Core Facility Proteomics & Mass Spectrometry

Glossary

Collision-Induced Dissociation or Collisionaly-Activated Dissociation, both terms are synonyms for the process whereby precursor ions are energetically activated upon which they collide with residual inert gas (He) present in a collision cell (typically an iontrap) and fragment.

Shotgun LC-MS/MS acquisition modes where DDA stands for Data-Dependent Acquisition and DIA for Data-Independent Acquisition. With the latter mode, the mass spectrometer is programmed to isolate larger m/z ion packages for fragmentation and cycles repeatedly through many such isolation windows covering the entire m/z range of interest during a chromatographic run (all peptide ions fragmented all the time), while with DDA only peptide ions seen in a full MS survey scan are isolated in a narrow m/z window and subjected for fragmentation.

ElectroSpray Ionization, a process where analytes dissolved in liquid are ionized by spraying this liquid into a fine mist under an electrical field. Analytes become often multiply charged, depending on the ionizable chemical groups present in the molecule.

Electron Transfer Dissociation, a process whereby positively charged ions are reacted with negatively charged radical ions. The radical transfers its free electron to the analyte ion rendering it unstable, thus induces its fragmentation.

Higher Energy Collision-Induced Dissociation, is a fragmentation process essentially like CID but using larger inert gas molecules (Ar or N2) at higher energy (typically in a quadrupole).

Big proteins can produce per se more peptides upon digestion with a protease than small proteins. The intensity of large proteins overestimates therefore the effectively present molar amount of such a protein in a proteome. iBAQ values are calculated by summing all peptide intensities belonging to same protein followed by normalization by the number of theoretically observable peptides. The number of observable peptides of a protein depends on the protease used to digest the protein and correlates with the protein size (number of amino acids).

Protein quantification made possible by using the chromatographic and spectral information provided by LC-MS/MS analysis of protein digests. No special treatment of the protein samples is required. However, it requires fastidious attention to all steps during sample preparation and LC-MS/MS in order to have the highest reproducibility as possible.

Liquid Chromatography coupled to two stage Mass Spectrometry, meaning that the mass spectrometer cycles between full, or survey scan mode followed by isolation and fragmentation mode of precursor ions present in the full scan during acquisition. The LC part is usually a reversed phase HPLC/UPLC setup.

Label-Free Quantitation intensity as calculated by MaxQuant software. Absolute protein intensities across several samples are normalized across all samples with a sophisticated algorithm in order to compensate measuring errors during sample processing and loading onto the LC-MS/MS instrument.

Matrix Assisted Laser Desorption Ionization, a process to ionize analytes with a laser by the assistance of matrix molecules capturing the energy of the laser light, then evaporate as charged molecules together with the analytes into the gas phase. The formed ions are often singly charged and require a mass analyzer with a big mass window.

Peptide mixtures are fractionated by at least two orthogonal chromatographic techniques prior to mass spectrometry. The two dimensions can be coupled on-line or off-line. While many labs use ion exchange chromatography in the first dimension, we prefer isoelectric focusing due to its better separation power or reversed phase liquid chromatography at basic pH. The second dimension is usually reversed phase chromatography at acidic pH because it is directly compatible with ESI and mass spectrometry.

Analyte solution is delivered in the nano-liter flow regime to a very fine spraying emitter with inner diameters of a couple to a few tens of micro-meters.

Off-Gel fractionation based on the principle of isoelectric focusing where analytes are separated by their isoelectric point in a pH gradient.

Protein Match Score Summation, all scores from peptide spectral matches to one particular protein are added up irrespective of the fact if a peptide was identified several times, with or without modifications. PMSS is sort of an extension of SpS adding a spectral quality factor. The idea is that fragment spectra of peptides coming from high abundant proteins in the sample will result more often in good quality spectra than peptides from low abundant proteins. In the latter case there are less parent ions available for fragmentation what results in decreased fragment ion intensities or noisy spectra which will receive decreased scores. Please note: while PHENYX peptide scores reflect this fact well, other identification software algorithms produce inconsistent scores.

 

Parallel Reaction Monitoring is a targeted approach run on a quadrupole-orbitrap instrument. The peptide precursor ion is isolated with the quadrupole scanning device then fragmented and all fragment ions are read out in the orbitrap detector.

In contrast to PMSS, here only the best peptide scores of each peptide sequence identifying one particular protein, irrespective of its modification state, are added up.

Peptide Spectral Match, defines a potentially correct interpretation of a single fragment spectrum.

These are peptides explaining the most likely proteins from where they came from (issue of protein isoforms). The definition of a razor peptide is based on Ockham’s Razor theory. A razor peptide is one that can be assigned to more than one protein and thus is assigned to the protein with the most independent evidence.

A complex protein mixture, or an entire proteome, is digested by a protease and the resulting peptide mixture is directly analyzed by LC-MS/MS (Lyse'n go).

Spectrum Summation, counting of how many times one particular protein was identified by peptide spectral matches. Others call it spectrum counting.

Selected Reaction Monitoring is a targeted acquisition mode only possible to run on a triple quadrupole instrument. Peptide precursor ions under investigation are isolated in the first quadrupole, fragmented with HCD in the second, and one particular fragment ion is then scanned out to the detector with the third quadrupole. MRM stands for multiplexed reaction monitoring whereby several SRM on the same peptide are performed to scan for different fragment ions.

Stable isotopes are introduced to proteins or peptides (after digestion of proteins) by way of chemical modifications or metabolic means during cell growth. The control sample receives the same treatment but with chemicals containing naturally occurring isotopes. Samples are mixed and relative protein expression is read out from the mass spectra by means of the mass difference between the introduced isotopes. The advantage over label-free methods relies in the possibility that different samples (e.g. wild type and knock-out, healthy and diseased) are treated in parallel and analyzed at the same time. Disadvantages are additional sample treatment steps (which result often in protein losses and incomplete reactions), the need to have always a control present, and the costs for reagents.

In shotgun type of experiments proteomes are investigated in a hypothesis free manner, or exploratively, in order to answer the question “what is there?”. In a targeted approach the mass spectrometer is programmed to isolate and fragment only precursor ions of interest. Targeted approaches are more sensitive than shotgun analysis, as the signal-to-noise ratio is dramatically increased. Targeted techniques are SRM/MRM or PRM. DIA together with the use of a fragment spectrum library can be considered as a hybrid between shotgun and targeted approach.

A Top3 protein intensity is calculated by the sum of the three most intense peptides coming from the same protein. The Top3 approach compensates protein intensity bias introduced by the size of a protein (see explanations about iBAQ).