The aim of proteomics is the detection and quantification of proteins and their post transla- tional modifications. In top-down proteomics, proteins are detected directly by LC-MS. MS mass accuracy is calculated as a percentage of the mass measured, so the larger the mass, the greater the degree of error. Proteins are therefore poor candidates for analysis because of their large masses. Large numbers of heavy carbon and nitrogen isotope containing amino acids produce a broad isotopic mass range further complicating identification. In bottom-up proteomics, proteins are enzymatically cleaved into peptides of 6 to 20 amino acids. These peptides have small enough masses to enable high mass accuracy. Their small isotopic series means the mono-isotopic peak can be detected, further increasing the accuracy. Peptides are fragmented within the MS to give a fragmentation spectra from which the peptide sequence can be deduced and the protein identity inferred.
Human cells contain more than 10000 proteins each. Digesting these into short peptides drastically increases the complexity of the samples to be analysed. Peptides in proteomic samples are therefore always separated by LC upstream of MS.
Currently most MS operate in a serial fashion, only able to do one measurement at a time. In discovery proteomics or data dependent acquisition (DDA), a top 10 or top 20 approach
is usually used. 10-20 ms2 scans are performed for every ms1 scan. During a ms2 scan only
one peptide is being analysed, all other ions entering the MS are lost. The retention time of a peptide usually ranges from 10-60s. It would therefore need to be detected within this time or it would be lost. In a complex sample there is usually not sufficient time to analyse all
the peptides present, therefore only the most intense peptides are selected for ms2 scans. Not
detecting a protein or peptide does not mean that is was not in the sample, only that the levels were to low to see in the time available.
Figure 2.4: Components of the Thermo scientificTMQ ExactiveTMhybrid quadrupole-
orbitrap mass spectrometer (Q Exactive)92, Ions enter the ion source are focussed by the stacked-ring ion guide (S lens) and then passed through a bent flatapole, eliminating unchanged species. A select range of ions is allowed to pass through the quadrapole, they are then collected in the curved linear ion trap (c-trap). The c-trap focusses the ions and passes them either to the Orbitrap of detection m/z or it can pass them to the high-energy collisional dissociation (HCD) cell for fragmentation, after which the ions are returned to the c-trap and then passed to the orbitrap for detection
Q Exactive The Q Exactive used in this study, is a hybrid MS, with a quad, c-trap, high-
energy collisional dissociation (HCD) cell and an orbitrap
ion source throughout the LC gradient. In ms1 mode the quad allows all ions within 300 m/z
to 1750 m/z into the c-trap. The c-trap collects ions for a defined fill time or until it reaches a set automatic gain control (AGC) threshold. The AGC is determined by the number of charges filling the c-trap. The c-trap focuses the ions which are then passed to the orbitrap for detection. Ions spin around the spindle of the orbitrap oscillating from side to side, the frequency of oscillation is measured. Using fourier transform an accurate m/z is determined for the ions. The time of flight between the c-trap and the orbitrap is also taken into consideration
to increase accuracy. In ms1 mode the the Q Exactive is operated at a resolution of 70 000 full
width at half maximum (FWHM), at 200 m/z and takes 256ms for each scan166 (Figure 2.4).
Ten peptide ions with the greatest intensity are selected for a ms2 mode scan. Each of
the peptides is isolated and collected individually in the c-trap. This is achieved by reducing the mass range of the quad to 2 Dalton (Da) on either side of the selected peptide m/z. The AGC and fill time threshold on the c-trap is reduced, since with a narrow m/z range less ions are required. The isolated peptide ions are collected, transferred to the HCD cell, where the peptide ions are fragment through collision with nitrogen gas. The fragments are transferred back to the c-trap and then to the orbitrap for detection.
Peptide ions fragmented in the Q Exactive HCD preferentially produce a b/y ion fragmen-
tation series (Figure 2.3). This fragmentation series is later used to generate an amino acid
sequence for the isolated peptide through peptide spectral matching (PSM). All ten selected peptide ions are scanned one after the other and placed on a dynamic exclusion list. If the
dynamic exclusion time is set to 30s, these peptide ions will not be selected for ms2 for the
next 30 seconds. The scan resolution in ms2 mode is 35 000, resulting in a scan time of 64ms,
with an additional 10ms for transfer and HCD time. The top ten cycle time is therefore ap-
proximately 1 second. Once all ten ms2 scans are complete another ms1 scan is performed, the
ten peptide ions with the highest intensity not on the dynamic exclusion list are selected for
another round of ms2 scans.
Using upstream fractionation processes it is possible to increase peptide separation. With less peptides being eluted simultaneously, a greater percentage of the total number eluting
at any one time can be identified. It is therefore possible to dig deeper into the proteome. Measuring time and cost is however increased. Measuring time and sample quantity are the two main limiting factors on the depth of analysis.