Services
Who are CPR?
14/05/15 09:45
We are the Centre for Proteome Research, located on the ground floor of the Biosciences Building. The group is led by Claire Eyers and Edward Emmott, both of whom have independent, research programmes. In addition, we are the Proteomics Shared Research facility (SRF) that is part of the Technology Directorate at the University. This means that we can also conduct proteomics experiments for colleagues in the Faculty, and beyond..
What capabilities do you have?
14/05/15 09:43
We have an extensive suite of instrumentation. These instruments have all been brought into CPR by grants awarded to Rob and Claire (with other colleagues) and are primarily directed towards the research programmes that they have to support. However, we are very willing to engage with other groups, as collaborators or in the context of the Shared Rsearch Facility.
• 2006: Waters GC-TOF Premier GS/MS system
• 2009: Waters Xevo QqQ/nanoAcquity
• 2009: Waters Xevo QqQ/nanoAcquity
• 2010: Thermo Velos Orbitrap/nanoAcquity (upgraded to Elite in 2015 for metabolomics)
• 2010: Waters Synapt G2/nanoAquity high resolution ion mobility QToF
• 2010: Bruker Amazon high speed ion trap/nanoAcquity
• 2010: Bruker Ultraflex Extreme 1kHz MALDI-TOF/TOF
• 2012: Thermo QExactive Orbitrap instrument/Dionex u3000 nano
• 2013: Waters G2si IM-QTOF for intact protein research
• 2013: Waters G2si IM-QTOF/ nanoAquity for proteomics
• 2013: Waters Xevo TQS QqQ/nanoAcquity
• 2014: Waters MALDI-Synapt G2si imaging system
• 2014: Waters LAESI-Synapt G2si imaging system, upgrade to include DESI in 2015
• 2015: Thermo Fusion tribrid mass spectrometer
• 2015: Thermo QExactive HF mass spectrometer
• 2015: Access to Fluidigm CyTOF mass cytometer
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• 2006: Waters GC-TOF Premier GS/MS system
• 2009: Waters Xevo QqQ/nanoAcquity
• 2009: Waters Xevo QqQ/nanoAcquity
• 2010: Thermo Velos Orbitrap/nanoAcquity (upgraded to Elite in 2015 for metabolomics)
• 2010: Waters Synapt G2/nanoAquity high resolution ion mobility QToF
• 2010: Bruker Amazon high speed ion trap/nanoAcquity
• 2010: Bruker Ultraflex Extreme 1kHz MALDI-TOF/TOF
• 2012: Thermo QExactive Orbitrap instrument/Dionex u3000 nano
• 2013: Waters G2si IM-QTOF for intact protein research
• 2013: Waters G2si IM-QTOF/ nanoAquity for proteomics
• 2013: Waters Xevo TQS QqQ/nanoAcquity
• 2014: Waters MALDI-Synapt G2si imaging system
• 2014: Waters LAESI-Synapt G2si imaging system, upgrade to include DESI in 2015
• 2015: Thermo Fusion tribrid mass spectrometer
• 2015: Thermo QExactive HF mass spectrometer
• 2015: Access to Fluidigm CyTOF mass cytometer
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What is the lowest limit of detection you can attain?
14/05/15 09:41
We would hope to be able to reach 100 attomol for the lowest abundance in a discovery experiment.
To know whether that is enough, let's do some quick calculations.
A detection limit of 100 attomol is equivalent to injecting 60 million molecules into the mass spectrometer - that sounds like a lot. If the sample was derived from yeast cells, we would have loaded of the order of 200,000 yeast cell equivalents onto the same column. Thus, we can measure 60 million molecules, derived from 200,000 cells. From this, you will see that the limit of detection in an unprocessed sample is 60,000,000/200,000 = 300 copies per cell.
That sounds pretty good, right? But, if we have been using HeLa cells, the numbers are very different. The limit to what can be loaded on the hplc column is dictated by the total protein load - 200,000 cells gives us about 1000 nanograms of protein. However, each HeLa cell would contain 50 times as much protein as a yeast cell, approximately. Therefore, for a 1000 ng column capacity, we can only load about 4,000 HeLa cells equivalent onto the column. For the same detection limit of 100 attomol, we obtain a limit of detection of 60,000,000/4,000 copies per cell, or 15,000 copies per cell. Rather different!
To overcome such difficulties, it is necessary to resort to sample prefractionation and concentration steps, which have the potential to introduce 'lossy' steps but also increase the number of subsequent LC-MS/MS analyses that need to be conducted, and hence the cost.
To know whether that is enough, let's do some quick calculations.
A detection limit of 100 attomol is equivalent to injecting 60 million molecules into the mass spectrometer - that sounds like a lot. If the sample was derived from yeast cells, we would have loaded of the order of 200,000 yeast cell equivalents onto the same column. Thus, we can measure 60 million molecules, derived from 200,000 cells. From this, you will see that the limit of detection in an unprocessed sample is 60,000,000/200,000 = 300 copies per cell.
That sounds pretty good, right? But, if we have been using HeLa cells, the numbers are very different. The limit to what can be loaded on the hplc column is dictated by the total protein load - 200,000 cells gives us about 1000 nanograms of protein. However, each HeLa cell would contain 50 times as much protein as a yeast cell, approximately. Therefore, for a 1000 ng column capacity, we can only load about 4,000 HeLa cells equivalent onto the column. For the same detection limit of 100 attomol, we obtain a limit of detection of 60,000,000/4,000 copies per cell, or 15,000 copies per cell. Rather different!
To overcome such difficulties, it is necessary to resort to sample prefractionation and concentration steps, which have the potential to introduce 'lossy' steps but also increase the number of subsequent LC-MS/MS analyses that need to be conducted, and hence the cost.
Can you measure the mass of my protein?
14/05/15 09:41
In short, probably yes!
We have set up a semiautomated system for the mass measurement of intact proteins, coupled to a very high quality instrument (Synapt G2si). This system will measure the mass of a protein to about 1Da in 10,000Da, and requires only microgram quantities of protein. The mass is measured by electrospray ionisation mass spectrometry and thus, the protein molecule acquires a large and variable number of charges (protons). Each protein thus creates a multiply-charged envelope of ions that need to be deconvoluted by proprietary software using maximum entropy algorithms. The result is a true mass spectrum that can reveal the mass of the analyte protein and also, the mass of associated contaminants and possibly, fragments or modifications.
We have set up a semiautomated system for the mass measurement of intact proteins, coupled to a very high quality instrument (Synapt G2si). This system will measure the mass of a protein to about 1Da in 10,000Da, and requires only microgram quantities of protein. The mass is measured by electrospray ionisation mass spectrometry and thus, the protein molecule acquires a large and variable number of charges (protons). Each protein thus creates a multiply-charged envelope of ions that need to be deconvoluted by proprietary software using maximum entropy algorithms. The result is a true mass spectrum that can reveal the mass of the analyte protein and also, the mass of associated contaminants and possibly, fragments or modifications.
Proteomics as a service
14/05/15 09:38
How can I have proteomics samples run?
We are always willing to talk to colleagues about the potential for running new analyses. These can vary from simple 'quick look' analyses to complete and complex, fully biologically replicated analyses. In all circumstances, we adopt a model of 'defend the mass spectrometer form the sample'! In fact, mass spectrometers are remarkably robust; it is the delivery of peptides through a nanoflow high pressure chromatography system that causes the problems.
Biological samples can be delivered in exotic and complex matrixes, either reflecting the biological context of the sample or the sample work up chemistry imposed by the user m(detergent, PEG and glycerol might seem like a dream extraction buffer to you, but we are never going to run that sample for you!). We are very reluctant to receive samples that contain insoluble material, high concentrations of detergents, polymers such a polyethylene glycol (PEG), for example. A nanoflow high resolution column (75um diameter and 150mm long) costs, with trap, nearly £1,000 and is time consuming to exchange and optimise. You can see why we're reluctant to take anonymous samples!
It is far, far better if you come to talk to us before you attempt to prepare proteomics samples.
We are always willing to talk to colleagues about the potential for running new analyses. These can vary from simple 'quick look' analyses to complete and complex, fully biologically replicated analyses. In all circumstances, we adopt a model of 'defend the mass spectrometer form the sample'! In fact, mass spectrometers are remarkably robust; it is the delivery of peptides through a nanoflow high pressure chromatography system that causes the problems.
Biological samples can be delivered in exotic and complex matrixes, either reflecting the biological context of the sample or the sample work up chemistry imposed by the user m(detergent, PEG and glycerol might seem like a dream extraction buffer to you, but we are never going to run that sample for you!). We are very reluctant to receive samples that contain insoluble material, high concentrations of detergents, polymers such a polyethylene glycol (PEG), for example. A nanoflow high resolution column (75um diameter and 150mm long) costs, with trap, nearly £1,000 and is time consuming to exchange and optimise. You can see why we're reluctant to take anonymous samples!
It is far, far better if you come to talk to us before you attempt to prepare proteomics samples.