High performance mass spectrometry: applications for the Cambridge biological sciences community
Lead Research Organisation:
University of Cambridge
Department Name: Biochemistry
Abstract
The Cambridge Centre for Proteomics (CCP) is a well-established mass spectrometry core facility in the Department of Biochemistry, University of Cambridge. CCP primarily offers proteomics services. Proteomics is the study of all proteins in a sample which could be proteins extracted from plant leaves, human parasites, bacteria, fungi and clinical samples such as plasma and urine. Researchers generally wish to know what proteins are present in their samples, but importantly the abundance of the proteins and how this changes in response to stress, drug treatment or disease state. Also, researchers may want information on the post translational modifications in the proteome and how this changes upon perturbation, with phosphorylation being the most popular modification studied.
CCP uses mass spectrometry as its major tool to interrogate the proteome. This instrument measures the mass of ions introduced typically via a liquid chromatography device, together referred to as LC-MS. From these mass measurements, both the identity of proteins present and their amount can be calculated. Mass spectrometry can also be used to characterise other biomolecules such as sugars, lipids and nucleic acids.
CCP is a very popular and busy facility. It currently has access to two mass spectrometers. Its Thermo QExactive is 9 years old and is very unreliable. It is covered by a contract to fix it if it breaks, but its manufacturer will discontinue the contract for this instrument in the next 2 years. This instrument is insensitive compared with more modern instruments. CCP also has access to 55% of the time of a 5-year-old Thermo Fusion Lumos Tribrid, which is more sensitive and a very good instrument to carry out a form of quantitative proteomics known as isobaric tagging. This instrument lacks sensitivity compared with newer instruments and is able to separate ions introduced into the instrument based on their mobility in the gas phase. Together, these instruments do not provide CCP with the capacity to process samples submitted by its users.
The need for a more up to data mass spectrometer has become critical. CCP supports its staff and equipment through fee for service and grant funding. CCP simply cannot sustain its services and will be faced with significant cuts. Moreover, it has increasing demands from it users including those with limited sample amounts or who need more elaborate characterisation of post translational modification.
Other facilities in Cambridge are reserved for the institutes in which they sit and have no spare capacity to assist CCP.
The requested LC-MS, Bruker TimsTOF Pro 2 is much more sensitive than CCP's current instruments. It processes samples more quickly and hence will increase the capacity of CCP and reduce sample turnround significantly. It also is equipped with an ion mobility functionality which assists in both sensitivity and distinguishing very similar molecules based on their cross sectional area as gaseous ions. This instrument excels at quantitative label free proteomics approaches that do not require the use of isobaric tagging, and is highly complementary to what is currently available here. Its ion mobility function also helps distinguish other classes of biomolecules with similar masses but subtly different behaviours within ion mobility. There is no similar LC-MS instrument in CCP or indeed in Cambridge.
This LC-MS system will support all CCP users from across the University of Cambridge, outside research institutes, users form other academic institutions and users from industry.
The proposed equipment, will not only allow them to carry out their planned work, but enable new avenues of research which have been hitherto impossible with existing instrumentation. The support from the University of Cambridge means that this proposal requests less than half of the cost of the TimsTOF Pro 2, and thus represents value for money.
CCP uses mass spectrometry as its major tool to interrogate the proteome. This instrument measures the mass of ions introduced typically via a liquid chromatography device, together referred to as LC-MS. From these mass measurements, both the identity of proteins present and their amount can be calculated. Mass spectrometry can also be used to characterise other biomolecules such as sugars, lipids and nucleic acids.
CCP is a very popular and busy facility. It currently has access to two mass spectrometers. Its Thermo QExactive is 9 years old and is very unreliable. It is covered by a contract to fix it if it breaks, but its manufacturer will discontinue the contract for this instrument in the next 2 years. This instrument is insensitive compared with more modern instruments. CCP also has access to 55% of the time of a 5-year-old Thermo Fusion Lumos Tribrid, which is more sensitive and a very good instrument to carry out a form of quantitative proteomics known as isobaric tagging. This instrument lacks sensitivity compared with newer instruments and is able to separate ions introduced into the instrument based on their mobility in the gas phase. Together, these instruments do not provide CCP with the capacity to process samples submitted by its users.
The need for a more up to data mass spectrometer has become critical. CCP supports its staff and equipment through fee for service and grant funding. CCP simply cannot sustain its services and will be faced with significant cuts. Moreover, it has increasing demands from it users including those with limited sample amounts or who need more elaborate characterisation of post translational modification.
Other facilities in Cambridge are reserved for the institutes in which they sit and have no spare capacity to assist CCP.
The requested LC-MS, Bruker TimsTOF Pro 2 is much more sensitive than CCP's current instruments. It processes samples more quickly and hence will increase the capacity of CCP and reduce sample turnround significantly. It also is equipped with an ion mobility functionality which assists in both sensitivity and distinguishing very similar molecules based on their cross sectional area as gaseous ions. This instrument excels at quantitative label free proteomics approaches that do not require the use of isobaric tagging, and is highly complementary to what is currently available here. Its ion mobility function also helps distinguish other classes of biomolecules with similar masses but subtly different behaviours within ion mobility. There is no similar LC-MS instrument in CCP or indeed in Cambridge.
This LC-MS system will support all CCP users from across the University of Cambridge, outside research institutes, users form other academic institutions and users from industry.
The proposed equipment, will not only allow them to carry out their planned work, but enable new avenues of research which have been hitherto impossible with existing instrumentation. The support from the University of Cambridge means that this proposal requests less than half of the cost of the TimsTOF Pro 2, and thus represents value for money.
Technical Summary
We are requesting a TimsTOF Pro2 LC-MS. This system has highly complementary to mass spectrometry (MS) systems already available in the Cambridge Centre for Proteomics (CCP) and other MS facilities within Cambridge. The instrument will support a huge range of projects, including from 11 named co-applicants with significant BBSRC funding within key BBSRC priority areas. It has significant added benefits over existing LC-MS systems in terms of sensitivity, speed and ion mobility separation of analytes giving the opportunity to dissect apart positional isomers of different species, important to studies investigating the ramifications of subtle changes in post translation modifications. The impressive sensitivity of TimsTOF Pro2 will enable projects where low sample amounts severely limit the quantity and quality of data achievable of current CCP equipment. This system excels at data independent acquisition (DIA) workflows which are important to robust sampling of the proteome during shotgun proteomics experiments. In DIA, the pre-selection system of precursor ions is not based on intensity and allows for simultaneous processing of ions within a pre-defined mass range. The result is far fewer missing values between successive runs. This is very important to studies, such as those of PI Lilley and co-applicant, Theodoulou where hundreds of very similar samples are required to be sampled with no loss to performance or coverage of system during data collection. Furthermore, the PI already has in place empowering analytical workflows for this instrument developed lab through collaboration.
Importantly, it will provide critical capacity to CCP which has limited access to more cutting edge MS equipment, and whose main 'workhorse' instrument is ageing and failing. CCP is the only mass spectrometry/proteomics facility open to all Cambridge researchers, as other facilities are based in institutes that run at capacity and prioritise in-house samples.
Importantly, it will provide critical capacity to CCP which has limited access to more cutting edge MS equipment, and whose main 'workhorse' instrument is ageing and failing. CCP is the only mass spectrometry/proteomics facility open to all Cambridge researchers, as other facilities are based in institutes that run at capacity and prioritise in-house samples.
