavage site) or the sequence is not present in the Uniprot database, please email the sequence in FASTA format to mass-spec@leeds.ac.uk.

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<h2 class="heading-underline">Non-covalent mass analysis</h2>

<table cellpadding="0" cellspacing="0" style="width:500px;border: 0px solid white;margin-left:auto;margin-right:auto;" class="heading-underline"><tbody><tr><td style="border: 0px solid white"><img alt="Non-covalent (native) MS" src="/biologicalsciences/images/noncovalent.png" /></td><td style="text-align: left;vertical-align:text-top;border: 0px solid white"><strong>Useful for:</strong><ul><li>Determining complex stoichiometry</li><li>Assessing protein conformation</li><li>Characterising oligomerisation behaviour</li><li>Measuring CCS values using IMS-MS</li></ul></td></tr></tbody></table>

Mass spectrometry can be used to determine sub-unit stoichiometry and heterogeneity of protein complexes by using conditions that can preserve non-covalent interactions into the gas phase.

The success of a non-covalent MS measurement will depend upon the sample purity, concentration and the type of interaction predominating in the complex.

Electrostatic interaction tend to be enhanced in the absence of bulk solvent whereas hydrophobic interactions tend to be weakend.

Samples should be buffer exchanged into a volatile ammonium buffer by dialysis or using a spin column. 200 mM ammonium acetate is the recommended buffer but this will vary depending on the required ionic strength and pH. 

Buffer exchange columns, spin dialysis filters and button dialysis options are available in the Facility if required.

Sample concentration should be greater than 20 uM where possible and a minimal sample volume of 50 uL.

Sample submission

Samples can be submitted to the service at any time by bringing them to the Mass Spec Facility in lab 9.107 of the Astbury building.

You should download and complete a sample request form to bring along with your sample (please note that results of analysis cannot be made available until a grant code has been received).

Please note that by submitting samples to the MS Facility for analysis you agree to our conditions of service and publications policy. A copy of which can be found here.

Users external to the University can find additional information in the sample submission section.

Notes

The concentrations and volumes stated above are provided as a guide only.

We will still accept and attempt MS analysis on samples of lower (or unknown) concentration and/or volume.

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