Skip to the content
 

Electrospray

Bruker HCT Ultra ion trap with ETD and Bruker micrOTOFq

Highly sensitive with a superior combination of mass accuracy, mass resolution, and scan speed.

Powerful techniques in combination with online HPLC, we have the Agilent 1200 capillary HPLC or higher sensitivity achieved with the Dionex Ultimate 3000 nanoLC.

Theory

  • Ionisation is achieved through a nebulization process were the sample in solution is sprayed into a spray chamber containing a nebulizing gas and an electric field which disperses the sample into fine droplets of charged particles.
  • The charged analyte molecules are desolvated by a drying gas before entering the mass analyzer.
  • The ion trap consists of a ring electrode between two cap electrodes so the ions are accumulated prior to being ejected for analysis giving the instrument superior sensitivity.
  • The MicrOTOFq consists of a quadrupole and TOF chamber for maximal resolution and mass accuracy.
  • In ESI-MS peptide and proteins are ionized several times giving each molecule multiple charges. In peptides doubly and triply charged ions are most common. The detection of these multiply charged ions makes the spectra significantly more complex than MALDI spectra however given the limited mass scanning range such as 50-3000 m/z of ESI instruments it allows them to detect molecules of masses up to a very high range eg 60kDa.
  • Both the ion trap and QTOF are able to perform MSMS analysis and posses the ability to select precursor ions and induce fragmentation through CID, this fragmentation is more effective in yielding more complete sequence data than can be obtained on the MALDI.
  • The ion trap has an additional fragmentation mechanism called Electron transfer Dissociation (ETD) which adds an extra dimension to the ability to sequence and characterize PTM's.


Typical Experiments and Procedures

Peptide and Protein Mass Determination

  • The high level of accuracy makes the QTOF the instrument of choice for mass determination of peptides and proteins.
  • The spectrum bearing the m/z values of the different charged states observed is deconvoluted to yield mass values with 5ppm accuracy.
  • ESI mass spectrometry requires the samples to be relatively salt free so samples are usually purified/desalted with online liquid chromatography. For samples to be infused directly requires a good desalting procedure and thus often requires a large amount of sample.
  • The presence of PTM's such as glycosylation unless strongly homogenous can yield very complex spectra which would be almost impossible for the deconvolution software to process. In which case the results will be difficult to interpret with confidence.

LC-MSMS

  • For samples that are complex and require purification LC-MS is a technique that combines the separation power of HPLC, with the highly sensitive detection of the mass spectrometer.
  • The fast scanning speed of the ion trap allows a continuous sampling of eluting peptides for MSMS analysis for sequencing or identification.

Protein Identification.  LC-MSMS

  • Unlike MALDI which uses data obtained from detecting the maximal observable peptides in a tryptic digest, LC-MSMS requires as few as one or two peptides to confidently identify a protein due to the more efficient CID fragmentation yielding specific sequence information.
  • The quality of CID data obtained also results in more confident identifications than those obtained by MALDI-TOFTOF.
  • A further advantage is that from a simple mixture of proteins most proteins will be able to be identified. For more complex samples such as extracts, multi-dimensional chromatography can be used to increase the separation capacity.
  • Identification by MSMS sequencing is a highly sensitive and effective method but is costly and time consuming compared with a MALDI analysis.
  • We recommend this technique when:
    - The sample contains salts or buffers which are easily removed by the HPLC.
    - Your samples are relatively complex 3 or 4 proteins (ie. 1D gel spot).
    - You are working with organisms that are not well-characterized genome and would rely on sequence homology for identification.

Protein Quantitation: Isobaric mass tagging

  • The use of isobaric tags such as ITRAQ, ICAT, SILAC, Exactag etc allows relative the quantitation of individual proteins from two different samples treated with a different tag.
  • Quantitation determined by measuring the relative intensity of reporter fragments in the MSMS spectra.

Post translational Modification Analysis

  • The ion trap is a powerful instrument for PTM analysis with the combination of CID for peptide sequence analysis and ETD a fragmentation technique which leaves the PTM intact to enable definitive residue assignment on which the PTM is located.

Sample Information and Preparation Required

  • Sample origin and species are helpful in making protein assignments based on database searching results.
  • Knowledge of sample buffer is important because not all buffers and additives are compatible with HPLC or mass spectrometry.
  • If you are providing a gel, please indicate what the gel is stored in, known and possible modifications (i.e. cys alkylation, phosphorylation) are also very important in obtaining accurate results from database searching.
  • For Direct Injection:

    Samples must be pure and free of salts such as NaCl, K2HPO4 or buffers. The positive ions (Na+, K+, Ca2+) can substitute for H+ on the peptide, and complicate interpretation of the spectra and they can also be reduced in the mass spectrometer and plate out on the ion optics, damaging the instrument Negative ions (Cl-, SO42-) can form ion pairs with basic sites and neutralize the positive charge of the peptide rendering it undetectable. Furthermore, positive salt ions can.
  • Solvent components for direct injection = 50% H2O + 50% Organic Solvent + 0.1% Formic acid. 
  • Organic Acid such as formic acid or acetic acid is used to ensure to protonation of the analyte enabling them to be observed in the mass spectrometer. Although TFA is widely used in reverse phase HPLC and mass spectrometry, it can form strong ion pairs with the positively charged analyte, decreasing sensitivity.
  • Organic Solvent is typically acetonitrile as it is commonly used in reverse phase HPLC, but other organic solvents such as methanol, ethanol, n-propanol, isopropanol can be used. HPLC grade solvents are essential.
  • The 1:1 ratio between H2O and organic is not critical but an organic solvent is added to increase the solubility of the analyte and to promote desolvation of the charged droplets formed by the electrospray process.
  • All stabilizers such as glycerol or PEG and ionic and non-ionic detergents such as SDS and Triton-X must be completely avoided.

For HPLC-MS

  • Samples can contain any salts and buffers compatible with a reversed phase HPLC system.
  • The concentration of organic solvent if required, must be at a minimum, <5%, to avoid losses of peptides washing through the HPLC system.
  • Samples must not contain stabilizers such as glycerol or PEG and ionic and non-ionic detergents such as SDS and Triton-X are very difficult to remove with HPLC so should be avoided even during sample preparation.