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The Astbury Centre for Structural Molecular Biology

Mass Spectrometry Facility

Alison Ashcroft

Manager: Prof Alison E. Ashcroft
telephone: 0113 343 7273
Astbury Centre for Structural Molecular Biology
Astbury Building
University of Leeds


  1. Sample analysis facility
  2. Description of instrumentation
  3. Mass spectrometric tutorial (Opens in new window)
  4. Sample preparation
  5. Examples of applications
  6. Links to other sites

1. Sample analysis facility

The mass spectrometry facility is situated in Laboratory 9.107 of the Astbury Building at the University of Leeds. There are five electrospray ionisation mass spectrometers, the Platform II, the Q-Tof, an LCT Premier and two new Synapt HDMS instruments, one with a m/z 32,000 quadrupole for the MS/MS of macromolecular complexes and the other with a m/z 4000 quadrupole and HPLC facilities. There is also a SELDI/MALDI/ProteinChip instrument.

This range of mass spectrometers is capable of analysing a wide variety of samples from small organic molecules to large biomolecular complexes. The instruments have excellent mass accuracy, sensitivity and mass resolution, and the Q-Tof has additional features for structural elucidation:

Molecular Weight measurements:

Accurate mass measurements:


Structural Characterisation (Tandem MS):

Research work:

A modest charge is made for sample analyses to cover the cost of consumables; for details of these charges please contact Prof Alison E. Ashcroft or Dr James R. Ault (Mass Spectrometry Facility Service Manager). Samples should be submitted together with a completed "Sample request form", and will only be accepted after prior arrangement. You are always welcome to discuss the types of analysis available, your specific sample requirements, the instrumentation, and data processing and interpretation. Download a sample request form here

2.Description of instrumentation

Photo of Synapt HDMS Original

The first “original” Synapt HDMS (Waters) was purchased with funding from a BBSER REI grant in 2007, and has a m/z 32,000 quadrupole for the MS/MS analysis of macromolecular biomolecules and a NanoMate Triversa (Advion Biosciences) automated injection and nanoESI interface.

Photo of Synapt HDMS

The second Synapt HDMS (Waters) was purchased with SRIF4 funding in 2009. This instrument has a m/z 4000 quadrupole and an Ultimate 3000 HPLC system (Dionex). Both Synapt HDMS instruments are of quadrupole-orthogonal acceleration time-of-flight geometry and have an in-built travelling wave ion mobility spectrometry device for the separation of ions with the same mass, or m/z ratio, but different cross-sectional areas (e.g., protein conformers, structural isomers).

Platform 2
Platform II

The Platform II was purchased with a Wellcome Trust grant. It is operated with electrospray ionisation and samples can be introduced into the mass spectrometer by a number of different techniques. Reasonably pure samples can be injected directly; contaminated samples can be de-salted on-line to the mass spectrometer by being passed through a desalting cartridge; multi-component samples can be both separated and analysed by employing on-line HPLC-MS or CZE-MS. The Platform II has a single quadrupole analyser and a photomultiplier detector.


The Q-Tof has been purchased by a Joint Research Equipment Initiative grant from the BBSRC/HEFCE together with other financial contributions from the University of Leeds and Micromass UK Ltd. The Q-Tof is usually operated with nanospray ionisation, a low flow adaptation of electrospray that offers superior sensitivity whilst consuming smaller sample volumes. Additionally the sample signal is maintained for longer periods of time, thus enabling extra experiments to be carried out. On-line capillary HPLC-MS(/MS) can be used for complex or dirty samples. This is ideal for samples containing a mixture of peptide digest fragments originating from proteins excised from 2D-gels, which is the basis of proteomics research.

The Q-Tof is a tandem mass spectrometer (MS/MS) with two analysers: the first being a quadrupole analyser that is used as an ion guide in MS mode, but as a resolving analyser in MS/MS mode. The second analyser is a reflectron time-of-flight analyser placed orthogonally to the quadrupole. The final detector is a microchannel plate detector for high sensitivity. The accuracy and reproducibility of the time-of-flight analyser enable accurate mass measurements to be carried out with small organic molecules and the excellent resolution makes charge state identification routine. In MS/MS mode, the two analysers are used together for structural studies by monitoring fragmentation patterns in molecules. The fragmentation patterns of peptides are particularly well documented and MS/MS is used extensively in protein chemistry for this purpose.

lct premier
LCT Premier with the Advion NanoMate inlet

The LCT Premier was purchased in 2005 with a Wellcome Trust grant and further financial support from the University of Leeds. It has been specially modified for the analysis of non-covalently bound macromolecular complexes and has the highest m/z range available commercially (m/z 60,000), as well as collisional cooling facilities. The LCT Premier is also equipped with a NanoMate automated injection system and nanospray inlet for high reproducibility. The instrument has a time-of-flight analyser that can be operated in "V" mode (for high mass analysis) or "W" mode (for high resolution applications).

SELDI/MALDI/protein chip reader
SELDI/MALDI/ProteinChip Reader

The SELDI/MALDI/ProteinChip Reader instrument was purchased with funding from a JIF grant. It can be used as a standard matrix assisted laser desorption ionisation (MALDI) instrument or with a variety of chemical and biochemical sample targets (e.g. hydrophobic, hydrophilic, anion exchange, cation exchange, immobilized metal affinity, antibody-capture) for on-line protein sample preparation i.e. surface enhanced laser desorption ionisation (SELDI). This instrument has a time-of-flight analyser.

3. Mass spectrometric tutorial

4. Sample preparation

Electrospray ionisation and matrix assisted laser desorption ionisation mass spectrometry are "soft" ionisation techniques whereby samples are analysed to produce primarily molecular weight information.

Electrospray ionisation is equally suitable for the mass measurement of most small organic molecules as well as proteins and other high molecular mass biomolecules. In general, positive ionisation electrospray is used for the analysis of peptides, proteins, glycoproteins and small molecules containing amines and other functional groups capable of holding a positive charge. Negative ionisation electrospray is used for the analysis of oligonucleotides, saccharides, and small organic molecules containing acidic functionalities and other groups capable of holding a negative charge.

With electrospray ionisation, the sample is introduced in solution to the mass spectrometer and so the technique can be coupled directly with HPLC or CZE to generate on-line chromatographic data providing molecular weight information for all the components, thus avoiding the need for off-line separation followed by isolation and analysis of the individual fractions.

Matrix assisted laser desorption ionisation (MALDI) is also used for the analysis of peptides and proteins, as well as polymeric materials. This technique is not as useful as electrospray ionisation for the analysis of small (< 500 Da) molecules but is more tolerant to the presence of non-volatile buffers and detergents. The sample is pre-mixed with a UV-absorbing matrix compound in solution before being allowed to dry and then inserted into the mass spectrometer for analysis.

Samples can be submitted for mass spectrometric analysis in either solid form or as a solution in an appropriate solvent. Both MALDI and electrospray/nanospray mass spectrometry work well for samples in solution at concentrations in the region 1 to 20 pmol/µL. A minimum of 10 µL is required depending on the type of analysis.

Acceptable solvent systems can be formulated from the following list: water, acetonitrile, methanol, propan-2-ol, hexafluoropropan-2-ol and chloroform. Solvents to be avoided are the less volatile ones such as dimethylformamide and dimethylsulphoxide, and less stable ones such as tetrahydrofuran.

Additives which can be used successfully include formic acid (< 2%), acetic acid (< 2%), trifluoroacetic acid (< 0.1%), and triethylamine (< 1%). All salts (e.g. sodium, potassium, phosphates, citrates, perchlorates) should be avoided. Samples should also be free from surfactants and detergents.

Involatile buffers MUST be avoided, including TRIS, CHAPS, and HEPES. Often the required pH can be achieved by use of ammonium acetate, ammonium formate, or ammonium hydrogen carbonate buffers, and if so the concentration of the buffer should be < 50 mM.

SELDI-MS experiments require individual design and should be discussed in advance.

5. Examples of applications

  1. Detection of Transient Protein Folding Populations.
  2. The Analysis of Oligonucleotides.
  3. Horse heart Myoglobin.
  4. Analysis of Modified and Unmodified Class I Aldolase.
  5. LC-MS Separation and Analysis of SFE Extracted Alkaloids.
  6. Structural Elucidation of a Peptide by ESI-MS/MS.
  7. A Protein Capture SELDI/MALDI Experiment

1. Detection of Transient Protein Folding Populations.


Time evolution of the ES-MS spectra monitoring pulse-labeling studies of the protein lysozyme refolding. Deuterated lysozyme was denatured in guanidinium deuterochloride and refolded by dilution. At different times water was added to the refolding mixture. The three peaks illustrated (all of charge state n=11) represent molecules that are unprotected (not folded), fully protected (native-like), and partially protected (partially folded intermediates).

Related references:

Kulkarni, S. K., Ashcroft, A. E., Carey, M., Masselos, D., Robinson, C. V. & Radford, S. E. (1999) A near-native state on the slow refolding pathway of hen lysozyme. Protein Science, 8, (1), 35-44.

Coyle, J. E., Texter, F. L., Ashcroft, A. E., Masselos, D., Robinson, C. V. & Radford, S. E. (1999) GroEL accelerates the refolding of hen lysozyme without changing its folding mechanism. Nature Structural Biology, (6) 7, 683-690.


2. The Analysis of Oligonucleotides.



The upper trace depicts the negative ESI-MS m/z spectrum of a 23-mer RNA sample, with multiply charged ions from n =8 (m/z 950.5) to n = 15 (m/z 505.6). The lower trace shows the molecular mass profile produced from the m/z spectrum by Maximum Entropy data handling techniques. The mass accuracy was within 0.5 Da.

Related references:

Parrott, A. M., Lago, H., Adams, C. J., Ashcroft, A. E., Stonehouse, N. J. & Stockley, P. G. (2000) RNA aptamers for the MS2 bacteriophage coat protein and the wild-type RNA operator have similar solution behaviour. Nucleic Acids Research, 28, 2, 489-497.

3. An example of a Non-covalently Bound Complex: Horse heart Myoglobin.


Horse heart myoglobin can be analysed either with the haem molecule attached to the protein by non-covalent interactions using neutral aqueous conditions, or in the denatured state (without the haem molecule) using acidified organic solvents. Under neutral conditions (upper trace) the protein remains folded and so has only 8 or 9 sites which are exposed for ionisation; also the haem ligand remains bound and the mass measured is 17,564.9Da. Under denaturing conditions (lower trace) the protein is much more highly charged (n = 9 to 28) due to the higher number of sites exposed in the unfolded state, and as the haem ligand is no longer bound the mass measured is 16,951.6 Da.

Related references:

Ramoni, R., Vincent, F., Ashcroft, A.E., Accornero, P., Grolli, S., Valencia, C., Tegoni, M. & Cambillau, C. (2002) Control of domain swapping in bovine odorant binding protein. Biochem. J., 365, 739-748.

Ashcroft, A. E., Brinker, A., Coyle, J. E., Weber, F., Kaiser, M., Moroder, L., Parsons, M. R., Jaeger, J., Hartl, U. F., Hayer-Hartl, M. & Radford, S. E. (2002) Structural plasticity and non-covalent substrate binding in the GroEL apical domain: a study using electrospray ionisation mass spectrometry and fluorescence binding studies. J. Biol. Chem., 277, 33115-33126.

4. Analysis of Modified and Unmodified Class I Aldolase.


Monitoring the modification of a Class I aldolase (MW 37979.3 Da) (lower trace)was achieved by ESI-MS. The modification involved the covalent addition of dihydroxy acetone phosphate, thus increasing the molecular weight to 38,134.3 Da (upper trace).

Related reference:

Thomson, G. J., Howlett, G. J., Ashcroft, A. E. & Berry, A. (1998) The dhnA gene of E. Coli encodes a Class I fructose bisphosphate aldolase. Biochem. J., 331, 437-445.

5. LC-MS Separation and Analysis of SFE Extracted Alkaloids.


The LC-MS chromatogram (above, lower trace) was obtained from the isocratic elution of a C18 4.6 mm HPLC column with 1:1 (v/v) 0.05% aq. TFA : methanol using ES-MS detection. A number of components was detected, including colchicine (MW 399) at 16.10 minutes (above, upper trace). The mass spectrum of colchicine from this LC-MS run was dominated by protonated molecular ions at m/z 400 (below).



6. Structural Elucidation of a Peptide by ESI-MS/MS


The peptide sequence illustrated in the m/z spectrum was generated by ESI-MS/MS analysis by selectively fragmenting this peptide (measured mass 1325.8 Da) from an unseparated mixture of peptides originating from the tryptic digest of a protein excised from a 2D gel spot. The mass differences between adjacent fragments are characteristic of a particular amino acid residue and the sequence was identified from this spectrum as LLYGGSVTGATCK.

Related references:

Ashcroft, A. E., Venter, H., Keen, J. N., Henderson, P. J. F. & Herbert, R. B. (2002) Molecular dissection of membrane transport proteins: mass spectrometry and sequence determination of the galactose-H+ symport protein, GalP, of Escherichia coli and quantitative assay of the incorporation of [ring-2-13C]histidine and 15NH3. Biochem. J., 363, 243-252.

Korchazhkina, O. V., Ashcroft, A. E., Kiss, T. & Exley, C. (2002) The degradation of A-beta 25-35 by the serine protease plasmin is inhibited by aluminium. J. Alzheimer's Disease, 4, 357-367.


7. A Protein Capture SELDI/MALDI Experiment



Proteins have been extracted from milk samples by use of SELDI/MALDI targets pre-prepared with different ProteinChip surfaces. The protrein profiles of milk samples show proteins extracted using a weak cation surface (upper trace) and a strong anion surface (lower trace)

6. Links to other sites

British Mass Spectrometry Society
European Society for Mass Spectrometry International Mass Spectrometry Society
International Mass Spectrometry Society
International Mass Spectrometry Conference 2003
International Mass Spectrometry Conference 2006
American Society for Mass Spectrometry
Association of Biomolecular Resource Facilities

General interest:
International mass spectrometry web resource
Base peak mass spectrometry resource
SELDI-MS (Ciphergen)
PROWL: a resource for protein chemistry and MS
Capillary electrophoresis and capillary electrochromatography
Back-to-Basics Tutorial (Micromass)
MS web diamonds (Ion Source)
Introduction to electrospray (New Objective)

Proteomics and mass spectrometry:
Cambridge Proteomics Centre
The Proteome Works System
Mascot: identification of proteins by database searching (Matrix Science)
ExPASy: Peptide characterisation software
UCSF protein database search tools for MS