Quantitative Mass Spectrometry - Supplemental Information

Elucidation of the Transduction Cascade Triggered by B cell Receptors

  • The peptide mixture is separated by means of nano high performance liquid chromatography and ionised by so-called electrospray (ESI) directly at the entry to the mass spectrometer.The peptide mixture is separated by means of nano high performance liquid chromatography and ionised by so-called electrospray (ESI) directly at the entry to the mass spectrometer.

Over the past 20 years, bio-analytical mass spectrometry has developed into a key technology in life sciences. This has resulted in the establishment of proteomics as a new field of research. Proteomics is defined as the investigation of the proteome i.e. the totality of all the proteins in a certain state of cell or an organism.

Read more on the topic of:

  • Mass Spectrometry and Proteomics
  • Bio-analytical Mass Spectrometry
  • Quantification of Proteins
  • Special Applications: Clarification of the B cell Receptor Transduction Cascade

in our print issue of G.I.T. Laboratory Journal Europe 11-12/2012.

Supplemental information:

Full figure description of Figure 1:

Fig. 1: Analysis of a peptide mixture with a Q ToF (A and B) and an Orbitrap (C and D) ESI mass spectrometer (more information in the QR code or at http://goo.gl/KKaEu). The peptide mixture is separated by means of nanoscale high-performance liquid chromatography (HPLC) and ionised by electrospray (ESI) directly at the entry to the mass spectrometer.

A: In a Q ToF (Quadrupole Time of Flight) instrument, initially the molecular mass (more correctly: the mass-to-charge ratio) of the peptide (the "precursor" ion) is determined from its time of flight in high vacuum. A mass spectrum ("MS") is produced.

B: To determine the amino-acid sequence of a peptides, the corresponding precursor ion is separated from all the other peptides, which have different mass-to-charge ratios, in an ion filter (isolation quadrupole, 1st quadrupole). The precursor ion passes on into the collision cell (2nd quadrupole). In the collision cell the pressure is slightly higher, owing to the presence of an inert gas such as argon, helium or nitrogen. Collisions between the peptide and the gas result in the rupture of peptide bonds. The peptide fragments pass on further into the time-of-flight area, and their masses are measured as described above. The fragment spectrum is known as the "MSMS".

C: In an orbitrap mass spectrometer the mass-to-charge ratio is determined not from the time of flight of a peptide or precursor ion, but rather from the frequency with which the precursor ion or peptide moves in the electric field around an axial, spindle-shaped electrode.This method for the determination of the mass-to-charge ratio is very accurate.

By means of a Fourier transform, the different frequencies of several different precursor ions (peptides) which orbit the electrode simultaneously can be converted into their precise mass-to-charge ratios. Before the precursor ions enter the orbitrap analyser, they are first collected in an ion trap; they are then bundled in a second ion trap (C-trap) and subsequently passed into the orbitrap analyser at right angles.

D: The orbitrap mass spectrometer has several methods for fragmenting peptides: CID (collision-induced dissociation) and HCD (high-energy collision-induced dissociation). In CID mode, after precise determination of the molecular weight in the orbitrap analyser (see above), individual peptides are selected in the ion trap, where they are accelerated and fragmented by collisions with helium atoms. The masses of the fragments are measured by the two detectors in the linear ion trap. Alternatively, selected peptides can also be passed into the HCD collision cell, where fragmentation is induced by collisions with nitrogen molecules. The fragments are then passed into the orbitrap, where the various masses of the fragments can be accurately determined. CID has the advantage that the fragment spectrum can be recorded very quickly, almost in parallel with the determination of the molecular weight of the peptides in the orbitrap analyser. HCD has the advantage that very small fragment masses (< 200 Da) can be measured, and the quality of the fragment spectrum is higher than that which can be obtained with CID.

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