level: Mass Spectrometry
Questions and Answers List
Flashcards on the case and lecture on Mass Spectrometry
level questions: Mass Spectrometry
| Question | Answer |
|---|---|
| what are the general steps of mass spectrometry? | 1. ionization: the sample is ionized by a beam of electrons, because of which an electron is removed, creating cationic ions. 2. acceleration: all ions undergo acceleration through an electric or magnetic field, to have the same amounts of kinetic energy. 3. deflection: depending on the mass-to-charge ratio, the ions will undergo different amounts of deflection in the magnetic field. small ions and ions with a greater positive charge will get deflected more. 4. detection: a detector, such as an electron multiplier, will detect all cationic ions. neutral and anionic ions are removed by a vacuum. |
| what is MALDI? | MALDI means matrix assisted laser desorption ionization, which is an ion source. the principle of MALDI is as follows: 1. an organic, crystalline matrix is added in excess to the sample. 2. the sample is irradiated by a laser. 3. there is vaporization of the analyte molecules. 4. the analyte molecules are ionized. 5. single ion images are generated. |
| what is the purpose of the matrix in MALDI and which one can best be used? | the matrix layer absorbs the energy from the irradiation laser and induces desorption and ionization of the analytes. to not introduce any errors, the matrix crystal deposition should be homogenous to ensure high sensitivity, reproducibility and artefart-free imaging. for imaging proteins in the brain: 2,6-DHA matrix for imaging lipids: 1,5-DAN or CHCA. |
| what is the principle of electrospray ionization? | electrospray ionization (ESI) is an ion source. in electrospray ionization, ions are continuously generated at atmospheric pressure by guiding a solution-based sample through a small tube, to which a voltage is connected. the sample solution is then electrostatically sprayed to generate an aerosol of charged droplets, which make their way into the mass analyser. |
| what is the principle of secondary ion mass spectrometry (SIMS)? | SIMS is an ion source that works as follows; 1. a focused primary ion beam bombards the sample with primary ions. 2. because of this, secondary ions are ejected from the sample. 3. secondary ions are collected and analysed in the mass spectrometer. SIMS is mainly used to analyse the composition of solid surfaces and thin films. the application to biological samples is limited since SIMS has insufficient sensitivity and spatial resolution. |
| what is a time-of-flight (TOF) mass analyser? | a TOF tube is a type of mass analyser. the ions that are created through an ion source are separated based on their velocity. the ions are accelerated by a certain voltage in the TOF drift tube. ions with lower m/z ratios will achieve a higher velocity and will be detected first by the detector. by measuring the time it takes to reach the detector, the m/z ratio can be determined and the ions can be identified. |
| what are advantages and disadvantages to mass spectrometry? | advantages: high sensitivity and able to recognize small molecules, high selectivity, fast analysis, provides quantitative results, widely applicable (used to detect proteins, lipids, nucleic acids etc.), can be used for the identification of unknown molecules. disadvantages: expensive equipment, skilled personnel needed, sample preparation can be extensive and challenging (embedding and fixation may introduce artifacts), large proteins are difficult to analyse, ionization process can be destructive to sample. |
| what are advantages and disadvantages of MADLI specifically? | advantages: very little sample is wasted, singly charged analytes are usually generated, high troughput, matrix facilitates ionization, allows for molecular mass determination of the analytes while keeping their spatial resolution in tact. disadvantages: difficult to couple to certain mass analysers due to its sensitivity, matrix causes chemical noise in samples with low molecular weights |
| how does mass spectrometry allow for protein detection? | - peptide mass fingerprinting: the measured proteolytic peptide masses are matched to the theorectical proteolytic peptide masses of the proteins. this can be done because you know where trypsin is supposed to cut. if you find peptides that are very rare, proteins are easily identified. but if you find peptides that are very common, many different peptides are needed to identify the protein. - labelling: the sample is labelled with stable isotopes, which are visualised after analysis. |
| what are different ion sources, mass analysers and ion detectors that can be used in mass spectrometry? | ion sources: MALDI, (desorption) electrospray ionization ((D)ESI), direct analysis in real time (DART), fast atom bombardment (FAB), electron ionization (EI). mass analysers: time-of-flight (TOF), quadrupole, magnetic sector, ion trap. ion detectors: electron multiplier, faraday cup, array detectors, photomultiplier conversion dynode. |
| what can be seen in a mass spectrum? | the mass spectrum shows the intensity (y-axis) as a function of the mass-to-charge ratio (x-axis). the higher the peak of a certain m/z ratio, the higher the relative presence of the compound belonging to that peak in the sample. small molecules typically have a single charge, which means that their m/z ratio is the same as their mass. however, larger molecules often have a charge (z) greater than 1, which means that the m/z ratio is not the same as their mass. |
| what the differences between the genome and the proteome? | genome: static, can be amplified with PCR, little sample complexity, good solubility, only 4 base pairs. proteome: dynamic (variable with time), cannot be amplified, high sample complexity, various solubility. |
| what is mass spectrometry? | MS is an analytical technique that separates ionized particles by using differences in the ratios of their charges to their respective masses and can be used to determine the molecular weight of particles. MS is used to identify molecules that are present in solids, liquids and gases, to determine the quantity of each type of molecule and to determine which atoms comprise a molecule and how they are arranged. |
| what are bottom up and top down approaches of sample analysis? what are the differences? | in the bottom up approach, intact proteins are digested into peptides prior to introduction into the mass spectrometer. this approach is very user friendly, generates a high throughput and gives more information about proteins with 'extreme' properties. but posttranslational modifications and isoform information is lost. in the top down approach, intact proteins are introduced into the mass spectrometer. this keeps posttranslational modifications and protein isoforms intact, but has limited sensitivity and throughput and quite pure samples are required and expensive instrumentation is needed. |
| what is 2D electrophoresis, why is it used in relation to mass spectrometry and what are some pros and cons? | 2DE is a technique that is used to separate proteins prior to mass spectrometry. proteins are separated along one axis based on isoelectric point (pH at which the net charge becomes zero) and along a different axis based on molecular weight. proteins of interest can then be 'cut' out and introduced into mass spectrometry. pros: reflects changes in protein expression, isoforms or posttranslational modifications, is able to separate many proteins, inexpensive. cons: time consuming, multiple proteins can remain present in one spot, only detects high abundance proteins, large proteins cannot be seen. |
| what occurs when proteins are digested? | 1. reduction with DTT to break the disulfide bonds 2. alkykation with IAM to block cysteines in proteins (if present) to prevent forming disulfide bonds again. 3. digestion with trypsin, which breaks the peptide bonds between the carboxyl group of arginine or lysine and the amino group of the adjacent amino acid. |
| what are different protein labelling techniques? | stable isotope labelling metabolic (SILAC): label introduced in first stage of analysis, very accurate labelling. chemical labelling: an isotope coded affinity tag (ICAT) is added to cysteines. isobaric peptide tagging: labelling is done after protein extraction and denaturation (all tags have identical mass). |
| what are the differences in speed and spatial resolution between MALDI, DESI and SIMS? | the spatial resolution of SIMS is the best out of the three. then comes MALDI and then DESI. but the higher the resolution, the smaller the amount of sample that can be ionized at the time. DESI is the fastest out of the three. SIMS takes quite a lot of time, MALDI is quite fast but time is lost in preparing the matrix. |