Instrumentation

Mass spectrometry

Organic mass spectrometry is a versatile technique for determining both the structure and constitution of single compounds or mixtures.
Today the most important ionization techniques in peptide and protein analytics are electrospray (ESI), its miniaturized variant nanoelectrospray (nanoESI), and matrix-assisted laser desorption/ionization (MALDI). In our research group, for instance, the following two instruments are employed.

· Electrospray ionization (ESI) and nanoelectrospray ionization (nanoESI):

Quadrupole time-of-flight hybrid mass spectrometer Q-TOF (Waters/Micromass)
with nano-UHPLC U3000 RSLCnano (Dionex/Thermo Fisher)

Quadrupole time-of-flight hybrid mass spectrometer Q-TOF-2 (Waters/Micromass)
with nano-HPLC U3000 (Dionex/Thermo Fisher)

NanoESI source (left) and nanoESI emitter (right)

· MALDI-TOF and TOF/TOF mass spectrometry:

LC-MALDI-TOF system consisting of a nano-UHPLC U3000 RSLCnano, a Probot microfraction collector (Dionex/Thermo Fisher; left image) and a MALDI-TOF-MS Voyager DE-PRO (AB Sciex; right image)

Automated MALDI target spotting of reversed phase nano-HPLC fractions

Tandem mass spectrometry

Tandem mass spectrometry, also known as MS/MS or MS², is a technique which allows the fragmentation of preselected ions. The mass analysis involves two major steps. In the first step, gas-phase ions of the analyte under investigation are isolated in the first mass analyzer and then dissociated through collision with inert gas atoms or molecules (e.g. nitrogen, argon, or helium). During this process which is called collision-induced dissociation (CID) ions of lower mass are generated. These fragment ions are then seperated in a second analyzer and finally detected.

Schema of the formation of fragment ions of peptides using the example of a tripeptide

Tandem MS experiments of peptides using ESI mass spectrometers mainly yield in cleavages of the peptide backbone and generation of the so called b- and y-ions (see figure). These ions are very useful for the determination of the identity of the peptides. For this purpose different sequencing approaches can be employed.