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What is a mass spectrometer?

A mass spectrometer allows us to determine the mass of a molecule such as a peptide from within a heterogeneous mixture. To do this a mass spectrometer uses electromagnetic fields in order to control the movement of charged molecules (ions) within the instrument. These ions need to be in the gas-phase in order to move freely within the instrument in response to the imposed electromagnetic fields. To allow for largely uninhibited movement of the gas-phase ions within the instrument mass spectrometers are contained within a vacuum chamber that has a vacuum generated within it by rotary and turbo-molecular vacuum pumps. This removes the ambient air from the instrument and depending on the type of mass analyser can span a wide range of vacuum strengths. For heterogeneous samples some form of pre-fractionation is used prior to mass spectrometry to reduce the complexity and this is shown below as the inlet. For peptides this is usually reverse phase liquid chromatography. From the inlet the molecules need to become charged and in the gas-phase prior to entrance to the mass spectrometer. This takes place in the ion source where different techniques may be used to generate gas-phase ions. To become positively charged molecules (peptides) gain protons to become positively charged. For peptides previously separated by reverse phase chromatography this is usually electrospray ionisation (ESI). Once within the mass spectrometer the movement of the ions is controlled based upon two of their physical parameters: their charge state (z) and their mass (m). Therefore the mass spectrometer does not measure mass directly but measures a combination of these two parameters the mass-to-charge ratio (m/z). The separation of ions in the mass spectrometer takes place with the mass analyser(s). There are many different types of mass analysers which function based on different physical principles but these devices separate ions with different m/z to allow for their separate detection. The detector within a mass spectrometer is most commonly an electron multiplier were the collision of ions with a charged anode leads to a cascade of increasing number of electrons which can be detected by an electrical circuit connected to a computer (data storage system). However many other types of detectors exists which may be destructive or non-destructive such as the detection system of the orbitrap mass analyser. This data will lead to the generation of a mass spectrum which shows a graph of the relative intensity of all ions detected in the mass spectrometer within a defined period of time versus their m/z. The most intense peak in a mass spectrum is called the “base peak”. The software which displays the mass spectrum will allow the user to zoom in on individual regions or peaks to appreciate the entire dataset in each spectrum.

How do we ionise peptides in LC-MS?

To generate gas phase peptide ions from the online eluate of a reverse phase liquid chromatography (LC) column electrospray ionisation (ESI) is usually used to gerenerate either positive or negatively charged ions. In this method liquid eluting from the chromatography column is either part of or attached to a fused silica capillary emitter which has a pulled-tip of diameter less than 10 um. The liquid is forced through this constriction and for positive ions high voltage (>+1.5 kV) is applied either through a T-junction upstream of the emitter or by coating the emitter (to the tip and in contact with the liquid) in an electrically conductive substance such as silver or gold. This will induce the formation of a spray of positively charged droplets from a Taylor cone generated at the pulled tip and is largely independent of the back-pressure of liquid coming from the LC system. At this point in the process a number of competing models exist for how gas-phase ions are generated from the liquid phase ions using ESI and shown below is a combination model. As solvent evaporates from the charged droplet the repulsive forces due to the build-up of positive charge make the evaporation of gas-phase peptide ions energetically favourable. This force increases as more solvent is evaporated leading to a high proportion of liquid-phase peptide ions under-going phas-transition into the gas-phase. These positively charged gas-phase ions at atmospheric pressure are attracted to the ion source region of the mass spectrometer by a succession of slight negative charges and will be introduced to the mass spectrometer with the suction forces made by the vacuum generated in the vacuum chamber.

What is tandem mass spectrometry?

In order to generate structural information about a peptide ion tandem mass spectrometry (MS/MS) is used to first isolate, fragment and then determine the m/z of the fragment ions produced. Other MS/MS modes are also possible that allow for the detection of specific peptide modifications or specific peptide ion and fragment ion combinations. There are two methods to carry-out an MS/MS method within a mass spectrometer which is tandem in either space or tandem in time. In tandem in space MS/MS ions are controlled as a discrete packet within a single mass analyser. In tandem in time MS/MS which is the classic mode performed by ion-traps, ions of all m/z are first trapped, all but the specific ion which is to be fragmented is ejected from the trap, the trapped ion is then excited to undergo fragmentation (usually collision induced dissociation with Helium gas, CID) and its fragments sequentially ejected onto a detector to generate a mass spectrum of the fragments. This process is often called MS2. Unique to an iontrap this trapping and fragmentation process may be repeated several times in order to fragment the fragments and is called MSn where (n) is the number of MS/MS rounds performed.

How does a quadrupole work?